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GLOBAL GENE EXPRESSION ANALYSIS OF

CRANIAL NEURAL TUBES IN EMBRYOS OF


DIABETIC MICE

JIANG BORAN

NATIONAL UNIVERSITY OF SINGAPORE

2008
GLOBAL GENE EXPRESSION ANALYSIS OF
CRANIAL NEURAL TUBES IN EMBRYOS OF
DIABETIC MICE

JIANG BORAN

MBBS

A THESIS SUBMITTED FOR THE DEGREE OF


DOCTOR OF PHILOSOPHY

DEPARTMENT OF ANATOMY
YONG LOO LIN SCHOOL OF MEDICINE
NATIONAL UNIVERSITY OF SINGAPORE

2008
ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

I would like to express my deepest appreciation to my supervisor, Associate

Professor S Thameem Dheen, Department of Anatomy, National University of

Singapore, for his innovative ideas, invaluable guidance and constant

encouragement throughout this study.

I am greatly indebted to Professor Ling Eng Ang, Head of Department of

Anatomy, National University of Singapore, for his full support in providing me

with the first class research facilities and an excellent academic environment, as

well as for his valuable suggestions to my career.

I am also thankful to Associate Professor Samuel Tay Sam Wah and Dr.

S Dinesh Kumar, Department of Anatomy, National University of Singapore, for

their kind suggestions and constructive criticisms.

I am happy to acknowledge my gratitude to Mrs Yong Eng Siang

(Neuroscience Lab), Mrs Ng Geok Lan (Histology Lab), Miss Chan Yee Gek

(Confocal Microscopy Lab), Mdm Wu Ya Jun (Electron Microscopy Unit), Mr

Yick Tuck Yong (Multimedia Unit), Mr Poon Zhung Wei (Autoclave Room),

and Mr Lim Beng Hock (Animal House) for their technical assistance and Mdm

Ang Lye Gek, Carolyne, Ms Teo Li Ching Violet and Mdm Diljit Kour for

their secretarial assistance.

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ACKNOWLEDGEMENTS

I would like to express my thanks to Miss Evelyn Loh Wan Ting for her

kind help and collaboration in the research.

I would like to extend my thanks to all staff members, my fellow graduate

students in Department of Anatomy, National University of Singapore for their

help and support one way or another.

I would like to express my special thanks to my friends, Dr Jayapal

Manikandan and Dr Peter Natesan Pushparaj at the Department of Physiology,

National University of Singapore for their friendly help and advice.

Certainly, without the financial support from the National University of

Singapore in terms of Research Scholarship and Research grant

(R-181-000-038-112 to Associate Professor S T Dheen), this work would not have

been brought to a reality.

I would like to express my heartfelt thanks to my parents for their full and

endless support for my study.

Finally, I would like to thank my wife, Mdm Yin Na, for her full support,

encouragement and assistance during my study.

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ACKNOWLEDGEMENTS

This thesis is dedicated to


my beloved family

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TABLE OF CONTENTS

CONTENTS

ACKNOWLEDGEMENTS......i

TABLE OF CONTENTS........................................................iv

LIST OF ABBREVIATIONS.....................................................xi

SUMMARY... xiii

PUBLICATIONS. xvii

Chapter 1: Introduction......1

1. Diabetes Mellitus.. 2

1.1. Definition and classifications of diabetes mellitus........ 2

1.2. The complications of diabetes mellitus. 3

1.3. Maternal diabetes... 5

1.3.1. Congenital malformations in embryos of diabetic mother... 6

2. Neural tube development.. 7

2.1. Neurulation............................................................. 7

2.1.1. Neural tube defects............................................................... 9

2.2. Neurogenesis in the developing cranial neural tubes.... 10

2.2.1. Developmental control genes in the process of neurogenesis..11

2.2.1.1. Doublecortin (Dcx).....12

2.2.1.2. Brain lipid binding protein (Blbp)..12

2.2.1.3. Paired box gene 6 (Pax6)....................13

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2.2.1.4. Neurogenin 2 (Ngn2) 15

2.3. Glucose metabolism and hypoxia in the developing cranial neural tubes 16

2.3.1. Glycolysis pathway.17

2.3.2. Hypoxia in the developing cranial neural tubes..18

2.3.2.1. Hypoxia-inducible factor 1 (Hif-1) 19

2.3.2.2. Vascular endothelial growth factor (Vegf). 21

2.4. Choroid plexus development.... 21

2.4.1. Choroid plexus (CP)21

2.4.2. Development of CP in mouse embryos...22

2.4.3. Factors involved in CP development...... 23

2.4.3.1. Transthyretin (Ttr). 23

2.4.3.2. Insulin-dependent growth factor 2 (Igf-2)..24

3. Analysis of global gene expression profile using DNA microarray25

3.1. Overview of DNA microarray technology 25

3.2. Basic principle of DNA microarray.. 26

3.3. Applications of DNA microarray.. 28

3.4. Data analysis strategies..... 28

4. Animal models.... 29

4.1. Animal models of diabetes mellitus. 29

4.1.1. Mechanism of action of streptozotocin (STZ) 30

4.2. Animal model of maternal diabetes. 30

5. Hypotheses and Objectives......................................... 32

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TABLE OF CONTENTS

5.1. Specific aims. 35

Chapter 2: Materials and Methods. 37

1. Animals38

2. Induction of diabetes mellitus 40

2.1. Blood glucose test 41

2.2. Collection of embryos..41

3. Histology. 42

3.1 Fixation of embryos.. 42

3.2. Preparation of silane coated slides43

3.3. Cryosection of embryos44

3.4. Haematoxylin and Eosin staining.44

4. RNA extraction and quantification..45

5. Microarray analysis..49

5.1. Synthesis of double stranded cDNA from total RNA49

5.2. cDNA cleanup and precipitation.. 51

5.3. Synthesis of Biotin-labeled cRNA52

5.4. cRNA clean up and quantification... 52

5.5. Fragmentation the cRNA for target preparation54

5.6. Target hybridization..54

5.7. Post-hybridization washing, staining and scanning of arrays.. 55

5.8. Data analysis.... 57

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TABLE OF CONTENTS

5.8.1. Absolute data analysis57

5.8.2. Comparison data analysis...58

5.8.3. Gene filtration.58

5.8.4. Gene clustering and functional analysis based on Gene Ontology58

6. Real time reverse transcriptase polymerase chain reaction (Real time RT-PCR)... 59

7. Immunohistochemistry (IHC). 65

8. Immunofluorescence staining. 69

9. BrdU labeling analysis 70

10. Terminal Deoxynucleotidyl Transferase -mediated dUTP Nick-End Labeling

analysis (TUNEL). 73

11. In situ Hybridization..75

11.1. Plasmids for preparation of cRNA probes.76

11.2. Preparation of competent cells..76

11.3. Transformation of competent cells with plasmids 78

11.4. Linearization of the plasmid..79

11.5. Synthesis of digoxigenin-labeled RNA probe from the linear DNA81

11.6. Whole mount of in situ hybridization...82

12. Western blot analysis.87

Chapter 3: Results 92

1. Neural tube defects in embryos of diabetic mice 93

2. Gene expression profile of cranial neural tubes in embryos of control

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TABLE OF CONTENTS

and diabetic mice 94

3. Effect of maternal diabetes on expression of genes involved in glucose

metabolism and that respond to physiological hypoxia in cranial neural

tubes of mouse embryos. 95

3.1. Maternal diabetes altered the expression of genes involved in glycolysis in

cranial neural tubes of mouse embryos 95

3.2. Maternal diabetes altered the expression of genes that respond to hypoxia in

cranial neural tubes of mouse embryos 96

3.2.1. Expression of hypoxia-inducible factor 1 (Hif-1) in cranial neural

tubes of embryos from control and diabetic mice.. 96

3.2.2. Expression of Vegf in cranial neural tubes of embryos from control

and diabetic mice97

4. Maternal diabetes induces apoptosis in the neuroepithelium of cranial neural

tubes in mouse embryos.......................................................................................... 98

5. Maternal diabetes inhibits proliferation index in the neuroepithelium of cranial

neural tubes in mouse embryos... 98

6. Maternal diabetes alters the expression of genes involved in neuronal

migration and neurogenesis in cranial neural tubes of mouse embryos. 99

6.1. Expression pattern of developmental control genes is altered in

embryos of diabetic mice...100

6.1.1. Paired box gene 6 (Pax6). 100

6.1.2. Neurogenin 2 (Ngn2)100

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TABLE OF CONTENTS

6.2. Development of radial glial cell lineages and migration of neurons

are disrupted in the cranial neural tubes of embryos from diabetic mice.. 101

6.2.1. Doublecortin (Dcx).... 101

6.2.2. Brain lipid binding protein (Blbp)102

7. Development of choroid plexus is impaired in cranial neural tubes

of embryos from diabetic mice. 103

7.1. Transthyretin (Ttr).. 103

7.2. Insulin-like growth factor 2 (Igf-2).................... 104

7.3. The proliferation index in the choroid plexus and adjacent

neuroepithelia.... 105

Chapter 4: Discussion..106

1. Apoptotic cells are increased and mitotic index is decreased in cranial

neural tubes of embryos from diabetic mice..108

2. Expression of genes involved in neuronal migration and differentiation

is altered in cranial neural tubes of embryos from diabetic mice. 110

3. Expression of genes that are involved in glucose metabolism and that respond

to hypoxia is altered in cranial neural tubes of embryos from diabetic mice112

4. Development of choroid plexus is impaired in cranial neural tubes

of embryos from diabetic mice. 115

Chapter 5: Conclusion............................................ 118

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References.123

Figures and Figure Legends149

Tables:

Table 1.. 39

Table 2.. 63

Table 3.. 68

Table 4.. 70

Table 5 199

Table 6 200

Table 7 202

Table 8 203

x
LIST OF ABBREVIATIONS

LIST OF ABBREVIATIONS
1,3-BPG: 1,3-bisphosphoglycerate
2-PGA: 2-phosphoglycerate
3-PGA: 3-phosphoglycerate
ABC: Avidin-Biotin complex
ALD: aldolase
bHLH: basic helix-loop-helix
Blbp: brain lipid-binding protein
BNIP3: BCL2/adenovirus E1B 19 kDa interacting protein 3
BrdU: 5-bromo-2-deoxyuridine
CNS: central nervous system
CP: choroid plexus
CSF: cerebrospinal fluid
DAB: diaminobenzidine tetrahydrochloride
dc: diencephalon
Dcx: doublecortin
DEPC: Diethyl Pyrocarbonate
DHAP: dihydroxyacetone phosphate
DLHPs: dorsolateral hinge points
DM: diabetes mellitus
ENO: enolase
F-1,6-BP: fructose 1,6-bisphosphate
F-6-P: fructose 6-phosphate
FABP: fatty acid-binding protein
G-6-P: glucose 6-phosphate
GADP: glyceraldehyde 3-phosphate
GAPDH: glyceraldehyde-3-phosphate dehydrogenase
GPI: glucosephosphate isomerase
HCl: hydrochloric acid
Hif-1: Hypoxia-inducible factor 1
HK: hexokinase
IDDM: insulin-dependent diabetes mellitus

xi
LIST OF ABBREVIATIONS

Igf-2: insulin-like growth factor 2


LDH: lactate dehydrogenase
MAP: microtubule associated protein
MHP: the median hinge point
Ngn2: neurogenin 2
NIDDM: noninsulin-dependent diabetes mellitus
NTDs: neural tube closure defects
Pax6: paired box 6
PBS: phosphate-buffered saline
PCR: polymerase chain reaction
PEP: phosphoenolpyruvate
PFK: phosphofructokinase
PGK: phosphoglycerate kinase
PGM: phosphoglycerate mutase
PK: pyruvate kinase
PYR: pyruvate
rc: rhombencephalon
SDS: sodium dodecyl sulfate
STZ: streptozotocin
T4: thyroxine
TBS: Tris buffered saline
tc: telencephalon
TPI: triosephosphate isomerase
Ttr: transthyretin
TUNEL: Terminal Deoxynucleotidyl Transferase -mediated dUTP Nick-End Labeling
Vegf: vascular endothelial growth factor

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SUMMARY

SUMMARY

A high frequency of pregnancy complications including perinatal mortality and

congenital malformations such as neural tube anomalies has been shown in

women with diabetes mellitus. However, the underlying mechanisms contributing

to these malformations are not clear. The aim of this study was to investigate the

molecular and morphological changes in cranial neural tubes of embryos from

diabetic mice.

Morphological analysis revealed malformations in cranial neural tubes,

particularly in the telencephalon, diencephalon, and rhombencephalon as well as

the ventricular system of E11.5 embryos from diabetic mice. The neuroepithelia of

the forebrain and hindbrain and ventricles appeared to be distorted and fused. The

molecular changes were analyzed by oligonucleotide microarray which is a useful

tool to evaluate the expression patterns of thousands of genes involved in

morphogenesis, cellular functions as well as biochemical and metabolic pathways

in cranial neural tubes of embryos from diabetic mice. Overall, 1613 genes have

been found to be differentially expressed in cranial neural tubes of embryos from

diabetic mice. Among these genes, a total of 390 genes exhibiting greater than

2-fold changes has been placed into 8 main functional categories as follows: 1.

metabolism (27.7%); 2. cellular physiological process (20.3%); 3. cell

communication (12.1%); 4. morphogenesis (6.9%); 5. response to stimulus (3.8%);

6. cell death (2.3%); 7. cell differentiation (2.1%); 8. small subsets of genes

(5.4%).

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SUMMARY

Further, the microarray analysis shows that several genes involving cell cycle

progression, migration and differentiation of neuronal and glial cells in cranial

neural tubes were differentially expressed in embryos of diabetic pregnancy. A

majority of those genes including doublecortin (Dcx), doublecortin-like kinase

(Dclk), brain lipid binding protein (Blbp), insulin-like growth factor-2 (Igf-2),

paired box gene 6 (Pax6), and Neurogenin 2 (Ngn 2) were downregulated in

embryos of diabetic pregnancy. In addition, maternal diabetes altered the cell

cycle progression in cranial neural tubes by regulating the expression of genes

involved in apoptosis and cell proliferation. These results indicate the disruption

of neuronal migration, neurogenesis as well as axonal wiring, which may

subsequently result in malformations in developing brains of embryos from

diabetic mice.

The neural tube malformation appears to be the result of multiple causes

including metabolic abnormalities caused by maternal diabetes. Genes involved in

pathways related to glucose metabolism and hypoxia have been found to be

altered in cranial neural tubes of embryos from diabetic pregnancy. Maternal

diabetes has been shown to induce excessive physiological hypoxia and oxidative

stress, contributing to severe pathological conditions in embryos. In the present

study, the maternal diabetes-induced physiological hypoxia appeared to have

triggered an adaptive response in the cranial neural tube by activating the

expression of several genes encoding enzymes involved in aerobic and anaerobic

glycolysis pathways. However the adaptive response seems to be insufficient to

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SUMMARY

reverse the hypoxic state of the neural tissue in embryos from diabetic mice as

there was an upregulation of hypoxia-inducible factor-1 (Hif-1) which is

activated under hypoxia and rapidly degraded in the presence of oxygen. The

upregulation of Hif-1 expression is harmful to the development of cranial neural

tubes in embryos of diabetic pregnancy as it has been shown to promote apoptosis

and cell growth arrest. Taken together, the altered expression of genes that

respond to hypoxia appears to be associated with cranial neural tube

malformations in embryos from diabetic mice.

In addition, maternal diabetes appears to impair the development of choroid

plexus (CP) and ventricular system in embryos. CP produces cerebrospinal fluid

(CSF) which determines the shape of the developing brain and transfers nutrients,

proteins and other molecules required for neuroepithelial cell survival as well as

proliferation and neurogenesis during brain development. The CP produces

several proteins including insulin-like growth factor 2 (Igf-2), which functions as

a morphogen in inducing differentiation of CP epithelial cells and transthyretin

(Ttr) which is involved in the transport of thyroxine and retinol, that regulate

neuronal differentiation in the developing brain. Malformation of the CP and the

ventricular systems together with reduced production of Ttr and Igf-2 in cranial

neural tubes of embryos from diabetic mice appear to influence the neuroepithelial

survival, proliferation and neurogenesis. It is possible that these changes may

impede further development of functional domains of the brain and subsequently

contribute to intellectual impairment in the offspring of diabetic mothers.

xv
SUMMARY

However, an extensive follow-up study is required to understand the molecular

mechanisms and consequence of cranial neural tube malformations observed in

embryos of diabetic mice.

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PUBLICATIONS

PUBLICATIONS

Journals:

1. Boran Jiang, SD Kumar, WT Loh, J Manikandan, EA Ling, SSW Tay and

ST Dheen, Global gene expression analysis of cranial neural tubes in

embryos of diabetic mice. (Journal of Neuroscience Research,

E-publication ahead of print, 2008)

2. Boran Jiang, SD Kumar, WT Loh, EA Ling, SSW Tay and ST Dheen,

Hypoxic stress in the cranial neural tubes of embryos from diabetic mice.

(In preparation)

3. Wan Ting Loh, S Thameem Dheen, Boran Jiang, S Dinesh Kumar,

Samuel S W Tay, Molecular and morphological characterization of caudal

neural tube defects in embryos of diabetic Swiss Albino mice. (BMC

Developmental Biology, submitted)

Conferences:

1. Jiang B, Loh E, Kumar SD, Tay SSW, Ling EA, Dheen ST, Development

of Choroid Plexus in the Neural Tube of Embryos from Diabetic

Pregnancies. (3rd Singapore International Neuroscience Conference, 23-24

May 2006, Singapore)

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PUBLICATIONS

2. B Jiang, D Kumar, S S W Tay, EA Ling, S T Dheen, Global analysis of

gene expression patterns in the developing brain and malformation of

choroid plexus in embryos of diabetic mice. (15th International Society of

Developmental Biologists Congress, 3-7 September 2005, Sydney,

Australia)

3. Jiang Boran, Dheen S Thameem, Ling Eng Ang and Tay Samuel S W, A

large-scale oligonucleotide microarray analysis of gene expression patterns

in the developing brain of embryos of diabetic mothers. (8th NUS-NUH

Annual Scientific Meeting, 7-8 October 2004, Singapore)

4. S.T. Dheen, B.Jiang, E.A.Ling, S.S.W.Tay, Analysis of global gene

expression patterns in the developing brain of mouse embryos derived

from diabetic pregnancy using the oligonucleotide microarray. (Society for

Neuroscience 34th Annual Meeting, 23-27 October 2004, San Diego,

United States)

xviii
CHAPTER 1: INTRODUCTION

CHAPTER 1

INTRODUCTION

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CHAPTER 1: INTRODUCTION

1. Diabetes Mellitus

Diabetes mellitus is a metabolic disorder characterized by high blood glucose

levels which result from deficiency in secretion or action of insulin. It affects

various organ systems in the body. The prevalence of diabetes among all age

groups worldwide in 2000 was estimated to be 2.8%, which was approximately

171 million people. The total number of diabetics is projected to rise to 366

million which is 4.4% in the population by 2030 (Wild et al., 2004). In Singapore,

the incidence of diabetes is increasing while Singaporeans are becoming more

affluent, their lifestyles are more sedentary and population is ageing rapidly.

According to the findings from 1998 National Health Survey of Singapore, the

prevalence of diabetes is approximately 9% among Singaporeans aged from 18 to

69 years (Tan Bee Yian, Epidemiology and Disease Control Department, Ministry

of Health Singapore, 1998).

1.1. Definition and classifications of diabetes mellitus

The term diabetes is derived from Greek meaning pass through which refers to

excessive urine production. The word mellitus means "honey", a reference to the

sweet taste of the urine in Latin. Diabetes mellitus (DM) is a metabolic syndrome

characterized by hyperglycaemia with disturbances of carbohydrate, lipid and

protein metabolism which is caused by deficiency of insulin secretion, abnormal

resistance to insulin's action or both (American Diabetes Association, 2007). The

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CHAPTER 1: INTRODUCTION

chronic hyperglycemia is associated with cardiovascular diseases, chronic renal

failure, retinal damage, nerve damage, and microvascular damage.

Diabetes mellitus is classified into three major forms: type 1, type 2, and

gestational diabetes by The World Health Organization (Alberti and Zimmet,

1998). Type 1 diabetes occurs due to absolute insulin deficiency caused by

autoimmune destruction of the pancreatic -cells. This form of diabetes was

previously termed as insulin dependent diabetes mellitus (IDDM), or juvenile

onset diabetes, which accounts for only 5%-10% of those with diabetes. Type 2

diabetes is characterized by insulin resistance in target tissues.. This form of

diabetes was formerly termed non-insulin dependent diabetes (NIDDM) or

adult-onset diabetes mellitus and accounts for 90%-95% of those with diabetes.

Gestational diabetes (GDM) is defined as a state of glucose intolerance with

the onset of pregnancy (Carpenter and Coustan, 1982). GDM also involves insulin

resistance which is similar to type 2 diabetes. The glucose intolerance that occurs

during pregnancy is often normalized after delivery; however such patients are at

higher risk of developing diabetes in the future.

1.2. The complications of diabetes mellitus

Chronic hyperglycemia is a major initiator of vascular complications in diabetics.

Various hyperglycemia-induced metabolic and hemodynamic abnormalities,

including increased advanced glycation end product (AGE) formation (Nakamura

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CHAPTER 1: INTRODUCTION

et al., 1997), enhanced production of reactive oxygen species (ROS) (Du et al.,

2000), activation of protein kinase C (PKC) (Xia et al., 1995), stimulation of the

polyol pathway (Lee et al., 1995) and the renin-angiotensin system (RAS) (Gurley

and Coffman, 2007) lead to diabetic vascular complications, such as

cardiovascular disease (CVD), retinopathy , neuropathy, and nephropathy .

Diabetes is associated with a marked increase in the risk of CVD. The

incidence of CVD was 2-4 times higher in diabetic patients than in the general

population (Stamler et al., 1993). CVD is the predominant reason for death of

patients with diabetes of over 30 years. In addition, CVD is responsible for about

70% of all causes of death in patients with type 2 diabetes (Laakso, 1999).

Moreover, diabetic retinopathy, manifested as microaneurysm and hemorrhage in

the retina, is a leading cause of acquired blindness among the adult people (Klein,

2007). The prevalence of diabetic retinopathy increases with duration of diabetes.

With 30 years of diabetes, almost all patients have some degree of retinopathy and

the incidence of proliferative retinopathy is about 60%. The prevalence of diabetic

nephropathy is also markedly increased among individuals with diabetes mellitus.

Diabetic nephropathy which is a major cause of end-stage renal disease (ESRD),

develops to glomerular sclerosis accompanied with renal failure (Sharma and

Ziyadeh, 1995). About 80% of patients with type 1 diabetes for 15 years develops

diabetic nephropathy. 50% of those with diabetic nephropathy develops ESRD

over the next 10 years. About 20-40% of type 2 diabetic patients who are treated

develops overt albuminuria. Among these patients with overt albuminuria, 20%

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CHAPTER 1: INTRODUCTION

develops ESRD over the following 20 years (Ismail and Cornell, 1999).

Furthermore, diabetes mellitus causes chronic, widely distributed lesions in the

peripheral nerves, resulting in diabetic neuropathy which is responsible for

50-75% of non-traumatic amputations (Caputo et al., 1994). In a large prospective

study of diabetic patients, the incidence increased from 7.5 % at the time of

diagnosis of diabetes to 50 % after 25 years with diabetes (Palumbo et al., 1978).

1.3. Maternal diabetes

Maternal diabetes, which refers to pre-existing diabetes before pregnancy, not

only affects the general health conditions of pregnant women but also causes

congenital malformations in fetuses and spontaneous abortions or still birth in

severe cases (Hawthorne et al., 1997). The relationship between diabetes and

abnormal pregnancy was described by Bennewitz in the 18th century (Drury, 1961).

In general, spontaneous abortion and birth defects, such as neural tube defects,

cardiac anomalies and skeletal abnormalities, appear more frequently in diabetic

pregnant women than non-diabetic pregnant women. Interestingly, most of the

affected tissues are formed during the first trimester of pregnancy when

organogenesis takes place. The defects may be reduced or eliminated if

euglycemia is achieved during the first trimester of pregnancy (Dicker et al., 1988;

Mills et al., 1988). In addition, several clinical studies demonstrate that the

incidence and severity of maternal diabetes-induced defects are correlated with

poor glycemic control (Goldman et al., 1986; Kitzmiller et al., 1996; Kitzmiller et

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CHAPTER 1: INTRODUCTION

al., 1991; Steel et al., 1990).

1.3.1. Congenital malformations in embryos of diabetic mother

Maternal diabetes increases the risk of congenital malformations in fetuses

resulting in perinatal mortality and neonatal morbidity (Aucott, 1994; Connell et

al., 1985; Garner, 1995; Hanson, 1993; Ogata, 1995). Studies have shown that

there is a significant increase in fetal malformations in infants of diabetic mothers

(Aberg et al., 2001; Day and Insley, 1976; Farrell et al., 2002; Mills et al., 1979;

Mills, 1982; Ramos-Arroyo et al., 1992; Sharpe et al., 2005). According to

population based studies in Washington DC and Atlanta, USA, the incidence of

congenital malformations among infants of diabetic mothers ranges from 7.8% to

9.7%, while in infants of non-diabetic population, it is approximately 2.1%

(Becerra et al., 1990; Janssen et al., 1996).

The maternal diabetes-induced congenital anomalies include both structural

and functional abnormalities (Gabbe, 1977). The functional malformations include

macrosomia, cardiomyopathy, and hyperbilirubinemia (Cowett and Schwartz,

1982), whereas the structural abnormalities include skeletal malformations such as

sacral agenesis, absence of the femur, ventricular septal defect in the heart,

pulmonary artery stenosis and gastrointestinal tract abnormalities including

gastroschisis (Amankwah et al., 1981; Fields et al., 1968). Moreover, a variety of

central nervous system deformities such as anencephaly, exencephaly, spina bifida,

and hydrocephalus has also been reported in infants of diabetic mothers (Becerra

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CHAPTER 1: INTRODUCTION

et al., 1990; Mills et al., 1979).

2. Neural tube development

The nervous system in vertebrate develops from a simple epithelial sheet, the

neural plate, from which a variety of neuronal cell types required for the

construction of a functional nervous system is developed. Brain development

progresses in an orderly fashion that can be divided into several major stages: the

neurulation (formation of the neural tube), neuroepithelial cell proliferation and

migration with formation of transient subplate structures, neuroglial differentiation

and formation of the neuronal circuits (ten Donkelaar, 2006).

2.1. Neurulation

Neurulation is a multifactorial process through which the neural tube is formed

(Smith and Schoenwolf, 1997). In humans, it begins in the 3rd week after

fertilization by the appearance of the neural groove. Under the inductive influence

of the notochord, the neuroectoderm cells elongate into columnar cells which form

the prospective neural plate. The interaction between the neural plate and

surrounding ectoderm promotes the shaping of neural plate, which lengthens

along anterior-posterior axis and bends subsequently to form a neural tube (Smith

and Schoenwolf, 1989). The bending of the neural plate occurs along the midline

of the neural plate where the cells of the neural plate are called the medial hinge

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CHAPTER 1: INTRODUCTION

point (MHP) cells. The MHP cells are connected with the notochord beneath them

and act as a hinge, which forms a furrow at the dorsal midline. Subsequently, two

other hinge regions form near the connection of the neural plate with the lateral

ectoderm. The cells of these two regions are called the dorsolateral hinge point

(DLHP) cells. Each of the three hinges acts as a pivot to direct the rotation of the

cells around it (Schoenwolf, 1991; Smith and Schoenwolf, 1989, 1991). MHP

bending creates the neural groove, with a V-shaped cross section, while DLHP

bending creates longitudinal furrows that bring the lateral aspects of the neural

plate towards each other in the dorsal midline.

The neural folds meet in the midline and undergo fusion, forming the neural

tube. The first site of the fusion occurs at the junction of the caudal

rhombencephalon and cranial spinal cord and then proceeds in a zipper like

fashion cranially and caudally (continuous closure model). The open regions of

the neural tube are called the anterior (cranial) and posterior (caudal) neuropores.

The cranial neuropore closes approximately at 24 postovulatory days and caudal

neuropore closes approximately at 26 postovulatory days in the human. The

secondary neurulation forms the caudal end of the spinal cord below the sacral

level by epithelialized mesodermal cells (O'Rahilly and Muller, 2002; Sadler,

2005). It has recently been shown that the neural tube closure occurs at multiple

sites along the cranio-caudal neuraxis in human embryos (Nakatsu et al., 2000).

Disturbances during the neurulation lead to malformations resulting in a spectrum

of neural tube defects.

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CHAPTER 1: INTRODUCTION

2.1.1. Neural tube defects

Neural tube closure defects (NTDs), including anencephaly and spina bifida, are

common human birth defects (Campbell et al., 1986; McBride, 1979). The mouse

neural tube consists of several cranio-caudal zones (AD, Illustration 1) within

which elevation of neural plate passes as a wave longitudinally. The elevated

neural folds come into apposition, contact and fuse at 6 discrete initiation sites

within a cranio-caudal zone (Macdonald et al., 1989). From each initiation site

(closure 1, 2, 3 and 4, Illustration 1), the contact and fusion extends along the

length of the folds until colliding with fusion initiated from another zone. Failure

of neural folds to elevate and fuse at the midline causes neural tube closure defects.

Exencephaly is caused by failure of neural folds to elevate and fuse at the zone B

or zone B and C of the neural fold (Illustration 1). Failure of elevation of the

neural folds at other zones causes spina bifida (caudal zone D), craniofacial defect

of exencephaly with split face (zone A and zone B) and rachischisis (whole of

zone D). The human NTDs, anencephaly, spina bifida and rachischisis, are

anatomically similar to mouse NTDs, suggesting that they arise from failure of

elevation of similar elevation zones (Illustration 1).

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CHAPTER 1: INTRODUCTION

Illustration 1: Zonal pattern of neural fold elevation and corresponding categories


of neural tube defects in mouse and human. AD: elevation zones; 14: location
of initiation sites of neural fold. (Adapted from Juriloff and Harris, 2000)

2.2. Neurogenesis in the developing cranial neural tubes

During neurodevelopment in mouse, the neural tube forms three vesicles namely,

prosencephalon, mesencephalon, and rhombencephalon, which give rise to the

adult forebrain, midbrain and hindbrain, respectively. Before embryonic day 11

(E11), the three vesicles mainly contain undifferentiated neuroepithelial cells

acting as precursors for neuronal and glial fates (Morest and Silver, 2003). As the

cerebral vesicles enlarge, the primitive neuroepithelial cells elongate to become

radial glial cells which function as the neuronal precursor cells (Levitt et al., 1983)

and as the migratory substrates for postmitotic neurons (Gasser and Hatten, 1990;

Hatten and Mason, 1990). The correct specification of radial glial cells is therefore

essential for normal organization of the developing brain. The neuroepithelial cells

proliferating in the ventricular zone migrate along the processes of radial glial

cells through the overlying intermediate zone to marginal zone of pial surface.

During the process of migration, the neuroepithelial cells differentiate into

different types of cells which form distinct domains in the brain. Maintenance of

the ratio of cell proliferation and differentiation is important for proper

neurodevelopment. It is hypothesized that the impaired proliferation and

differentiation patterns in the neuroepithelia may contribute to the neural tube

anomalies.

10
CHAPTER 1: INTRODUCTION

2.2.1 Developmental control genes in the process of neurogenesis

Several signaling molecules and nuclear transcription factors are part of signaling

cascades that control proliferation, migration and differentiation of neuronal and

glial progenitors during neurogenesis. The inductive signaling molecules direct

cell activity by instructing the synthesis of different transcriptional factors, which

establish the genetic network necessary for the development of the neural tube.

However, the mechanisms by which these signaling cascades control the

neurogenesis are largely unknown.

In addition, several classes of transcription factors have been shown to

control the differentiation and specification of precursor cells in the neural tube.

Recent evidence suggests that combinations of transcription factors of the

homeodomain proteins such as paired box 6 (Pax6), and basic helix-loop-helix

(bHLH) proteins such as neurogenin 2 (Ngn2), establish molecular codes that

determine the fate of neuronal progenitors and glial progenitors by controlling the

proliferation, specification, and differentiation of neural stem cells (NSCs) during

development (Ross et al., 2003; Stoykova et al., 2000; Toresson et al., 2000).

Recently, it has been shown that a number of microtubule-associated proteins

(MAPs) such as doublecortin (Dcx) are involved in neurogenesis (des, P et al.,

1998) by regulating the key events of mitotic division and migration of neurons

during neurodevelopment.

11
CHAPTER 1: INTRODUCTION

2.2.1.1. Doublecortin (Dcx)

Doublecortin (Dcx) is a microtubule associated protein (MAP) which is expressed

in differentiating and migrating neuronal cells, but is not detected in mature

neurons under normal conditions (Francis et al., 1999; Gleeson et al., 1999). Dcx

is expressed primarily in post mitotic neurons during cortical development,

specifically during periods of neuronal migration as well as neurite formation

(Francis et al., 1999; Gleeson et al., 1999). Mutations in the human DCX gene are

associated with abnormal neuronal migration, epilepsy, lissencephaly and mental

retardation (LoTurco and Bai, 2006). Dclk, a gene that shares high homology with

Dcx controls the mitotic division and also determines the fate of neural

progenitors during neurogenesis (Shu et al., 2006).

2.2.1.2. Brain lipid binding protein (Blbp)

Brain lipid binding protein (Blbp) is a member of the fatty acid-binding protein

(FABP) family that has been shown to modulate transcription through their

interactions with nuclear receptors and to play roles in the metabolism

(Haunerland and Spener, 2004). Blbp is expressed in radial glial cells which

function as neural progenitors and as a scaffolding supporting neuronal migration.

Within the cell, Blbp is localized both in the cytoplasm and nucleus in vivo,

indicating that Blbp is involved in the trafficking of a ligand to a nuclear receptor

(Feng et al., 1994). In addition, Blbp regulates the migration of developing

12
CHAPTER 1: INTRODUCTION

neurons into cortical layers (Feng et al., 1994). Pathologically, Blbp is

overexpressed in patients with Downs syndrome, and this overexpression has been

suggested to contribute to the associated neurological disorders (Sanchez-Font et

al., 2003).

Blbp is expressed in radial glia cells of the developing brain and plays a role

in mediating neuronal-glial signaling. Further it has been shown that Blbp function

is required for radial glial morphological changes in response to neuronal cues

(Anton et al., 1997; Feng et al., 1994) and for regulating the morphology and

axonal interactions of Schwann cells (Miller et al., 2003). Radial glial cells serve

as the neuronal as well as astrocyte progenitors (Campbell and Gotz, 2002; Gotz

et al., 2002; Merkle et al., 2004). The dual roles played by radial glia as both

migratory scaffolding and neuronal progenitors have indicated that there may be

intimate links between the signaling pathways that control radial glial cell

development, neurogenesis and neuronal migration (Hatten, 1999; Noctor et al.,

2001; Parnavelas, 2000).

2.2.1.3. Paired box gene 6 (Pax 6)

Pax 6 encodes a transcription factor containing both a paired domain and a

homeodomain. It is highly conserved across diverse species. In mammals, it is

expressed in the eye, specific regions of the CNS, the nasal placodes, olfactory

epithelium and in the pancreas during development (Grindley et al., 1995;

13
CHAPTER 1: INTRODUCTION

St-Onge et al., 1997; Walther and Gruss, 1991). In mice, Pax 6 begins to be

expressed on embryonic day 8.5 (E8.5) in the developing eyes, nasal structures,

spinal cord and forebrain, including the telencephalon (Grindley et al., 1997;

Mastick et al., 1997; Stoykova and Gruss, 1994). Within the telencephalon, Pax 6

expression is restricted to the ventricular zone where neurogenesis primarily

occurs and to the subventricular zone in the dorsal telencephalon where

gliogenesis primarily occurs (Caric et al., 1997; Gotz et al., 1998). Pax 6

expression persists in both of these regions throughout neurogenesis and

gliogenesis, indicating that it may play a vital role in these processes. In addition,

Pax 6 haploinsufficiency (Pax6+/-) in the mouse results in the Small eye (Sey)

phenotype (Hill et al., 1991). Homozygotes (Pax6-/-) die perinatally with no eyes

and multiple brain abnormalities. PAX 6 haploinsufficiency also causes eye and

brain defects in humans (Estivill-Torrus et al., 2001; Sisodiya et al., 2001; Ton et

al., 1991).

Recent studies have suggested that the Pax 6 is important for regulation of

cell proliferation, migration and differentiation at various sites of the CNS. This

gene is widely expressed in the developing CNS, including the embryonic cerebral

cortex, and it has been shown to be required for radial glial cell development and

neuronal migration (Warren et al., 1999).

14
CHAPTER 1: INTRODUCTION

2.2.1.4. Neurogenin 2 (Ngn2)

The proneural genes that encode basic helix-loop-helix (bHLH) transcription

factors play a vital role in establishing the fates of neural progenitors (Bertrand et

al., 2002; Kageyama and Nakanishi, 1997). The main mouse proneural genes are

Mash1 (Ascl1), neurogenins (Ngn) and Math1 (Atoh1). These genes have dual

functions: a) promoting the differentiation of individual progenitors, and b)

selecting the neuronal or glial lineages. In addition, several lines of evidence have

shown that they are also involved in specifying neuronal subtype identities. It has

been reported that Mash1, Ngn1 and Ngn2 are expressed in a complementary

pattern in the developing telencephalon and spinal cord and specify distinct

neuronal subtype identities (Parras et al., 2002).

The expression of Ngn2 in forebrain progenitor cells promotes the generation

of glutamatergic neurons (Berninger et al., 2007; Parras et al., 2002). In the

cerebral cortex, Ngn2 is regulated by Pax 6 to maintain the correct molecular

identity of cortical progenitors (Stoykova et al., 2000; Toresson et al., 2000; Yun et

al., 2001). In the spinal cord, Ngn2 promotes cell cycle arrest and neuronal

differentiation of neuroepithelial cells (Mizuguchi et al., 2001; Novitch et al.,

2001; Scardigli et al., 2001). Ngn2 also acts with Olig2, bHLH transcription factor

to contribute to the specification of motor neuron progenitors in the spinal cord

(Mizuguchi et al., 2001; Novitch et al., 2001).

15
CHAPTER 1: INTRODUCTION

2.3. Glucose metabolism and hypoxia in the developing cranial neural tubes

In glucose metabolism, glucose is oxidized to synthesize ATP (energy) by

glycolysis pathway during which the glucose is cleaved into pyruvate. Further

series of reaction occurs by one of two different pathways: anaerobic which

occurs in the cytoplasm and aerobic which takes place in the mitochondria. In

anaerobic glycolysis, the pyruvate is reduced to lactate whereas in aerobic

glycolysis, pyruvate is transported inside mitochondria and oxidised to acetyl

coenzyme A. Glucose metabolism is characterized by a high rate of lactic acid

production in rat embryos during early organogenesis on embryonic day 10

(Shepard et al., 1970). Glucose also appears to be the predominant substrate for

the developing brain during embryogenesis. Studies in experimental animals and

humans indicate that glucose utilization of brain initially is low and increases with

maturation with increasing regional heterogeneity and increasing functional

activity (Dyve and Gjedde, 1991; Settergren et al., 1980).

The developing brain requires enormous supplies of energy to support

neuronal and glial development during pre-and post-natal development. Murine

and human brains consume over half the energy available to the organism as a

whole during this critical period (Gibbons, 1998), when undernutrition may result

in permanent intellectual deficit (Chase and Martin, 1970). However, the energy

resources for the developing brain are not known. Insulin enhances energy and

substrate utilization by peripheral tissues, but does not seem to be involved in the

regulation of brain metabolism as very little insulin is synthesized in the brain

16
CHAPTER 1: INTRODUCTION

(Baskin et al., 1987). Recently, it has been shown that endogenous brain

insulin-like growth factor 1 (Igf-1) serves an anabolic, insulin-like role in

developing brain metabolism (Cheng et al., 2000).

Maintenance of normal energy metabolism is critical for organogenesis in

mammalian embryos (Freinkel et al., 1983). It has been shown that glucose uptake

is increased in embryonic cells without downregulation of glucose transporter,

Glut1 protein in the diabetic environment and the increased glucose uptake

induces metabolic overload in the embryonic mitochondria (Yang et al., 1995).

Further, maternal diabetes-induced glucose uptake into the fetal brain cells has

been suggested to result in many congenital malformations in embryos (Reece et

al., 1996).

2.3.1. Glycolysis pathway

Glycolysis is the metabolic process that converts glucose into pyruvate in the

cytosol of the cell. The sequence of reactions in the glycolysis pathway is

catalyzed by ten enzymes which include: Hexokinase (HK), Glucose Phosphate

Isomerase (GPI), Phosphofructokinase (PFK), Aldolase (ALD), Triosephosphate

Isomerase (TPI), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH),

Phosphoglycerate Kinase (PGK), Phosphoglycerate Mutase (PGM), Enolase

(ENO), Pyruvate Kinase (PK). The activity of the pathway is regulated at key

steps to ensure that glucose consumption and energy production match the needs

of the cell. In mammals, HK, PFK & PK catalyze the key steps which determine

17
CHAPTER 1: INTRODUCTION

the rate of the energy production in the glycolysis pathway (Masters et al., 1987).

Under anaerobic conditions, pyruvate is converted to lactate by the enzyme

lactate dehydrogenase (LDH). In the brain, lactate which is a significant energy

source for neurons is released from astrocytes, which surround and protect

neurons (Pellerin, 2005). The lactate is taken up by adjacent neurons and

converted to pyruvate, which is oxidized via Krebs cycle (Bouzier-Sore et al.,

2003; Itoh et al., 2003). Impaired energy production may lead to lethal

consequences in cells and hence manipulation of metabolism may provide a

therapeutic strategy.

2.3.2. Hypoxia in the developing cranial neural tubes

Hypoxia is a well-known teratogen contributing to congenital malformations in

mouse embryos (Curley and Ingalls, 1957). Physiological hypoxia, caused by the

increasing mass of the avascular early postimplantation embryo, plays a critical role

during normal embryogenesis. The embryo is relatively in a state of partial

hypoxia which is essential for proper morphological development (Chen et al.,

1999). This physiological hypoxia activates the hypoxia-inducible factor 1-

(Hif-1), a heterodimeric transcription factor to induce expression of genes that in

turn trigger hematopoiesis and development of structures forming the circulatory

system, thereby increasing O2 delivery to embryonic tissues and reducing the

hypoxic state (Adelman et al., 1999; Iyer et al., 1998). Increased O2 delivery may be

critical for the development of organ systems including nervous system. Thus

18
CHAPTER 1: INTRODUCTION

failure to increase O2 delivery at this stage of development may impair activation of

genes needed for formation of the various organs including the neural tube. It has

been shown that excessive hypoxia in embryos causes abnormal development of

the brain (Giusti et al., 2008), heart (Tintu et al., 2007) and cleft lip (Paulozzi and

Lary, 1999). In human beings, hypoxia has been shown to cause intrauterine

growth retardation, developmental delay, and intrauterine death (Ergaz et al.,

2005). Further, the central nervous system is known to be critically affected in the

prenatalperinatal period by hypoxicischemic insults, which produce several

disorders such as loss of neural projections, increased susceptibility to seizures,

apoptosis and an imbalance in normal activity of glutamatergic and GABAergic

neurons, resulting in acute cell excitotoxicity (Rodriguez Gil et al., 2000).

2.3.2.1. Hypoxia-inducible factor 1 (Hif-1)

Hypoxia-inducible factor 1 (Hif-1) is the master regulator of cellular responses to

the state of hypoxia (Poellinger and Johnson, 2004; Semenza, 1999). Hif-1 is a

heterodimeric basic helixloophelixPAS domain (bHLH-PAS) transcription

factor, composed of Hif-1 and Hif-1 subunits (Wang et al., 1995). HIF-1,

which is the aryl hydrocarbon receptor nuclear translocator (ARNT) (Hoffman et

al., 1991), is a common subunit of multiple bHLH-PAS proteins. Hif-1 is the

unique O2-regulated subunit that determines Hif-1 activity (Semenza, 1999).

Levels of Hif-1 protein and Hif-1 DNA-binding activity are increased

exponentially when O2 concentration decreased (Jiang et al., 1996).

19
CHAPTER 1: INTRODUCTION

Hif-1 regulates the expression of a wide range of genes, protein products of

which allow metabolic adaptation to low oxygen conditions. These genes include

genes encoding erythropoietin (EPO) (Wang and Semenza, 1993), transferrin (Lee

and Andersen, 2006), inducible nitric oxide synthase (iNOS) (Palmer et al., 1998),

and vascular endothelial growth factor (Vegf) (Forsythe et al., 1996). In addition,

Hif-1 activates transcription of genes encoding glycolytic enzymes, such as ALD

1A, ENO 1, LDHA, PFK and PGK 1 (Wenger and Gassmann, 1997). These

proteins play important roles in systemic, local or intracellular O2 homeostasis:

EPO increases blood O2-carrying capacity by stimulating erythropoiesis (Wang

and Semenza, 1993); transferrin delivers iron to the bone marrow for

incorporation into hemoglobin (Primosigh and Thomas, 1968); iNOS synthesizes

NO which modulates vascular tone (Worthington et al., 2000); and induction of

glycolytic enzymes allows for increased anaerobic ATP synthesis (Scheuer, 1972);

Vegf mediates vascularization, particularly during embryonic development (Breier

et al., 1992).

On the other hand, HIF1 signal regulation could be detrimental instead of

adaptive in some circumstances. For instance, HIF1 induces expression of BNIP3,

a proapoptotic member of the BCL2 family, which induces apoptosis (Bruick,

2000; Sowter et al., 2001). Moreover, HIF1 also induces expression of IGFBP-3

(Feldser et al., 1999; Liu et al., 1992) and P21 (Goda et al., 2003) which

negatively regulate cell proliferation and cell cycle.

20
CHAPTER 1: INTRODUCTION

2.3.2.2. Vascular endothelial growth factor (Vegf)

Since hypoxia induces angiogenesis as a compensatory mechanism to supply

adequate oxygen, hypoxia is one of the most potent inducers of several

well-known angiogenic factors including vascular endothelial growth factor (Vegf)

(Mazure et al., 1996; Plate et al., 1992). Vegf is considered as a key regulator of

angiogenesis particularly during embryonic development (Breier et al., 1992;

Dumont et al., 1995; Millauer et al., 1993). Several studies have shown that

development of the cardiovascular system is exquisitely dependent on normal

levels and appropriately timed expression of Vegf. Loss of a single Vegf allele is

lethal in the mouse embryo between days 11 and 12 (Ferrara et al., 1996).

Conversely, even a modest upregulation of Vegf levels during development leads

to severe abnormalities in heart development and embryonic lethality (Miquerol et

al., 2000).

2.4. Choroid plexus development

2.4.1. Choroid plexus (CP)

CP is a specialized secretory organ situated within the ventricles of the brain. CP

is composed of a simple epithelial layer with underlying connective tissue and

blood vessels. The CP epithelial cells produce cerebrospinal fluid (CSF) which

fills the ventricular system, enters the subarachnoid space and circulates around

the brain and spinal cord (Speake et al., 2001). Subsequently, CSF is absorbed into

21
CHAPTER 1: INTRODUCTION

blood vessels through arachnoid villi. The CP and CSF play important roles

(nutritive, mechanical, metabolic and immune) in brain physiology and

neurological diseases (Emerich et al., 2005; Engelhardt et al., 2001; Strazielle and

Ghersi-Egea, 2000). The CSF-borne molecules appear to pass through the CP

epithelium and regulate the brain functions. CP also serves as an important

channel for the entry of therapeutic drugs as it is a major component of the

blood-CSF and blood-brain barriers which segregate CNS environment from the

circulating blood (Johanson et al., 2005). During brain development, CP has been

shown to function as a signaling centre (Yamamoto et al., 1996). Recently, it has

shown that the regulatory factors found in the CSF promote cell survival,

proliferation, and neurogenesis during brain development (Parada et al., 2005).

2.4.2. Development of CP in mouse embryos

The CP first begins to appear shortly after the neural tube closure in the cerebral

ventricles of the developing brain of mouse embryos (Dziegielewska et al., 2001).

It develops in the order of fourth ventricles at E10-10.5, lateral ventricles at E11

and third ventricles afterwards in the mouse brain (Sturrock, 1979). The CP

consists of a single layer of epithelial cells around the mesenchymal (stromal)

tissue containing blood vessels (Dziegielewska et al., 2001). The epithelial cells of

the CP express and transfer proteins and other metabolically important molecules

essential for brain development, such as transthyretin (Ttr) and insulin-like growth

factor 2 (Igf-2). Furthermore, the CP has been implicated as a secondary signaling

22
CHAPTER 1: INTRODUCTION

center during brain development (Yamamoto et al., 1996). It also provides an

essential expanding mechanism that determines the shape of the brain during its

development (Dziegielewska et al., 2001). It is hypothesized that the impairment

of CP development causes adverse effects on the patterning and shaping of the

developing brain.

2.4.3. Factors involved in CP development

2.4.3.1. Transthyretin (Ttr)

Transthyretin (Ttr) is a 55-KD tetrameric carrier protein which was first detected

in CSF and serum. It was originally called prealbumin. In the blood, Ttr binds to

thyroid hormones with high affinity (Blake and Oatley, 1977) and retinol via

retinol-binding protein (Muto and Goodman, 1972). The function of Ttr is to

transport thyroid hormones from the blood to brain (Schreiber et al., 1990).

In the brain, Ttr is expressed in CP epithelial cells and is regarded as a

marker of CP epithelium (Murakami et al., 1987). Ttr mRNA expression has been

shown to be localized in the primordial CP epithelium prior to CP morphogenesis

and in the developing CP of the fourth and lateral ventricles of the rat embryos at

E13 (Cavallaro et al., 1993; Makover et al., 1989). In addition, Ttr protein is first

detected in the tela choroidea which is the developmental forerunner of the CP. Ttr

protein continues to express in the CP of fetal rat at the E14 days as well as at the

E18 days (Kato et al., 1986). The level of Ttr mRNA expression in CP is increased

23
CHAPTER 1: INTRODUCTION

during the first half of gestation and stayed constant thereafter until birth (Tu et al.,

1990). Ttr plays an important role in vitamin A and thyroid hormone metabolism

in the developing embryo (Makover et al., 1989). Particularly, it serves to

transport thyroxine (T4) or retinol (metabolite of vitamin A) across the

blood-retina barrier, thereby facilitating their effects on cell differentiation and

brain morphogenesis.

2.4.3.2. Insulin-like growth factor 2 (Igf-2)

Insulin-like growth factor 2 (Igf-2) is a polypeptide hormone with structural and

functional homology with Igf-1 and pro-insulin. Igf-2 acts as a growth factor

during fetal development (Baker et al., 1993; Heyner and Garside, 1994). Igf-2

levels in the circulation are high in the fetus and decline rapidly after birth.

Targeted disruption of the Igf-2 gene in mice leads to a deficiency in growth and

differentiation factor in the central nervous system (Kiess et al., 1994).

Igf-2 mRNA expression is localized in many tissues including the liver and

CP in the brain. In the rat, Igf-2 mRNA expression begins at E13 and increases

gradually as morphogenesis proceeds. In the CP stroma (mesenchyme), Igf-2

mRNA is abundant prior to CP morphogenesis but decreases as embryogenesis

proceeds and is absent in the adult (Cavallaro et al., 1993), suggesting that Igf-2

acts as a morphogen in inducing differentiation of CP epithelial cells during brain

development (Cavallaro et al., 1993)

24
CHAPTER 1: INTRODUCTION

3. Analysis of global gene expression profile using DNA microarray

Human Genome Project (HGP), which has been launched for more than 15 years,

has established the blueprint of genome sequences of human and provided a solid

basis for translational researches from genomics to post-genomics. In the

post-genomics era, researchers are focusing on the function of each gene in the

genome on the biological processes. This new area is called functional genomics

which is the functional analysis of gene expression profile using DNA microarray

technique (Lander, 1999)

3.1. Overview of DNA microarray technology

Each array consists of thousands of genes attached to a solid support. Fluorescent

labeled RNA or DNA is hybridized to complementary DNA on the array and

detected by a laser scanner. After the hybridization intensities for each DNA on

the array are determined, the microarray data can be further analyzed to identify

expression patterns and variation that correlate with biological processes. There

are two types of DNA microarray. One is the spotted microarray on which the

pre-synthesized DNA samples are bound, and the other is oligonucleotide

microarray. In oligonucleotide microarray, the DNA fragments synthesized are

spotted on glass surface using the photolithographic method.

25
CHAPTER 1: INTRODUCTION

3.2. Basic principle of DNA microarray

The basic principles of spotted microarray and oligonucleotide microarray are

similar. Firstly, total RNA or messenger RNA isolated from samples of either

control or experimental groups is reversely transcripted to cDNA or converted to

cRNA by incorporating fluorescent dyes. The labeled cDNA or cRNA is

hybridized to microarray chips for a period of time, after which the excess cDNA

or cRNA is washed off and the microarray chips are scanned under a laser scanner

(Heller, 2002).

Spotted microarray:

Although the basic principles of these two types of microarray are similar, they

also have their own characteristics. On spotted arrays, the DNA fragments are

longer than several hundred base pairs. The cDNA samples of control and test

groups are labeled with different fluorophores, cy3-dUTP and cy5-dUTP and then

hybridized onto the array. Relative amount of a particular gene transcript in the

two samples is determined by measuring the signal intensities detected by the two

fluorophores and calculating signal ratios (Ferea and Brown, 1999).

Oligonucleotide array:

On oligonucleotide microarray, each gene is presented by 15-20 different

oligonucleotides with 25 base pairs. For cross hybridization control, each gene has

a mismatch (MM) probe set which is identical to the perfect match transcript

26
CHAPTER 1: INTRODUCTION

except for one base pair in the middle. Based on the signal of mismatched

oligonucleotide, cross-hybridization and local background is estimated and

subtracted from the perfect match signal (PM). In the oligonucleotide array,

biotinylated cRNA is transcribed from mRNA of control and test samples and the

cRNA of each sample is hybridized to a separate array. Differences in mRNA

levels between samples are determined by comparison of hybridization patterns

produced on separate arrays. (Lipshutz et al., 1995; Lockhart et al., 1996; Pease et

al., 1994)

Differences between the 2 arrays and advantage of oligonucleotide array:

There are two main differences between spotted microarray and oligonucleotide

microarray: 1) For spotted microarrays, each probe has its own hybridization

character, since the DNA fragments are synthesized individually and the length of

them is also different. However, for the oligonucleotide microarray, all probes are

designed to have similar hybridization temperature and binding affinity, which

allow minimization of the hybridization differences among the target genes. 2)

The spotted microarray measures two samples together and provides relative

amount of each target gene between two samples. The oligonucleotide array

measures each sample separately and gives the absolute amount of target gene in

each sample, which allows flexibility in sample comparisons. Since

oligonucleotide array has similar probe hybridization character and may be used

in research with more than two samples. In the present study, oligonucleotide

27
CHAPTER 1: INTRODUCTION

microarray was used.

3.3. Applications of DNA microarray

DNA microarray can be applied in many aspects of basic and clinical research. In

basic research, DNA microarray is normally applied to identify novel genes (Certa

et al., 2001), determine gene function (Certa et al., 2002), generate expression

profile (Lukiw, 2004) and elucidate signaling pathways (Carrasquillo et al., 2002).

DNA microarray can also be used in a wide variety of clinical research, such as

drug target discovery (Gerritsen et al., 2002), biomarker determination (Flach et

al., 2006), prognostic tests development (De et al., 2006), and disease-subclass

determination (Koehler et al., 2004).

3.4. Data analysis strategies

There are three steps for data analysis: data normalization, data filtering and

pattern identification. Initially data are normalized before comparing expression

values. Following this, uninformative genes are filtered out from the data. The

final step is to find patterns and groups of genes that have similar biological

meaning. The methods for the final steps are basically two types: One is to list the

increased and decreased genes based on threshold, and the other is sophisticated

clustering (Claverie, 1999) which uses a progressive combination of elements that

are the most similar.

28
CHAPTER 1: INTRODUCTION

4. Animal models

4.1. Animal models of diabetes mellitus

Animal model is a useful tool for researchers to investigate the disease states in

ways which may not be accessible in human patients. Rodents, such as rats and

mice, are most commonly used as diabetic animal models since they are small in

size, manageable and cost effective. The rodent animal models of diabetes display

the similar symptoms of human disease although rodents may not adequately

reflect the human situation.

According to their clinical similarity to human disease, the animal models of

diabetes can be classified into two types: animal models of insulin-dependent

diabetes (IDDM) and non-insulin dependent diabetes (NIDDM). The animal

models of IDDM can be induced by viral infection (Craighead, 1981), chemical

destruction of pancreatic -cells (Like and Rossini, 1976), partial pancreatectomy

(Freyse et al., 1982; Reiser et al., 1987), or immunologic mechanisms which

inactivate insulin or destroy pancreatic islets (Toreson et al., 1968), resulting in

destruction of islet -cells that lead to an absence of intrinsic insulin secretion.

Type 1 diabetes mellitus in humans is characterized by a specific destruction

of the cells in the pancreas. Therefore, toxin-mediated damage to the

insulin-producing pancreatic -cells is a useful tool in the study of the

consequences of hyperglycaemia, such as diabetic complications (Flyvbjerg et al.,

1995; Reagan et al., 1999; Yu et al., 2001). Streptozotocin (STZ) is one of the

29
CHAPTER 1: INTRODUCTION

most commonly used chemicals producing hyperglycaemia by selective

destruction of cells in pancreatic islets (Junod et al., 1969).

4.1.1. Mechanism of action of streptozotocin (STZ)

Streptozotocin (STZ) is an N-nitroso derivative of D-glucosamine isolated from

Streptomyces achromogenes. STZ has broad-spectrum antibiotic and

anti-neoplastic activity (Bono, 1976) and it has been shown to disturb glucose

transport (Wang and Gleichmann, 1998), glucokinase function (Zahner and

Malaisse, 1990) and induce multiple DNA strand breaks in rat pancreatic cells

(LeDoux et al., 1986). Subsequently, this chemical acts as diabetogen as it

selectively destroys pancreatic cells in several species (Junod et al., 1967;

Rakieten et al., 1963). A single large dose of STZ (200-250 mg/kg body weight) or

multiple low doses of STZ (40 mg/kg body weight) on five consecutive days can

induce diabetes in rodents by producing progressive loss of cells and

hyperglecaemia associated with intense pancreatic insulitis (Junod et al., 1967;

Like et al., 1978).

4.2. Animal model of maternal diabetes

The diabetic animal models induced by STZ have been widely used for the study

of teratogenic effects of maternal diabetes on fetal development and possible

mechanisms of maternal diabetes-induced congenital malformations (Cederberg et

30
CHAPTER 1: INTRODUCTION

al., 2003; Giavini et al., 1986; Hiramatsu et al., 2002; Liao et al., 2004; Sakamaki

et al., 1999; Wentzel et al., 2005). Initially, alloxan, another diabetogenic agent

injected into pregnant mice between gestational day 8.5 and13.5 caused a high

maternal death rate (Watanabe and Ingalls, 1963), together with a wide range of

congenital malformations, high embryo death rate and delayed development

(Baker et al., 1981; Deuchar, 1977; Eriksson et al., 1983). Subsequently it has

been demonstrated that STZ induced diabetes is severe and showed low fertility

and high preimplantation losses, low fetal weight and high placental weight

(Giavini et al., 1986). The reliability of this animal model was further confirmed

by comparing the results of in vivo experiments with the results of clinical practice

in the following aspects:

1. The frequency of congenital malformation in the offspring of diabetic rats

or mice is around 6 to 8 times higher than the normal rate. Similar rate is

reported in diabetic women (Becerra et al., 1990; Janssen et al., 1996).

2. The most frequent malformations observed are: defects in cardiovascular

and central nervous systems and caudal regressions in the offspring of

both animal models and diabetic women (Aberg et al., 2001; Eriksson,

1984).

3. The increased frequency of embryo resorption in diabetic rats mimics the

high rate of abortions in diabetic women (Greene, 1999; Moley, 2001).

31
CHAPTER 1: INTRODUCTION

5. Hypotheses and Objectives

It is well established that diabetic women exhibit higher incidences of pregnancy

complications such as spontaneous abortion, still birth and congenital

malformations in various organ systems including the central nervous system

(Becerra et al., 1990; Greene et al., 1989; Hawthorne et al., 1997; Mills et al.,

1979). Several studies have attempted to demonstrate the molecular and

biochemical mechanisms of maternal diabetes-induced congenital malformations

in rodents. Recently, it has been shown that altered expression pattern of some

genes involved in regulating the forebrain patterning and neural tube closure,

leads to forebrain patterning defects and NTD in embryos of diabetic pregnancy

(Liao et al., 2004; Phelan et al., 1997). Further, results obtained from in vitro

analysis indicated that high glucose impairs the proliferation and cell fate

specification of NSCs and this impairment may form the basis for neural tube

anomalies in embryos of diabetic pregnancy (Fu et al., 2006). A number of studies

described that the development of malformations in various organs (including the

neural tube) in diabetic pregnancy could be due to metabolic derangements caused

by a deficiency of polyols, oxidative stress and physiological hypoxia (Baker et al.,

1990; Chang et al., 2003; Goldman et al., 1985; Hashimoto et al., 1990; Hiramatsu

et al., 2002; Li et al., 2005). These reports did not fully explore the molecular

mechanisms of neural tube patterning defects as the neural tube development is a

complex process and is regulated by a number of signaling molecules and

transcription factors. Global analysis of expression patterns of thousands of genes

32
CHAPTER 1: INTRODUCTION

simultaneously by microarray technology in the cranial neural tube of embryos

from normal and diabetic mice may unravel the molecular changes which

contribute to brain anomalies in infants of diabetic pregnancies.

During development, the neural tube forms three vesicles namely,

prosencephalon, mesencephalon, and rhombencephalon, which give rise to the

adult forebrain, midbrain and hindbrain, respectively. Before embryonic day 11

(E11), the three vesicles mainly contain undifferentiated neuroepithelial cells

acting as precursors for neuronal and glial fates (Morest and Silver, 2003). As the

cerebral vesicles enlarge, the primitive neuroepithelial cells elongate to become

radial glial cells which function as the neuronal precursor cells (Levitt et al., 1983)

and as the migratory substrates for postmitotic neurons (Gasser and Hatten, 1990;

Hatten and Mason, 1990). The correct specification of radial glial cells is therefore

essential for normal organization of the developing brain. The neuroepithelial cells

proliferating in the ventricular zone migrate along the radial glial cells to marginal

zone and differentiate into different types of cells which form distinct domains in

the brain. Maintenance of the ratio of cell proliferation and differentiation is

important for proper brain patterning. It is hypothesized that the impaired

proliferation and differentiation patterns in the neuroepithelia contribute to brain

anomalies in infants of diabetic pregnancies.

Another component of the brain, i.e., choroid plexus (CP) which is a

specialized secretory organ situated within the ventricles of the brain also plays an

important role in early brain development. The CP first begins to appear shortly

33
CHAPTER 1: INTRODUCTION

after the neural tube closure in the ventricles of cranial neural tube of mouse

embryos (Dziegielewska et al., 2001). It develops in the order of 4th ventricles at

E10-10.5, lateral ventricles at E11 and 3rd ventricles afterwards in the mouse

brain (Sturrock, 1979). The CP consists of a single layer of epithelial cells

around the mesenchymal tissue containing blood vessels and produces the cerebral

spinal fluid (CSF) which provides an essential expanding mechanism that

determines the shape of the brain during its development (Dziegielewska et al.,

2001). The epithelial cells of the CP transfer proteins and other metabolically

important molecules essential for brain development. Furthermore, the CP has

been implicated as a secondary signaling center during brain development

(Yamamoto et al., 1996). It is hypothesized that the impairment of CP

development causes adverse effects on the patterning and shaping of the

developing brain.

The objective of this study was to investigate the molecular and

morphological changes in the neuroepithelia of the cranial neural tube including

telencephalon, diencephalaon and, rhombencephalon and choroid plexus in

embryos from diabetic mice. The molecular changes were analyzed by using

oligonucleotide microarray which is a useful tool to evaluate the expression levels

of thousands of genes involved in morphogenesis, cellular functions as well as

biochemical and metabolic pathways in cranial neural tubes of embryos from

diabetic mice.

34
CHAPTER 1: INTRODUCTION

5.1. Specific aims

1. Morphology of cranial neural tubes in embryos of diabetic mice was analyzed

histologically using Haematoxylin and Eosin (H&E) staining.

2. Global gene expression profile of the cranial neural tubes in embryos from

diabetic mice and normal mice was analyzed using Affymetrix oligonucleotide

microarray. Genes that were found to be differentially expressed between

control and experiment groups were hierarchically clustered using

bioinformatics software and the functions of these genes were analyzed based

on Gene Ontology Consortium database [http://www.geneontology.org/].

3. The differentially expressed genes selected according to their functions in

neurogenesis, neuronal migration, glucose metabolism and physiological

hypoxia were further examined by in situ hybridization, real time RT-PCR,

immunohistochemistry and western blot.

4. The proliferation index and apoptosis in the cranial neural tube of embryos

from diabetic and normal mice were investigated by immunohistochemical

detection of BrdU incorporation and TUNEL assay, respectively.

5. Morphology of choroid plexus in the cranial neural tube of embryos from

diabetic mice was examined histologically using H&E staining and specific

markers of choroid plexus, such as Ttr and Igf-2, were studied using

immunohistochemistry.

Analysis of gene expression profiles may help identifying novel key molecules

35
CHAPTER 1: INTRODUCTION

involved in the cranial neural tube malformations of embryos from diabetic mice.

This may provide better insights into the development of therapeutic options for

the fetal malformations in diabetic pregnancy and the determination of biomarkers

to identify the possible fetal abnormalities in utero.

36
CHAPTER 2: MATERIALS AND METHODS

CHAPTER 2

MATERIALS AND METHODS

37
CHAPTER 2: MATERIALS AND METHODS

1. Animals

Swiss albino mice (8-10 weeks) were used for the present study. The mice were

purchased from the Laboratory Animal Centre (National University of Singapore,

Singapore) and housed in clean cages (not more than 4 mice in one cage) in a

temperature-controlled room with 14h light and 10h dark schedule. Altogether,

271 embryos collected from 64 diabetic mice and 305 embryos from 64 normal

mice were used for this study (Table 1). All procedures involving animal handling

were in accordance with the guidelines of the Institutional Animal Care and Use

Committee (IACUC), National University of Singapore.

38
CHAPTER 2: MATERIALS AND METHODS

Table 1: Animals used in this study


Species Age Sex Number Experiments
Swiss Albino Mouse 8-10 weeks Female 64 Diabetic pregnancy
Swiss Albino Mouse 8-10 weeks Female 64 Normal pregnancy
Swiss Albino Mouse 8-10 weeks Male 35 Used for mating
3 from DP (n=3)
E11.5
3 from NP (n=3)
Swiss Albino Mouse Nil Histological analysis
3 from DP (n=3)
E13.5
3 from NP (n=3)
227 from DP Malformation analysis
Swiss Albino Mouse E11.5 Nil
261 from NP in whole mount embryo

3 from DP (n=3)
Swiss Albino Mouse E11.5 Nil Microarray
3 from NP (n=3)
4 from DP (n=4)
E11.5
4 from NP (n=4)
Swiss Albino Mouse Nil Real time RT-PCR
4 from DP (n=4)
E13.5
4 from NP (n=4)
3 from DP (n=3)
E11.5
3 from NP (n=3)
Swiss Albino Mouse Nil Immunohistochemistry
6 from DP (n=6)
E13.5
6 from NP (n=6)
3 from DP (n=3)
E11.5
3 from NP (n=3)
Swiss Albino Mouse Nil BrdU labeling analysis
3 from DP (n=3)
E13.5
3 from NP (n=3)
6 from DP (n=6)
Swiss Albino Mouse E11.5 Nil In situ hybridization
6 from NP (n=6)
3 from DP (n=3)
Swiss Albino Mouse E11.5 Nil TUNEL
3 from NP (n=3)
3 from DP (n=3)
Swiss Albino Mouse E11.5 Nil Western blot
3 from NP (n=3)

E: embryonic day; DP: diabetic pregnancy; NP: normal pregnancy; n: number of pregnant adult

39
CHAPTER 2: MATERIALS AND METHODS

2. Induction of diabetes mellitus

STZ (an N-nitroso derivative of glucosamine) is one of the most commonly used

diabetogen, inducing insulin-dependent diabetes mellitus in rodents by causing

selective destruction of insulin-secreting -cells in pancreatic islets (Junod et al.,

1969; Rossini et al., 1977). These experimental diabetic animal models have been

widely used to study the diabetes-induced changes in various adult tissues (Dheen

et al., 1994; Kamal et al., 1991) and to understand the molecular mechanisms of

maternal diabetes-induced malformations in various organs including the neural

tube in embryos (Eriksson et al., 2003; Fine et al., 1999; Liao et al., 2004; Phelan

et al., 1997).

Materials

Streptozotocin (STZ) (Cat. No. S0130, Sigma, USA)

0.01M sodium citrate buffer:

Deionised water 100ml

Sodium citrate 2.94g

The pH was adjusted to 4.5 by adding 0.01mol/l citric acid.

Procedure

The STZ was freshly dissolved in 0.01M sodium citrate buffer at a concentration

of 24mg/ml. Diabetes mellitus was induced in 8-10 week-old female Swiss albino

mice with an intraperitoneal injection of STZ (90mg/kg body weight) on three

successive days under aseptic conditions.

40
CHAPTER 2: MATERIALS AND METHODS

2.1. Blood glucose test

Blood glucose level was measured three days after STZ injection. Mouse was kept

in a mouse-restrainer and the tip of its tail protruded out of the restrainer was cut

by a sharp sterile blade. One drop of blood was placed onto a blood glucose strip.

After 20 sec, blood glucose level was displayed on a LCD monitor of blood

glucose meter (Medisense Precision Q.I.D. Blood Glucose Meter, UK). Only

those mice with non-fasting blood glucose level exceeding 300mg/dl were used as

experimental diabetic mice. Age-matched control mice used in the present study

showed non-fasting blood glucose level less than 130mg/dl.

2.2. Collection of embryos

Materials

0.1M phosphate-buffered saline (0.1M PBS):

Di-sodium hydrogen phosphate heptahydrate 13.3g

Sodium chloride 8.5g

Deionized H2O was added to make up to 1 liter.

The pH was adjusted to 7.4 with 1N HCl solution.

5-bromo-2-deoxyuridine (BrdU) solution

BrdU (Cat. No. B5002, Sigma-Aldrich, USA) 10mg

Sterile H2O 1ml

The BrdU powder was freshly dissolved in sterile H2O at a

41
CHAPTER 2: MATERIALS AND METHODS

concentration of 10mg/ml.

Procedure

Four diabetic female mice were placed in a cage with one normal male mouse

overnight. Noon on the day when a copulation plug was observed was considered

day 0.5 of pregnancy. Embryos were collected on embryonic day 11.5 (E11.5) or

embryonic day 13.5 (E13.5) separately. Eight pregnant mice were injected

intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) (100g/g body weight,

Sigma, USA), 2 h before the collection of embryos on either E11.5 or E13.5.

Embryos were collected after Caesarean section of pregnant mice anaesthetized

with intraperitoneal injection of pentobarbital (80mg/kg body weight), cleaned

from placental membrane by sharp sterile scissors and stored in 0.1M PBS at 4C.

3. Histology

3.1 Fixation of embryos

Materials

0.1M phosphate buffer (pH7.4):

NaH2PO4.H2O 2.76g

Na2HPO4.2H2O 14.24g

Deionized H2O was added to make up to 1 liter.

The pH was adjusted to 7.4 with 1N HCl solution.

42
CHAPTER 2: MATERIALS AND METHODS

4% paraformaldehyde (4% PF):

Paraformaldehyde 4g

0.1M phosphate buffer (pH 7.4) 100ml

0.1M phosphate buffer with 30% sucrose:

Sucrose 30g

0.1M phosphate buffer (pH 7.4) was added to make up to 100ml.

Procedure

The fixative used in this study was freshly prepared before use. The 4% PF was

prepared by dissolving paraformaldehyde powder in 0.1M phosphate buffer (pH

7.4) on a hot plate. The fixative was then cooled down to room temperature. The

whole embryos were fixed in 4% PF at 4C overnight and subsequently

cryoprotected with 30% sucrose in phosphate buffer at 4C for 6h. The whole

embryos were photographed using a stereomicroscope (Leica MZ APO, Germany)

equipped with a digital camera. Embryos with neural tube defects from diabetic

mice and normal embryos from non-diabetic mice were used as the experimental

and control groups respectively.

3.2. Preparation of silane coated slides

Materials

2% silane solution:

Silane 2ml

Absolute alcohol 98ml

43
CHAPTER 2: MATERIALS AND METHODS

Procedure

The slides were first cleaned by soaking in absolute alcohol for 2 min and

air-dried. Dried slides were soaked in the silane solution for 2 min and then rinsed

with distilled water for 2 times. The slides were air-dried at room temperature and

stored in a 37C oven.

3.3. Cryosection of embryos

Embryos with neural tube defects from diabetic mice and normal embryos from

non-diabetic mice were mounted on a metal chuck using M-1 embedding matrix

(Lipshaw; Pittsburgh, USA) and quickly frozen by immersing in liquid nitrogen.

Transverse sections of 30m thickness were cut using a cryostat (Leica CM 3050;

Leica Microsystems, Germany), mounted on silane coated slides and then allowed

to dry at room temperature for 2h.

3.4. Hematoxylin and Eosin staining

Materials

Hematoxylin & Eosin

Serially diluted alcohol and xylene

Permount mounting medium (Cat. No. SP15, Fisher Scientific, USA)

Procedure

For morphological studies, the tissue sections were stained with hematoxylin and

44
CHAPTER 2: MATERIALS AND METHODS

eosin. Frozen sections were rinsed in deionized water before staining with

hematoxylin for 10 min at room temperature. Differentiation was achieved by

dipping the mounted sections in acidified 70% ethanol (containing hydrochloric

acid). The sections were then washed in deionized water and counter-stained

with aqueous eosin for 5 min. Finally, the sections were dehydrated in an

ascending series of alcohol and passed through xylene before being mounted with

Permount.

For counting the number of epithelial cells in choroid plexus, four embryos

from control and three embryos from diabetic mice were used. In each embryo, at

least two sections cut through the cranial neural tube containing the choroid

plexus were examined and the number of epithelial cells was scored under the

light microscope fitted with a digital camera (BX51, Olympus, Tokyo, Japan).

4. RNA isolation and quantification

Principles

RNA was isolated using RNeasy mini kit (Qiagen, Germany). This technology

utilizes the selective binding properties of a silica-gel-based membrane as well as

the speed of microspin technology. RNA longer than 200 bases binds to the

silica-gel membrane by a specialized high-salt buffer system. Cells or tissues are

lysed in a highly denaturing buffer with guanidine isothiocyanate (GITC) which

rapidly inactivates RNases to ensure the integrity of RNA. Ethanol is added to

45
CHAPTER 2: MATERIALS AND METHODS

make proper binding conditions, and then the extract of homogenized sample is

loaded to an RNeasy mini column to let the total RNA bind to the membrane.

Contaminants efficiently are washed off from the sample extract by short spin.

High-quality RNA is then eluted from the membrane in RNase-free water. With

this procedure, all RNA molecules longer than 200 nucleotides are purified while

most RNAs shorter than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, and

tRNAs, are selectively excluded.

Materials

RNeasy mini kit (Cat. No. 74106, Qiagen, Germany)

-Mercaptoethanol (Cat. No. 63689, Fluka, USA)

Spectrophotometer (Eppendorf, Germany)

10X MOPS buffer

MOPS 41.8 g

DEPC-treated H2O 700 ml

The pH was adjusted to 7.0 with 2 N NaOH

1 M sodium acetate 20 ml

0.5 M EDTA (pH 8.0) 20 ml

The volume was made up to 1 liter with DEPC-treated H2O

1X MOPS buffer

10X MOPS buffer 100 ml

DEPC-treated H2O 900 ml

46
CHAPTER 2: MATERIALS AND METHODS

RNA loading solution

Formaldehyde 45l

Formamide 45l

10X MOPS buffer 5l

EB (10mg/ml) 3.5l

0.1M EDTA (pH 7.5) 1.5l

Bromophenol blue dye (in 50% glycerol) 8l

Procedure

The cranial neural tube containing the forebrain, midbrain and hindbrain was

dissected out by cutting the neural tube below the level of caudal hindbrain (Fig

1A, B) using a surgical scalpel under a stereomicroscope, rapidly frozen in liquid

nitrogen and stored at -80C. Total RNA was extracted from the cranial neural

tubes in embryos from diabetic and normal mice using the RNeasy mini kit

according to the manufacturers instruction. Neural tubes were lysed in 700l of

RNeasy Lysis Buffer (RLT) with 7l -Mercaptoethanol (-ME). The lysate was

homogenized using a homogenizer and mixed with 700l of 70% ethanol. The

mixture was added into a RNeasy mini spin column, and then centrifuged at

13,000rpm for 15 sec. The flow-through was discarded. Buffer RW1 (700l) was

added to the column and centrifuged again at 13,000rpm for 15 sec. The flow

through was discarded. RPE (500l) was added to wash the column by

centrifugation at 13,000rpm for 15 sec. Finally, the tube with column was

centrifuged at 13,000rpm for 1 min and the flow through was discarded. To elute

47
CHAPTER 2: MATERIALS AND METHODS

the total RNA, the RNeasy column was transferred to a new 1.5ml collection tube

and 30l of water (RNase free) was added directly to the membrane in the column.

The tube was allowed to stand for a min and then centrifuged for 1 min at

13,000rpm.

The concentration and purity of the extracted RNA were evaluated at 260 nm

and 280 nm using a spectrophotometer (Eppendorf, Germany). A value within the

range of 1.9-2.1 was considered an acceptable degree of purity. The integrity of

the extracted RNA was determined by electrophoresis on a denaturing agarose gel.

Mini-gel box, gel tray and combs were washed with deionized water. 1.2g of

agarose was added to 87.5ml of deionized water and heated in a microwave oven

for 2 min. After the agarose solution was cooled down, 5 ml of 10X MOPS buffer

and 7.5ml of 12.3M formaldehyde was added into the agarose solution in a

fume-hood. The solution was poured onto a gel-tray and allowed to solidify for 1h

at room temperature. Solid gel was submerged in a gel box with 1X MOPS buffer.

1g of the extracted RNA was added in 10ul RNA loading solution and then

heated at 70C for 10 min. The RNA sample was rapidly chilled on ice for 1 min

and then loaded into gel. The denaturing gel was run at 50V until the

bromophenol blue dye migrated at least 3 cm in the gel.

48
CHAPTER 2: MATERIALS AND METHODS

5. Microarray analysis

Principles

DNA microarrays provide a platform to analyse thousands of genes at once in

nucleic acid samples (Michael and Patrick, 1999). In general, microarray

experiment involves some basic steps as follows: first, RNA was extracted from

biological samples; secondly, the RNA is in vitro transcripted, while incorporating

either fluorescent nucleotides or a tag that is later stained with fluorescence; and

thirdly, the labelled RNA is hybridized to its corresponding strand spotted on

microarray. Finally the microarray is scanned under laser light and analyzed.

5.1. Synthesis of double stranded DNA from total RNA

Materials

GeneChip One-Cycle cDNA Synthesis Kit (Cat. No. 900431, Affymetrix, USA)

includes:

T7-(dT)24 primer 5-GGCCAGTGAATTGTAATACGACTCACTAT

AGGGAGGCGG-(dT)24 - 3 (50M)

SuperScript II Reverse Transcriptase (200U/l)

5X First-strand cDNA buffer

E. coli DNA ligase (10U/l)

E. coli DNA polymerase I (10U/l)

49
CHAPTER 2: MATERIALS AND METHODS

RNase H (2U/l)

T4 DNA polymerase (5U/l)

5X second strand buffer

dithiothreitol (DTT) (0.1M)

dNTP mix (10mM)

EDTA (0.5M)

RNase free water

Procedure

First-Strand cDNA synthesis

2l of T7-(dT)24 primer, 5.0g of RNA and variable DEPC treated water were

mixed in 1.5ml of RNase free polypropylene tube and incubated at 70C for

10min in a water bath. The tube was centrifuged briefly at about 10,000 rpm for

10s and placed on ice. 4 l of 5X first-strand cDNA buffer, 2l of 0.1M DTT, and

1l of 10mM dNTP were added in the tube and mixed well. The mixture was

incubated at 42C for 2min. 1l of SuperScript II Reverse Transcriptase (200U/l)

was added, mixed well by pipetting and incubated at 42C for 1h. Subsequently,

the tube was centrifuged briefly at about 10,000 rpm for 10s and placed on ice.

Second-Strand cDNA synthesis

30l of 5X second-strand reaction buffer, 3l of 10mM dNTP mix, 1l of 10U/l

E. coli DNA ligase, 4l of 10U/l DNA polymerase I, 1l of 2U/l RNase H, and

50
CHAPTER 2: MATERIALS AND METHODS

91l of DEPC treated water were added to the first strand cDNA synthesis

reaction. The mixture was incubated at 16C for 2h. 2l of T4 DNA polymerase

(5U/l) was then added into the tube, mixed well and incubated for 5min. The

reaction was terminated by adding 10l of 0.5M EDTA.

5.2. cDNA cleanup and precipitation

Materials

GeneChip Sample Cleanup Module (Cat. No. 900371, Affymetrix, USA)

Includes: cDNA cleanup spin columns

cDNA binding buffer

cDNA wash buffer, 6 ml concentrate

cDNA elution buffer

Fragmentation buffer, 5X

Collection tubes, 1.5ml, 2ml

Procedure

600l of cDNA binding buffer was added to the synthesized cDNA and the

mixture was transferred into a cDNA cleanup spin column placed in a 2ml

collection tube. The tube was centrifuged at 13,000rpm for 1min and the

flow-through was discarded. Then the spin column was washed with 750l of

cDNA wash buffer by centrifuging at 13,000rpm for 1min. Finally, the spin

column was centrifuged for 5min with column cap open. To elute the cDNA, 14l

of cDNA elution buffer was added directly onto the column membrane and the

51
CHAPTER 2: MATERIALS AND METHODS

spin column was centrifuged at 13,000rpm for 1min.

5.3. Synthesis of Biotin-labeled cRNA

Materials

GeneChip IVT Labeling Kit (Cat. No. 900449, Affymetrix, USA) includes:

10X reaction buffer

Biotin-labeled ribonucleotides (dNTPs with Bio-UTP and Bio-CTP)

IVT Labeling Enzyme Mix

20X T7 RNA polymerase (Cat. No. 2085, Ambion, USA)

Procedure

12l of sample cDNA, 4l of 10X reaction buffer, 12l of biotin-labeled

ribonucleotides, 4l of IVT Labeling Enzyme Mix, 1l of 20X T7 RNA

polymerase, and nuclease-free water to provide a final reaction volume of 40l

were added to a 1.5ml RNase free tube. The tube was briefly vortexed, centrifuged,

and then incubated at 37C for 5h. After that, the tube was immediately chilled on

ice to terminate the reaction.

5.4. cRNA cleanup and quantification

Materials

RNeasy mini kit (Cat. No. 74106, Qiagen, Germany)

Spectrophotometer (Eppendorf, Germany)

52
CHAPTER 2: MATERIALS AND METHODS

5X Fragmentation Buffer:
In an RNase-free Falcon tube, the followings were mixed thoroughly and

filtered through a 0.2m vacuum filter.

1M Tris-acetate, pH 8.1 (adjusted with glacial acetic acid) 4.0 ml

Magnesium Acetate (MgOAc) 0.64 g

Potassium Acetate (KOAc) 0.98 g

DEPC-treated water to final volume 20mL

Procedure

cRNA cleanup using Qiagen RNeasy mini kit

First, the volume of in vitro transcription (IVT) product was adjusted to 100l

with RNase free water. Then 350l of buffer RLT was added and mixed

thoroughly by pipetting up and down. Subsequently, an additional 250l of 100%

ethanol was added and mixed thoroughly. The mixture was transferred to a mini

spin column and centrifuged at 13,000rpm for 15 sec. The column was washed by

buffer RPE twice and transferred to a new RNase free vial. Finally, 50l of RNase

free water was added directly to the membrane in the column and centrifuged for

1min at 13,000rpm to elute the purified cRNA.

Quantification of the cRNA (IVT Product)

RNA yield was determined spectrophotometrically. The ratio of the absorbance at

260nm/280nm was maintained between 1.9 and 2.1 for the purity of RNA. For

quantification of cRNA, an adjusted cRNA yield was calculated to reflect

carryover of unlabeled total RNA using a formula given below:

53
CHAPTER 2: MATERIALS AND METHODS

Adjusted cRNA yield = RNAm - (total RNAi)(y)

RNAm = amount of cRNA measured after IVT (g)

Total RNAi = starting amount of total RNA (g)

y = fraction of cDNA reaction used in IVT (in this study, y=1)

5.5. Fragmentation of the cRNA for target preparation

Hybridization of the cRNA targets to Gene chips is improved if the cRNA size is

fragmented to 35 to 200 bases using metal-induced hydrolysis at high temperature.

15g of the purified cRNA was mixed with 6l of 5X fragmentation buffer in a

tube and the reaction was made up to 30ul with RNase free water. The mixture

was incubated at 94C for 35min and then immediately cooled down to 4C. The

fragmented sample RNA was stored at -20C.

5.6. Target hybridization

Materials

GeneChip Eukaryotic Hybridization Control Kit (Cat. No. 900299,

Affymetrix) includes: Control oligonucleotide B2, 3nM & Eukaryotic

hybridization control, 20X

Herring sperm DNA (Cat. No. D1811, Promega)

Acetylated bovine serum albumin (BSA), 50 mg/ml (Cat. No. 15561020,

Invitrogen)

Hybridization buffer, 1X, 2X

Mouse Expression Array 430A and 430A 2.0

54
CHAPTER 2: MATERIALS AND METHODS

Procedure

10g of fragmented cRNA was mixed with 3.3l of 3nM control oligonucleotide

B2, 10l of 20X eukaryotic hybridization control, 2l of 10 mg/ml herring sperm

DNA, 2l of 50mg/ml acetylated BSA, 100l of 2X hybridization buffer, and

nuclease-free water to provide a final volume of 200l, incubated at 99C for

5min and subsequently incubated at 45C for 5min. The insoluble material was

removed by centrifuging at 13,000rpm for 5min. Meanwhile, the probe array was

filled with 160l of 1X hybridization buffer and incubated at 45C for 10min.

After removing the buffer, the probe array was filled with 130l of hybridization

mixture and incubated in the oven heated to 45C for 16h.

5.7. Post-hybridization washing, staining and scanning of arrays

Materials and Equipments

GeneChip Scanner 3000 (Cat. No. 00-00212, Affymetrix, USA)

GeneChip Fluidics Station 450 (Cat. No. 00-0079, Affymetrix, USA)

Wash Buffer A (non-stringent wash buffer) (Cat. No. 900721, Affymetrix, USA)

Wash Buffer B (stringent wash buffer) (Cat. No. 900722, Affymetrix, USA)

SAPE stain solution


2X MES Stain Buffer 600l
acetylated BSA (50mg/ml) 48l
(Cat. No. 15561-020, Invitrogen, USA)

SAPE (1mg/ml) 12l


(Cat. No. S-866, Molecular Probes, USA)
DEPC-treated water to 1200l

55
CHAPTER 2: MATERIALS AND METHODS

Antibody solution
2X MES Stain Buffer 300l
acetylated BSA (50mg/ml) 24l
(Cat. No. 15561-020, Invitrogen, USA)

normal goat IgG (10mg/ml) 6l


(Cat. No. I-5256, Sigma, USA)

biotinylated anti-Streptavidin antibody (0.5mg/ml) 3.6l


(Cat. No. BA-0500, Vector Labs, USA)
DEPC-treated water to 600l

Procedure

After 16h of hybridization, the hybridization cocktail was removed and 160l of

non-stringent wash buffer was filled into probe array. Bubbles were carefully

removed to avoid hybridization background. The fluidics protocol for antibody

amplification started with two post hybridization washes, 10 cycles of 2 mixes

with non-stringent wash buffer at 30C and 6 cycles of 15 mixes with stringent

wash buffer at 50C. Subsequently three stain protocol was used. First, each array

was stained with 600l of streptavidin-phycoerythrin (SAPE) solution for 5min at

35C. Then the arrays were washed with non-stringent wash buffer (10 cycles of 4

mixes) at 30C. 600l of antibody solution was used in second stain for

amplifying signals. 600l of SAPE solution was used again in third stain. Finally

the arrays were washed with non-stringent wash buffer (15 cycles of 4 mixes) at

35C. When the protocol was finished, the arrays were filled with non-stringent

wash buffer and temperature was maintained at 25 C. Care was taken to make the

arrays free of bubbles for proper scanning.

Arrays were scanned once under 570nm wavelength with 3um pixel value in

56
CHAPTER 2: MATERIALS AND METHODS

a GeneChip Scanner 3000 according to Affymetrix protocol. Scanned output

image files (.dat) were analyzed with GeneChip Operating Software (GCOS).

Arrays were scaled globally to an average intensity of 500 and analyzed

independently.

Microarray data quality was assessed using Affymetrix guidelines. The

boundaries of the probe area were identified by the hybridization of the B2

oligonucleotide which serves as a positive hybridization control and was used by

the software to place a grid over the image. The hybridization controls were added

into the hybridization samples and were used to evaluate sample hybridization

efficiency on the arrays. -actin and GAPDH were used to assess RNA sample

and assay quality. Specifically, the signal values of the 3 probe sets for -actin

and GAPDH were compared to the signal values of the corresponding 5 probe

sets. The ratio of the 3 probe set to the 5 probe set is generally not more than 3

for the 1-cycle assay.

5.8. Data analysis

5.8.1. Absolute data analysis

In single array analysis which is also called the absolute analysis, .chp file is

created using .cel image file. The image file is automatically generated from .dat

file by Affymetrix Gene Chip Operating Software (GCOS). Single array analysis

acts as a launch pad by providing initial dataset required to perform comparison

analysis. This dataset is considered as baseline if it is used as the control and as

57
CHAPTER 2: MATERIALS AND METHODS

the experiment if it is treated.

5.8.2. Comparison data analysis

In comparison analysis, two samples are compared against each other in order to

detect and quantify changes in gene expression (Chen, 2007). In our present study,

comparison files were generated from cel file by GCOS for each group, using

control as the baseline and treated as the experiment.

5.8.3. Gene filtration

The following criteria were set for determining which genes were differentially

expressed between diabetic and control groups. Firstly, all of the measurements on

each chip were divided by the 50th percentile value (per chip normalization).

Secondly, each gene was normalized to the baseline value of the control sample

(per gene normalization) using median. Genes from "all genes" with expression

control signal from 75 to 18,872 in at least 3 of 6 conditions were selected. Genes

Present or Marginal in at least 3 of 6 samples were then selected. The genes

were filtered using the "Filter on Fold Change" option in GeneSpring software. A

minimum 1.5 fold change in gene expression defined differential expression for

this data set.

5.8.4. Gene clustering and functional analysis based on Gene Ontology

Genes filtered by the above criteria were subjected to agglomerative

average-linkage hierarchical clustering with Gene Spring 7.3 software (Agilent,

58
CHAPTER 2: MATERIALS AND METHODS

CA, USA) using standard correlation as similarity matrix. Further data mining for

functional classification of genes based on Gene Ontology (GO), which is widely

accepted as the standard for describing the biological process, molecular function

and cellular component for genes was carried out using DAVID (The Database for

Annotation, Visualization and Integrated Discovery).

6. Real time reverse transcriptase polymerase chain reaction (Real time

RT-PCR)

Principles

RT-PCR

Reverse transcription combined with polymerase chain reaction (RT-PCR) is a

technique to purposely amplify the number of copies of a specific region of DNA.

The process of PCR includes denaturing, annealing and synthesis of DNA, which

are repeated for 30 to 40 cycles. During these processes, the double strand of the

sample DNA is heated to open as a single stranded DNA which is then hybridized

with a set of 5 and 3 end primers, short fragments of DNA that are designed to

base pair to a specific region of single stranded sample DNA (Mullis and Faloona,

1987). RT-PCR is currently the most sensitive technique for mRNA detection and

quantitation in compared to the two other commonly used techniques such as

Northern blot analysis and RNase protection assay. RT-PCR can be used to

quantify mRNA levels from much smaller samples and even from a single cell.

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CHAPTER 2: MATERIALS AND METHODS

Real time RT-PCR

The real time RT-PCR is a recent advanced quantitative RT-PCR technology with

high sensitivity. Real time RT-PCR incorporates a DNA-binding fluorescent dye,

such as SYBR green I, which binds in the minor groove of double-stranded DNA,

to detect and quantify the PCR product after each cycle of the reaction (Morrison

et al., 1998). In the process of DNA denaturing, the dye is unbound and exhibits

very little fluorescence in the reaction system. When DNA is extended, the

fluorescent dye intercalates into the newly formed double-stranded DNA,

resulting in an increase in the fluorescence signal. Thus, the increase in

fluorescence intensity at the end of the extension step of every PCR cycle is

directly proportional to the increase in amount of amplified DNA. After a few

cycles of PCR, the fluorescent signal of the PCR product is measured to be

statistically significant above background signal. This point is described as

threshold cycle (Ct), which occurs during the exponential phase of PCR cycle

(Gibson et al., 1996). In addition, the specificity of the amplification can be tested

by a melting curve of the PCR product (Ririe et al., 1997). DNA melts at a

specific temperature called the melting temperature (Tm). The melting

temperature of a DNA depends on its GC/AT ratio, therefore the DNA that has

more GC base pairs have a higher Tm than those rich in AT base pairs.

Fluorescent dye is released upon melting of the double stranded DNA, providing

accurate Tm data for every single amplified product, thus desired products can be

distinguished from undesirable products, such as primer dimers.

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CHAPTER 2: MATERIALS AND METHODS

Data analysis of real time RT-PCR

There are two kinds of Real time RT-PCR data analysis, absolute quantification

and relative quantification. The absolute quantification determines the copy

number of the target transcript by relating the PCR signal to the standard curve.

The relative quantification determines the change in gene expression level of

experimental samples relative to another set of untreated controls or samples at

time zero in a time-course study (Livak and Schmittgen, 2001). In addition, the

target gene is normalized with an endogenous reference gene for the relative

quantification. Housekeeping genes and non-regulated genes like

glyceraldehydes-3-phosphate dehydrogenase (GAPDH), albumin, actin, tubulin,

cyclophilin, 18S rRNA and 28S rRNA are frequently used as the reference genes

(Marten et al., 1994; Thellin et al., 1999).

The 2-Ct method (Livak and Schmittgen, 2001) was used in this study to

analyze the real time RT-PCR data and determine the relative changes in gene

expression. The data are calculated as the fold change of target gene expression

normalized to an endogenous reference gene, relative to a calibrator. For the

treated samples, evaluation of 2-Ct indicates the fold change of gene expression

relative to the untreated control. For this method to be valid, the amplification

efficiencies of the target and reference must be approximately equal.

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CHAPTER 2: MATERIALS AND METHODS

Materials

Oligo (dT) primer (Cat. No. C1101, Promega, USA)

dNTP (Cat. No. U1240, Promega, USA)

RNase inhibitor (Cat. No. N2111, Promega, USA)

Molony Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) with

5X Reaction buffer (Cat. No. M1701, Promega, USA)

LightCyclerTM-FastStart DNA Master SYBR Green I (Cat. No.

03515869001, Roche, Germany)

LightCyclerTM real time PCR machine (Roche, Germany)

Primers of target genes (Table 2)

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CHAPTER 2: MATERIALS AND METHODS

Table 2: Primers and thermal profiles for real time RT-PCR


Annealing Extension
Gene Product
Accession No Primers temp./time temp./time
symbol size (bp)
(C/s) (C/s)
5-gaagagctatgagctgcctga-3
NM_007393.1 -actin 103 60/10 72/5
5-ggattccatacccaagaagga-3
5-ccttcgactcttcctcctttg-3
NM_013697 Ttr 112 60/10 72/5
5-gacagcatccaggactttgac-3
5- tctccttatcccaagcacctt-3
NM_010514 Igf2 138 60/10 72/5
5-ttctccttgggttctttcacc-3
5- tccagtcagcaaaggtaagga-3
NM_010025 Dcx 146 60/10 72/5
5-ccaagagagaacagcaaacca-3
5- gcctcagactcagccactatg-3
NM_007523 Bak1 146 60/10 72/5
5- gagtgaaggtggggttcaagt-3
5- ccagggctagacgaagagaat-3
NM_011249 Rbl1 104 60/10 72/5
5- cacatctggcttgaacttgtg-3
5- agctaaggttacccacgaacc-3
NM_009760 Bnip3 106 60/10 72/5
5- caaaactgaccacccaaggta-3
5'- gcacactgtgagaagaccaca-3'
NM_016669 Crym 149 60/10 72/5
5'- aaacagtgttgagctgtgatgg-3'
5'- tctaagcttgtgagagctgctg-3'
NM_026998 Snx6 104 60/10 72/5
5'- tctcttctgcctcactgtttca-3'
5'-atcagcttggtggtgtctttg-3'
NM_013627 Pax6 144 60/10 72/5
5'-gcacctggacttttgcatct-3'
5'-aatctgagactctggagttgaagg-3'
NM_009718 Ngn2 124 60/10 72/5
5'-agctcctcgtcctcctcct-3'
5-aggaagctagggctgaaacaa-3
NM_010431 Hif-1 127 60/10 72/5
5-gccatgtaccagaatcaaacc-3
5-gtccaacttctgggctcttct-3
NM_001025250 Vegf 138 60/10 72/5
5-ttccttctcttcctcccctct-3
5'- caccaaagtgtaccgtgtcct-3'
NM_010438 Hk1 112 60/10 72/5
5'- tcttaggcgttcgtagggtct-3'
5'- ggagctaccacacaccctaca-3'
NM_013820 Hk2 117 60/10 72/5
5'- gaagttggttcctccaaggtc-3'
5'-tcagcatggcagactatgtgt-3'
NM_008826 Pfkl 106 60/10 72/5
5'-ggaacgcaggaacagtagtga-3'
5'- ccaagtatgaggcgagctatg-3'
NM_019703 Pfkp 115 60/10 72/5
5'- aacattccaggtcaggtgatg-3'
5'- agcaaaagagtaggggctgag-3'
NM_011099 Pkm2 134 60/10 72/5
5'- tgcagtgacagaagtggagtg-3'
5'- ggctccccagaacaagattac-3'
NM_010699 Ldha 135 60/10 72/5
5'- atctcgcccttgagtttgtct-3'

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CHAPTER 2: MATERIALS AND METHODS

Procedure

Synthesis of cDNA

The synthesis of cDNA was carried out in a total volume of 25l reaction mix

containing 2g of total RNA, 2.5mol/l of Oligo(dT) primer, 200U of M-MLVRT,

2mmol/l of each dNTPs, 5U of RNase inhibitor and RNase free water. Initially,

2g of RNA with 1l Oligo(dT) primer was heated at 70C for 5min in a

microcentrifuge tube to denature the template. Then the tube was chilled on ice

and other components were added in the reaction. The mixture was incubated at

42C for 1h and the reaction was stopped by heating for 5 min at 95 C.

The real time RT-PCR

Based on the probe sequences information from Affymetrix NETAFFY analysis

center (https://www.affymetrix.com/analysis/netaffx/), primers were designed

using Primer 3 software (http://frodo.wi.mit.edu/). The primer sequence and PCR

thermal profiles are listed in Table 2. The reaction mix (20l) containing 5l of

cDNA, 4l of LightCycler FastStart DNA MasterPLUS SYBR Green I master mix,

1l (0.5M) of each primer and 9l of PCR grade water was pre-incubated at 95

C for 10 min. Subsequently, the PCR was performed with 45 cycles of

denaturation at 95 C for 15sec, annealing at temperatures corresponding to the

genes (Table 2) and elongation at 72 C for PCR product size/25sec. The

expression level of specific gene was quantified, expressed as Ct, the cycle number

at which the LightCycler System first recorded the upstroke of the exponential

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CHAPTER 2: MATERIALS AND METHODS

phase of PCR product amplification, and normalized by the level of -actin

expression in each individual sample (Livak and Schmittgen, 2001).

7. Immunohistochemistry (IHC)

Principles

Immunohistochemistry (IHC) is a widely-used method for localizing target

proteins in tissues or cells using the principle of antigen-antibody specific binding.

A series of technical developments in IHC has created various sensitive detection

systems, including the enzyme-conjugated system, such as peroxidase conjugated

system, for light microscope; the electron-dense particles-labeled system for

electron microscope; and the fluorescent dye-tagged system, such as FITC, Cy3,

Rhodamine labeled system, for fluorescence or confocal laser scanning

microscopy.

IHC on tissue sections is routinely performed with the following steps:

fixation, blocking, incubation with primary antibody and detection of the primary

antibody. Fixation of tissue is required to stabilize and protect the tissues or cells

from rigors of subsequent processing and staining. Blocking of nonspecific

background staining enhances the specificity of the primary antibody. Antibody

molecules are labeled or flagged to permit their visualization.

The detection system can be divided into direct conjugate-labeled antibody

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CHAPTER 2: MATERIALS AND METHODS

method and indirect conjugate-labeled antibody method. The direct method is a

one-step staining method in which the labeled antibody binds directly to the target

antigen in tissues. In the indirect method, the primary antibody that is raised

specifically against the target antigen is added to the sample, followed by addition

of the labeled secondary antibody, which has specificity against an antigenic

epitope present on the primary antibody. Thus, the secondary antibody serves to

label the primary antibody, which, in turn, is bound to the antigen in tissue. The

indirect method is frequently used since the signal could be amplified through

several secondary antibody reactions with different antigenic sites on the primary

antibody.

Materials

0.1M phosphate-buffered saline (PBS):

Di-sodium hydrogen phosphate heptahydrate 13.3g

Sodium chloride 8.5g

Distilled water to make up to 1000ml

The pH was adjusted to 7.4 with 1N hydrochloric acid (HCl) in the

solution.

0.1M PBS containing 0.1% Tween-20 (PBST):

Tween-20 1ml

0.1M PBS 1000ml

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CHAPTER 2: MATERIALS AND METHODS

0.05M Tris buffered saline (TBS; pH 7.6):

Tris base (Trihydroxymethylaminomethane) 6g

Sodium chloride 8g

Distilled water to make up to 1000ml

The pH was adjusted to 7.6 with 1N hydrochloric acid (HCl) in the

solution.

0.1% ammonium nickel sulphate Tris buffered saline (TBS; pH 7.6):

Ammonium nickel sulphate 1g

TBS 1000ml

3,3 diaminobenzidine tetrahydrochloride (DAB) solution:

DAB 50mg

30% hydrogen peroxide 33l

TBS 100ml

0.3% Hydrogen Peroxide (0.3% H2O2):

33% Hydrogen Peroxide 1ml

0.1M PBST 100ml

Permount mounting medium (Cat. No. SP15, Fisher Scientific, USA)

Procedure

Frozen sections mounted on silane coated slides were air dried for 2h. The slides

were rinsed for 30min in 0.1% PBST solution. Sections were treated with 0.3%

H2O2 to inhibit endogenous peroxidase activity. Sections were then blocked by

5% normal serum derived from host species of secondary antibodies for 1h, and

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CHAPTER 2: MATERIALS AND METHODS

subsequently incubated with the respective primary antibodies at the proper

dilution overnight at room temperature. Details of the various primary antibodies

used are listed in Table 3. After incubation with the primary antibodies, tissue

sections were rinsed with 0.1% PBST 3 times for about 15min and incubated with

biotinylated secondary antibodies for 1h at room temperature. Details of the

secondary antibodies used are list in Table 3. The sections were rinsed with PBS,

and incubated with Avidin-Biotin complex (ABC reagent, Vector Lab., USA) for

1h. Immunostaining was visualized with DAB (Sigma, USA) as the peroxidase

substrate. The reaction product was intensified with 0.1% ammonium nickel

sulphate. The tissue sections were finally counter-stained with 1% methyl green,

dehydrated in graded concentrations of alcohol and xylene and coverslipped with

Permount.

Table 3: List of primary and secondary antibodies used for immunohistochemistry

Catalogue
Antibody Name Dilution Host Company
No
Primary antibody
Santa Cruz Biotechnology,
anti-transthyretin 1:100 Goat SC8104
USA
anti-insulin like growth Santa Cruz Biotechnology,
1:100 Rabbit SC5622
factor 2 USA
Secondary antibody
biotinylated anti-goat IgG 1:200 Rabbit Vector Lab, USA BA5000
biotinylated anti-rabbit IgG 1:200 Goat Vector Lab, USA BA1000

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CHAPTER 2: MATERIALS AND METHODS

8. Immunofluorescence staining

Materials

Mouse IgG blocking reagent (Vector Laboratories)

4, 6- diamidino-2-phenylindole dihydrochloride (DAPI) (Molecular Probes, USA)

Fluorescent Mounting Medium (Cat. No. S3023, Dako, Denmark)

0.1M phosphate-buffered saline (PBS)

0.1M PBS containing 0.1% Tween-20 (PBST)

Procedure

Frozen sections mounted on saline coated slides were air dried for 2h. The slides

were rinsed for 30min in 0.1% PBST solution. Sections were then blocked by 5%

normal serum derived from host species of secondary antibodies for 1h, and

subsequently incubated with the primary antibodies at the appropriate dilution

overnight at room temperature. Dilution factor for the various primary antibodies

used are listed in Table 4. Subsequently, sections were washed with 0.1% PBST

and incubated with Cy3-conjugated anti-rabbit IgG for Blbp staining and

biotinylated anti-guinea pig IgG for doublecortin (Dcx) staining (Table 4) for 1h

at room temperature. For Dcx staining, the sections were incubated with

Fluorescein Avidin D for 1h at room temperature. Finally, sections were

counterstained with DAPI (1g/ml) for 5min, and mounted with fluorescent

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CHAPTER 2: MATERIALS AND METHODS

mounting medium. The photo-images were captured using an Olympus FV1000

confocal microscope.

Table 4: Primary and Secondary antibodies used for immunofluorescence staining

Catalogue
Antibody Name Dilution Host Company
No
Primary antibody
anti-doublecortin 1:5000 guinea pig Chemicon, USA AB5910
anti-brain lipid binding protein 1:3000 rabbit Chemicon, USA AB9558
Secondary antibody
Cy3-conjugated anti-rabbit IgG 1:200 sheep Sigma, USA C2306
biotinylated anti-guinea pig IgG 1:200 goat Vector Lab, USA BA7000
Immunofluorescence
Fluorescein Avidin D 1:300 Vector Lab, USA A2001

9. BrdU labeling analysis

Principles

5-bromo-2-deoxyuridine (BrdU) is a synthetic thymidine analog that gets

incorporated into DNA strands of proliferating cells during the S-phase of the cell

cycle. Specific antibody against BrdU can be used to visualize the incorporated

BrdU in the cell immunohistochemically. Denaturation of the DNA by exposing

the cells to acid or heat is required to facilitate the binding of the antibody. The

proliferation index of the cells in a tissue section is analyzed by quantifying the

percentage of BrdU positive cells.

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CHAPTER 2: MATERIALS AND METHODS

Materials

5-bromo-2-deoxyuridine (BrdU) (Cat. No. B5002, Sigma, USA)

Mouse anti-BrdU monoclonal antibody (Cat. No. B2531, Sigma, USA)

Cy3-conjugated goat anti-mouse IgG (Cat. No. AP181C, Chemicon, USA)

Mouse IgG blocking reagent (Cat. No. PK2200, Vector Lab., USA)

biotinylated anti-mouse IgG (Cat. No. BA2000, Vector Lab., USA)

Avidin-Biotin complex (ABC reagent) (Cat. No. PK4002, Vector Lab.,

USA)

Diaminobenzidine (DAB) (Cat. No. D8001, Sigma, USA)

4, 6- diamidino-2-phenylindole dihydrochloride (DAPI) (Cat. No. D1306,

Molecular Probes, USA)

Fluorescent Mounting Medium (Cat. No. S3023, Dako, USA)

0.1M phosphate-buffered saline (PBS)

0.1M PBS containing 0.1% Tween-20 (PBST)

Permount (Cat. No. SP15, Fisher Health Care, USA)

Procedure

Pregnant mice from the control and diabetic groups (n=3 in each group) were

injected intraperitoneally with BrdU (100g/g body weight) 2 h before the

collection of embryos by Caesarean section. The whole embryos were fixed in 4%

PF at 4C overnight and cryoprotected with 30% sucrose in phosphate buffer at

4C for 4h. Embryos with neural tube defects from diabetic mice and normal

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CHAPTER 2: MATERIALS AND METHODS

embryos from non-diabetic mice were used as the experimental and control

groups respectively. Transverse sections of 30m thickness through cranial neural

tubes of the embryos were cut using a cryostat (Leica CM 3050, Leica

Microsystems, Germany).

Immunofluorescence labeling was carried out for the localization of BrdU

positive cells in the neural tube of E11.5 embryos. Sections were rinsed in PBST,

treated with 2N HCl at 37C for 30 min, blocked with mouse IgG blocking serum

for 1h, and incubated with mouse anti-BrdU monoclonal antibody (1:500)

overnight at room temperature. Subsequently, sections were incubated with

Cy3-conjugated goat anti-mouse IgG (1:200) for 1h. Finally, sections were

counter-stained with DAPI (1g/ml) and mounted with Fluorescent Mounting

Medium. Photo-images of the sections were captured in a confocal microscope

(Olympus FV1000, Olympus, Japan).

Immunohistochemistry labeling was carried out for the localization of BrdU

positive cells in choroid plexus epithelium in cranial neural tubes of E13.5

embryos. After rinsed in PBST, sections were treated with 2N HCl at 37C for

30min. Then sections were blocked with mouse IgG blocking serum for 1h and

incubated with mouse anti-BrdU monoclonal antibody (1:500) overnight at room

temperature. Subsequently, sections were incubated with biotinylated anti-mouse

IgG (1:200) for 1h at room temperature. The sections were rinsed with PBS and

incubated with Avidin-Biotin complex for 1h. Immunostaining was visualized

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CHAPTER 2: MATERIALS AND METHODS

with DAB as a peroxidase substrate. The reaction product was intensified with

0.1% ammonium nickel sulphate. The tissue sections were finally counter-stained

with 1% methyl green, dehydrated in graded concentrations of alcohol and xylene

and mounted with Permount.

The number of BrdU-positive cells in the cranial neural tube was quantified

using a total of 12 sections (one each from the telencephalon, diencephalon,

rhombencephalon and choroid plexus of each embryo) from 3 embryos of each

group (control and diabetic). The percentage of BrdU-positive cells was calculated

in relation to the total number of cells in the neuroepithelium of diabetic and

control mouse embryos. Results were expressed as mean SE of the percentage

of BrdU positive cells.

10. Terminal Deoxynucleotidyl Transferase -mediated dUTP Nick-End

Labeling analysis (TUNEL)

Principles

Apoptosis is the most common form of programmed cell death and is essential in

many processes, such as cancer and embryogenesis. Inappropriate regulation of

apoptosis may play an important role in many pathological conditions including

brain diseases (Ceccatelli et al., 2004; Johnson, Jr. and Deckwerth, 1993;

Oppenheim et al., 1999). In general, apoptotic cells display a characteristic pattern

73
CHAPTER 2: MATERIALS AND METHODS

of structural changes in nucleus and cytoplasm, including rapid blebbing of

plasma membrane and nuclear disintegration which is associated with extensive

chromatin damage and fragmentation of DNA. During apoptosis, DNAse activity

generates double-stranded, low-molecular-weight DNA fragments (mono- and

oligonucleosomes), and introduces strand breaks ("nicks") into the

high-molecular-weight DNA. The process of DNA fragmentation can be identified

by labeling the free 3-OH termini of DNA nick ends with fluorescein-dUTP using

the enzyme, Terminal deoxynucleotidyl Transferase (TdT), which attaches labeled

nucleotides to all 3OH-ends. Labeling with fluorescein is followed by

immunohistochemical detection using anti-fluorescein-specific antibodies that are

conjugated to horse-radish peroxidase or alkaline phosphatase. Apoptotic cells can

be visualized using the substrate DAB in a light microscope. This method is very

sensitive and widely used for detection of apoptosis (Gavrieli et al., 1992).

Materials

In situ cell death detection kit (Cat. No. 11684817910, Roche, Germany)

0.1M phosphate-buffered saline (PBS)

0.1M PBS containing 0.1% Tween-20 (PBST):

4% paraformaldehyde (PF)

3, 3 diaminobenzidine tetrahydrochloride (DAB) solution

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CHAPTER 2: MATERIALS AND METHODS

Procedure

Detection of apoptosis in the cranial neural tube of embryos from diabetic and

control mice by TUNEL was carried out with an in situ cell death detection kit

according to the manufacturers instruction. Frozen sections mounted on silanized

slides were air dried for 2h and then washed twice with PBS. The sections were

fixed with freshly prepared 4% PF for 20 min at room temperature, washed with

PBS for 30 min, incubated with 3% H2O2 in methanol for 10 min and then

permeabilized with PBST for 2min on ice. Subsequently, the sections were

incubated with TUNEL reaction mixture containing the TdT and

fluorescein-labeled nucleotides for 60min at 37C in a humidified box in the dark.

The section were rinsed 3 times with PBS and then incubated with anti-fluorescein

antibody conjugated with horseradish peroxidase for 30min at 37C. Finally, the

reaction products were visualized with DAB. The sections were counter-stained

with Haematoxylin and the TUNEL-positive cells were examined under a light

microscope (BX51, Olympus, Japan) at 20X magnification.

11. In situ Hybridization

Principles

In situ hybridization is widely used to determine the spatial expression pattern of a

gene. This involves applying a labeled antisense RNA probe, complimentary

nucleotide sequence to the mRNA of the gene of interest, either to tissue sections

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CHAPTER 2: MATERIALS AND METHODS

or to whole-mount specimens. Labeled probes are prepared by incorporating

radioisotopes or non-reactive digoxigenin (DIG)-dUTP into nucleic acids. The

labeled probes can be detected by autoradiography or anti-DIG IgG which is

usually conjugated with alkaline phosphatase. Since there are several drawbacks

in using radiolabeled probes, including long exposure time, rapid radioactive

decay leading to a limited visualization window, and disposal problems associated

with radioactive material, currently non-reactive DIG-labeled probes have been

widely used. This method was originally described by Hemmati-Brivanlous

group (Hemmati-Brivanlou et al., 1990) and then modified by various groups.

11.1. Plasmids for preparation of cRNA probes

Plasmids containing cDNA of paired box gene 6 (Pax6) and neurogenin 2 (Ngn2)

were kindly provided by Dr. Guillermo Oliver (St. Jude Children's Research

Hospital, Tennessee, USA) and Dr. Francois Guillemot (The National Institute for

Medical Research, London, UK), respectively.

11.2. Preparation of competent cells

Materials

TFB1 solution:

RbCl2 1.2g

MnCl2.4H2O 0.99g

76
CHAPTER 2: MATERIALS AND METHODS

CaCl2 0.15g

Glycerol 15.0g

3M KAc 1ml

The solution was made up to 100 ml with sterilized water. The pH

was adjusted to 5.8 by glacial acetic acid.

TFB2 solution:

0.5M MOPS 2ml

RbCl2 0.12g

CaCl2.2H2O 1.1g

Glycerol 15.0 g

The solution was made up to 100ml with sterilized water. The pH

was adjusted to 6.8 with NaOH.

Luria-Bertani (LB) medium:

Trypton 10g

Yeast extract 5g

NaCl 10g

H2O 1L

LB medium was purchased from the NUMI store of the National

University of Singapore.

Procedure

XL Blue strain cells of E. Coli taken from -80C freezer were spread on the

pre-warmed LB plate and incubated at 37C overnight. The next day, a single

77
CHAPTER 2: MATERIALS AND METHODS

clone was transferred into 5ml of LB medium and shaken at 250rpm in an orbital

shaker (Forma, USA) at 37C overnight. The culture was transferred to 250ml of

LB at 37C, and shaken at 250rpm till the OD reached 0.6 (=600). The cells were

centrifuged at 3500rpm for 10min at 4C. The pellet was resuspended in 50ml of

ice cold TFB1 solution, incubated on ice for 5min, and then centrifuged at

3500rpm for 10min at 4C. The pellet was resuspended again in 10ml of TFB2

solution and incubated on ice for 30min. Finally, the cells were aliquoted as 100l

per vial, rapidly immersed into liquid nitrogen, and stored in -80C freezer.

11.3. Transformation of competent cells with plasmid

Materials

Plasmid mini and midi kits (Cat. No. 12123 and 12143, Qiagen, Germany)

Procedure

100ng of plasmid of Pax6 or Ngn2 was added into 100l of competent cells and

incubated on ice for 30min. Cells were heat shocked at 42C for 90 sec and then

chilled on ice for 2min. Then, 500l of LB was added into each tube, which was

shaken in an orbital shaker at 200rpm, 37C for 1h. The cells were dispersed and

striped evenly on LB plate (with ampicillin) and incubated at 37C with the lid

facing up for 5 min and then incubated with the lid facing down overnight.

Five colonies were selected and each of them was transferred into a separate

tube containing 5ml of LB with 100g/ml ampicillin, and the tubes were shaken at

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CHAPTER 2: MATERIALS AND METHODS

250rpm, 37 C overnight in an orbital shaker. Cells from 2ml of E. coli culture

were harvested and the plasmids were extracted using a plasmid mini kit (Qiagen,

Germany). The plasmid incorporation was confirmed with restriction enzyme

analysis.

1ml of E. coli culture made from confirmed single colony was inoculated in a

flask containing 100ml of LB with 100g/ml of ampicillin and shaken at 250rpm,

37C overnight. The cells were harvested and the recombinant plasmids were

extracted using a plasmid midi kit according to manufacturers instruction.

Generally, the Qiagen plasmid purification technique involves an alkaline

lysis procedure, followed by binding of plasmid DNA to anion-exchange resin

under appropriate low salt and pH conditions. RNA, proteins, dyes and

low-molecular-weight impurities were washed off by a medium-salt buffer and

then the plasmid DNA was eluted in a high-salt buffer. Finally, the plasmid DNA

was concentrated and desalted by isopropanol precipitation.

11.4. Linearization of the plasmid

Materials

BamHI restriction enzyme (Cat. No. R6021, Promega, USA)

Tris-saturated phenol (pH 8.0)

4M Lithium chloride (LiCl)

70% ethanol

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CHAPTER 2: MATERIALS AND METHODS

100% ethanol

RNase free water

TE buffer (pH8.0) 10ml

1M Tris pH8.0 1ml

H2O 9 ml

0.5 M EDTA 20ul

Procedure

Plasmids were linearized with specific restriction enzyme (BamHI for Pax6 and

Ngn2). Restriction digests were performed at 37C for 2h in a 100l reaction

containing 5g of plasmid DNA with 25 Units of restriction enzyme and 1X

buffer. The linearized DNA was loaded onto 0.8% low melting agarose gel. The

band containing the linearized DNA was cut from the gel on the UV

transilluminator and weighed. Equal volume of TE buffer was added to the gel in

the tube and kept at 55C for 5min till the gel was liquefied. Equal volume of

Tris-saturated phenol (pH 8.0) was added into the tube and mixed well. The tube

was centrifuged at 12000rpm for 10min at 4C and the aqueous phase was

transferred into a new tube. One tenth volume of ice cold 4M LiCl and 2.5 volume

of ice cold 100% ethanol was added into aqueous phase and kept in -80C for 1h.

After centrifugation at 12000rpm for 15min and washing the pellet with 70%

ethanol, the pellet of linearized plasmid DNA was dried and dissolved in RNase

free water.

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CHAPTER 2: MATERIALS AND METHODS

11.5. Synthesis of digoxigenin-labeled RNA probe from the linear DNA

Materials

DIG RNA labeling mix (Cat. No. 1277073, Roche Applied Science,

Germany)

T3 RNA polymerase (Cat. No. 600111, Stratagene, USA)

T7 RNA polymerase (Cat. No. 600123, Stratagene, USA)

RNase inhibitor (Cat. No. N2111, Promega, USA)

DNase I (Cat. No. AM2222, Ambion, USA)

RNase-free water

0.2M EDTA

4M LiCl

70% & 100% ethanol

Procedure

The reaction mixture containing 1g of linear DNA, 2l of 10X Reaction buffer,

2l of Dig RNA labeling mix, 1l of T3 (for Pax6) or T7 (for Ngn2) RNA

polymerase, 1l of RNase inhibitor and RNase-free water (make up to total

volume of 20l) was incubated at 37C for 2h. At the end of incubation, 2l of

DNase I was added and incubated at 37C for 15min to remove template DNA.

The reaction was stopped with 2l of 0.2M EDTA. RNA transcripts were

precipitated with 0.1 volume of 4M LiCl and 2.5 volume of ice cold 100% ethanol

at -80C for 1h. After centrifugation at 12000rpm at 4C for 15min and washing

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CHAPTER 2: MATERIALS AND METHODS

the pellet with ice cold 70% ethanol, the pellet of the precipitated RNA transcripts

was dried, dissolved in RNase-free water and stored at -80C.

11.6. Whole mount in situ hybridization

Materials

DEPC treated Water:

H2O 1000ml

DEPC 1ml

Stirred overnight and autoclaved

20X SSC

NaCl 87.6g

Sodium citrate 44.1g

DEPC-H2O was added up to 500ml and pH was adjusted to 7.0.

50mg/ml Heparin

Heparin (Cat. No. H3400, Sigma, USA) 50mg

H2O 1ml

10mg/ml yeast tRNA

yeast tRNA (Cat. No. R6625, Sigma, USA) 10mg

H2O 1ml

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CHAPTER 2: MATERIALS AND METHODS

prehybridization solution

Boehringer Block 0.5 g

Formamide 25 ml

20X SSC (pH 7) 12.5 ml

heated to 65C for about 1h. Once dissolved, followings were added:

10mg/ml yeast tRNA 5ml

50mg/ml Heparin 100 l

20% Tween-20 250 l

10% CHAPS 500 l

0.5 M EDTA 500 l

H2O 6 ml

Hybridization solution

Prehybridization solution 500l

0.1g/l DIG-labelled RNA Probe 5l

Heated at 95C and chilled on ice for 5 min before use.

Maleic Acid Buffer (MAB)

Maleic acid 11.6g

NaCl 8.8g

DEPC-H2O was added up to 1L and pH was adjusted to 7.5 with

1N NaOH.

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CHAPTER 2: MATERIALS AND METHODS

10% boehringer blocking reagent

Boehringer block (Cat. No. 11096176001, Roche, Germany) 1g

PBST 10ml

Blocking antibody buffer

Goat serum 1ml

10% Boehringer blocking reagent 1ml

PBST 8ml

The mixture was heated at 65 C until total dissolution, filtered through

4.5 micron filters, and chilled on ice.

Preabsorbed AP-anti-Digoxygenin Antibody

AP-anti-Digoxygenin antibody (Cat. No. 1093274, Roche, Germany) 3l

Antibody buffer 15ml

The above solution was rocked at 4C for 2h

Alkaline phosphatase (AP) buffer:

1M Tris, pH9.5 1ml

1M MgCl2 0.5ml

5M NaCl 0.2ml

Tween 20 10l

DEPC water was added up to 10ml.

Proteinase K (Cat. No. 03 115 836 001, Roche, Germany)

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CHAPTER 2: MATERIALS AND METHODS

BM purple (Cat. No. 11 442 074 001, Roche, Germany)

3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)

(Cat. No. C-3023, Sigma, Germany)

Procedure

Preparation of mouse embryos

The embryos were dissected free of extra embryonic membranes and fixed in

freshly prepared 4% PF at 4C overnight. The next day the embryos were washed

in PBST (PBS with 0.1% Tween-20) twice for 10min each. Then the embryos

were dehydrated with a methanol series in PBST (25%, 50%, 75% and 100%) and

stored in 100% methanol at -80C for further use.

Hybridization

The embryos were rehydrated through 75%, 50% and 25% methanol series in

PBST. The embryos were washed 3 times with PBST for 5min each, bleached by

incubating with 6% H2O2 for 1hr, and washed 3 times with PBST again for 5min

each. Then the embryos were treated with 5g/ml of proteinase K in PBST for

15min and the reaction was stopped by washing in freshly prepared 2mg/ml

glycine in PBST. After washed 2 times with PBST for 5min each, the embryos

were refixed with fresh 4% PF + 0.2% glutaraldehyde in PBST for 15min, and

washed with PBST 3 times for 10min each. Prehybridization of embryos was

carried out with 1ml of prehybridization solution at 65C for 3h. For hybridization,

the prehybridization solution was replaced with hybridization solution containing

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CHAPTER 2: MATERIALS AND METHODS

1g/ml digoxigenin-labelled RNA probe, and the embryos were incubated at 70C

overnight.

Post-hybridization washes and antibody incubation

After hybridization, the embryos were washed with 800l of prehybridization

solution for 5min at 70C. Then the embryos were washed three times with

addition of 400l of 2X SSC, each time without removing prehybridization

solution. After removing the solution, the embryos were washed twice in 2X SSC

with 0.1% CHAPS at 70 C for 30 min each. The embryos were washed in Maleic

acid buffer (MAB) twice at room temperature for 10 min each and at 70 C for 30

min twice. After washed in PBST 3 times for 5 min each, the embryos were

preblocked in 1ml blocking antibody buffer for 2h at 4C with rocking. The

embryos were incubated with preabsorbed anti-Digoxygenin-AP antibody

overnight at 4C with rocking.

Post-antibody washes and color development

The next day, embryos were washed 6 times with 0.1% BSA in PBST for 45min

each to remove nonspecific staining. After washed in PBST twice for 30 min each,

the embryos were washed again in AP buffer at room temperature twice for 10min

each. Then embryos were incubated with 1ml BM purple in a dark chamber till the

color developed to the desired extent. The staining reaction was stopped by

washing in PBS with several frequent changes. After staining, embryos were fixed

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CHAPTER 2: MATERIALS AND METHODS

in 4% PF.

The whole mount embryos were photographed using a stereomicroscope

equipped with camera (Leica MZ APO, Germany). Some embryos were sectioned

(30m) using the cryostat (Leica CM 3050, Germany) and the sections were

photographed using Olympus X51 light microscope (Olympus, Japan).

12. Western blot analysis

Principles

Western blot (or immunoblot) is a commonly used method to detect and determine

the relative amount of specific proteins in tissue homogenate or extract (Towbin et

al., 1979). Firstly, protein samples are extracted from tissues or cells using

assorted detergents, salts, and buffers which help lysis of cells and solubilize

proteins. Protease and phosphatase inhibitors are also added into the homogenate

to prevent the degradation of the sample by its own enzymes. Secondly, the

mixture of protein is separated on a gel by electrophoresis. Polyacrylamide gels

and buffers with sodium dodecyl sulfate (SDS) are the most common type of gel

electrophoresis. SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains

polypeptides in a denatured state once they are denatured with strong reducing

agents to break their secondary and tertiary structure (e.g. S-S disulfide bonds to

SH and SH) which allows separation of proteins by their molecular weight. After

separation, proteins are transferred to a membrane which can be either

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CHAPTER 2: MATERIALS AND METHODS

nitrocellulose or polyvinylidene fluoride (PVDF). Primary antibodies are used to

detect specific protein antigens on the membrane. Detection of antibody-antigen

immune complexes on a membrane is accomplished using secondary antibodies

covalently conjugated with horseradish peroxidase (HRP) enzyme. The

chemiluminescence of HRP substrate is developed on a standard X-ray film.

Materials

Protease inhibitor cocktail kit (Cat. No. 78410, Pierce, USA)

Mammalian protein extraction reagent (Cat. No. 78503, Pierce, USA)

Protein assay kit (Cat. No. 5000002, Bio-Rad, USA)

10% resolving gel:

H2O 7.9ml

30% acrylamide mix 6.7ml

1.5 M Tris (pH 8.8) 5.0ml

10% SDS 0.2ml

10% ammonium persulfate 0.2ml

Tetramethylethylenediamine (TEMED) 0.008ml

5% stacking gel:

H2O 5.5ml

30% acrylamide mix 1.3ml

1.0 M Tris (pH 6.8) 1.0ml

10% SDS 0.08ml

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CHAPTER 2: MATERIALS AND METHODS

10% ammonium persulfate 0.08ml

TEMED 0.008ml

1x SDS gel-loading buffer:

50mM Tris.Cl (pH 6.8)

100mM dithiothreitol

2% SDS

0.1% bromophenol blue

10% glycerol

Tris-glycine electrophoresis buffer:

25mM Tris

250mM glycine

0.1% SDS

Transfer buffer:

25mM Tris

250mM glycine

20% Methanol

1X TBS:

Tris base 2.42 g

NaCl 0.8 g

Made up to 1L with H2O; pH was adjusted to 7.6 with 2N HCl

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CHAPTER 2: MATERIALS AND METHODS

1X TBST:

1X TBS 1 liter

0.1% Tween 20

Mouse anti-Hif1 monoclonal antibody (Cat. No. MAB5382, Chemicon,

USA)

Rabbit anti-Vegf polyclonal antibody (Cat. No. SC-152, Santa Cruz, USA)

Goat anti-rabbit IgG conjugated with horseradish peroxidase (Cat. No.

7074, Cell Signaling Technology, USA)

Mouse anti--actin monoclonal antibody (Cat. No. A1978, Sigma-Aldrich,

USA)

Goat anti-mouse IgG conjugated with horseradish peroxidase (Cat. No.

31430, Pierce, USA)

SuperSignal West Pico Chemiluminescent Substrate (Cat. No. 34080,

Pierce, USA)

96 well plate (Cat. No. 3860-096, Asahi Techno Glass, Japan)

Procedure

Protein extracts from cranial neural tube of individual embryo from control and

diabetic mice were prepared separately using protein extraction reagent, which

was added with protease inhibitor cocktail and EDTA solution. Briefly, the cranial

neural tube was lysed with 600l of protein extraction reagent in a homogenizer.

The supernatant was collected by centrifugation at 13,000g at 4C for 15min and

stored at -20C. The concentration of protein extracts was determined using a

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CHAPTER 2: MATERIALS AND METHODS

protein assay kit. Four different concentrations (0.5, 0.25, 0.125, and 0.0625mg/ml)

of BSA were prepared as protein standards. 10l of each standard or sample and

200l of dye reagent were added together to each well of a 96-well plate, mixed

thoroughly and incubated at room temperature for 10min. The absorbance of

protein standard or sample was measured at 595nm using a microplate reader

(GENios, Tecan, Switzerland). The concentration of protein extract was

determined according to the standard curve of BSA.

Aliquots of protein extracts (10g each) were heated to 95C for 5min in 5x

SDS gel-loading buffer to denature the proteins before the proteins were separated

by 10% SDS-PAGE. Next, the proteins were transferred from the gel to the 0.2m

PVDF membranes using a semi-dry electrophoretic transfer cell (Bio-Rad, USA).

Membranes were washed with TBST buffer and then blocked with 5% non-fat

milk in TBST for 1h at room temperature. Subsequently, the membranes were

incubated with mouse anti-Hif1 antibody (1:100) or rabbit anti-Vegf antibody

(1:200) overnight at 4C in a shaker. The next day, blots were incubated with

secondary anti-mouse IgG antibody (1:10000) or anti-rabbit IgG antibody

(1:10000) conjugated with horseradish peroxidase for 1h at room temperature.

Equal amount of protein loading was confirmed by reprobing the membrane with

anti- -actin mAb (1:5000). Immunopositive bands were visualized by

chemiluminescent substrate, and quantified by scanning densitometer and

Quantity One software, version 4.4.1 (Bio-Rad, USA).

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CHAPTER 3: RESULTS

CHAPTER 3

RESULTS

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CHAPTER 3: RESULTS

1. Neural tube defects in embryos of diabetic mice

It is well demonstrated that maternal diabetes induces congenital malformations in

various organs including neural tube during development. Neural tube defects

(NTDs) are one of the common anomalies in embryos of diabetic mice in this

study. About 12% of embryos (E11.5) from diabetic mice (n=20) showed neural

tube malformations, including excencephaly, syntencephaly and spina bifida as

reported previously (Liao et al., 2004). The exencephalic phenotype is

characterized by defect in closure of the forebrain, midbrain and hindbrain (Fig.

1A, B).

Histological analysis of transverse sections of different parts of the cranial

neural tube including the telencephalon (tc), diencephalon (dc) and

rhombencephalon (rc) from E11.5 embryos of control and diabetic mice clearly

demonstrated the cranial neural tube malformations with varying degrees of

severity in embryos of diabetic mice (Fig 2A-D). The neuroepithelia of

telencephalon, diencephalon and rhombencephalon from embryos of diabetic mice

were distinctly distorted and their walls were fused, leaving the ventricular system

including lateral ventricles (LV), third ventricle (III) and fourth ventricle (IV),

collapsed (Fig 2A-D). The size and volume of the ventricles appeared to be

reduced markedly in comparison to that of embryos from normal pregnancies.

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2. Gene expression profile of cranial neural tubes in embryos of control and

diabetic mice

In order to gain insights of molecular basis for morphological changes in the

cranial neural tube of embryos with NTDs derived from diabetic mice, gene

expression profile of the cranial neural tubes in E11.5 embryos of control and

diabetic mice was established by microarray analysis. The microarray analysis

was carried out using the Affymetrix oligonucleotide chip which helped to

compare global gene expression patterns of the cranial neural tubes of E11.5

embryos from control and diabetic mice.

Overall, 1613 genes have been found to be differentially expressed with the

magnitude of more than 1.5 folds change in the developing brain of E11.5

embryos from diabetic mice as compared to that of embryos from control mice.

These genes were hierarchically clustered using GeneSpring v7.3 (Agilent, CA,

USA) (Fig 3).

Among these genes, a total of 390 genes exhibited greater than 2-folds of

change with a cluster of 180 genes (46.2%) showing increased expression and the

rest (53.8%) showing decreased expression in the cranial neural tubes of embryos

from diabetic pregnancies. According to the Gene Ontology Consortium database

for biological processes [http://www.geneontology.org/], these genes have been

placed into 8 main functional categories as follows (Fig 4): (1) metabolism

(27.7%); (2) cellular physiological process (20.3%); (3) cell communication

(12.1%); (4) morphogenesis (6.9%); (5) response to stimulus (3.8%); (6) cell

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death (2.3%); (7) cell differentiation (2.1%); (8) small subsets of genes (5.4%).

About 19.4% of genes that were differentially expressed had no known functional

annotations. Microarray results were validated by the real time RT-PCR for

selected genes (Table 5).

3. Effect of maternal diabetes on expression of genes involved in glucose

metabolism and that respond to physiological hypoxia in cranial neural tubes

of mouse embryos

Embryonic dysmorphogenesis in diabetic pregnancies appears to be the result of

multiple causes including the maternal metabolic abnormalities. Microarray

analysis revealed that the expression of genes that are involved in pathways

related to glucose metabolism and that respond to physiological hypoxia was

altered in embryos of diabetic pregnancies.

3.1. Maternal diabetes altered the expression of genes involved in glycolysis in

cranial neural tubes of mouse embryos

Expression of majority of the genes involving glycolysis pathway was found to be

upregulated in cranial neural tubes of embryos from diabetic mice (Table 6 and

Fig 5). Expression of some of these genes (hk1, hk2, pfkl, pfkp, pkm2 and ldh)

which encode key enzymes of the glycolysis pathway has been validated by the

real time RT-PCR (Fig 6). The results obtained by RT-PCR confirmed the

microarray results.

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3.2. Maternal diabetes altered the expression of genes that respond to hypoxia in

cranial neural tubes of mouse embryos

Expression of genes that respond to hypoxia was also upregulated in cranial neural

tubes of embryos from diabetic mice (Table 6). For example, hypoxia-inducible

factor 1 (Hif-1) was found to be upregulated in cranial neural tube of embryos

from diabetic mice. Hif-1 plays a key role in the adaptive response to hypoxia,

transactivating many genes whose protein products are involved in pathways of

angiogenesis, glucose metabolism and cell proliferation (Semenza, 2003).

Morever, the upregulation of Hif-1 transactivates antiproliferative and

proapoptotic genes (Bacon and Harris, 2004). The expression of Igfbp3 and P21,

which are regulated by Hif1 and negatively regulate the cell proliferation and cell

cycle (Feldser et al., 1999; Goda et al., 2003), was found to be upregulated. In

addition, Bnip3, which is a hypoxically inducible gene and a proapoptotic member

of BCL2 family (Sowter et al., 2001) was also found to be upregulated.

3.2.1. Expression of hypoxia-inducible factor 1 (Hif1) in cranial neural tubes of

embryos from control and diabetic mice

Microarray analysis showed the increased expression of Hif1 gene in cranial

neural tubes of E11.5 embryos from diabetic mice in comparison to that of control

mice (Table 6). This result was further confirmed by the real time RT-PCR which

showed upregulation of Hif1 mRNA expression in cranial neural tubes of E11.5

embryos derived from diabetic mice (Fig 7). The Hif1 protein was detected in

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cranial neural tubes of E11.5 embryos from control and diabetic mice by western

blot (Fig 8A). Quantitative analysis showed that the Hif1 protein level was

increased in cranial neural tubes of embryos from diabetic mice when compared to

that of embryos from control mice (Fig 8B). This change in the Hif1 protein

expression correlated well with the change in the Hif1 mRNA expression in

cranial neural tubes of embryos from diabetic mice.

3.2.2. Expression of Vegf in cranial neural tubes of embryos from control and

diabetic mice

Hypoxia induces angiogenesis as a compensatory mechanism to supply adequate

oxygen by inducing several angiogenic factors including vascular endothelial

growth factor (Vegf) (Mazure et al., 1996; Plate et al., 1992). Vegf is considered

as a key regulator of angiogenesis particularly during embryogenesis (Breier et al.,

1992; Dumont et al., 1995; Millauer et al., 1993). Analyses by both microarray

(Table 6) and real time RT-PCR (Fig 9) showed that expression of Vegf gene was

significantly increased in cranial neural tubes of embryos from diabetic mice. In

addition, the protein expression of Vegf has also been analyzed in cranial neural

tubes of embryos from control and diabetic mice (Fig 10A). The quantity of Vegf

expression was significantly increased in cranial neural tubes of embryos from

diabetic mice, compared to that of embryos from control mice (Fig 10B). This

change in the Vegf protein expression was comparable with the change in the Vegf

mRNA expression in cranial neural tubes of embryos from diabetic mice.

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4. Maternal diabetes induces apoptosis in the neuroepithelium of cranial

neural tubes in mouse embryos

It has been previously shown that maternal diabetes induces apoptosis in the

neural tube of mouse embryos (Fine et al., 1999; Phelan et al., 1997). Microarray

analysis in the present study revealed that the expression levels of a majority of

the genes that regulate apoptosis including proapoptotic genes such as

transformation related protein 53 (p53), BCL2/adenovirus E1B 19 kDa interacting

protein 3 (Bnip3) and BH3 interacting domain death agonist (Bid) were increased

in cranial neural tubes of embryos from diabetic mice (Table 7).

TUNEL analysis further showed that numbers of apoptotic cells were

markedly increased in the neuroepithelia of forebrain (including the telencephalon

and diencephalon) and rhombencephalon in embryos from diabetic mice, in

comparison to that of embryos from normal mice (Fig 11).

5. Maternal diabetes inhibits proliferation index in the neuroepithelium of

cranial neural tubes in mouse embryos

The proliferation index in cranial neural tubes of E11.5 embryos from diabetic and

normal mice was examined by immunohistochemical detection of BrdU

incorporation (Fig 12A-D). The transverse sections through the telencephalon,

diencephalon and rhombencephalon of cranial neural tubes from embryos of

diabetic mice were compared with that of embryos from normal mice.

Immunofluorence staining showed that the percentage of BrdU labeled cells was

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CHAPTER 3: RESULTS

significantly decreased in neuroepithelia of telencephalon (40.6%3.2% vs.

63.7%4.1%, p<0.001), diencephalon (29.7%1.6% vs. 53.7%8.3%, p<0.02)

and rhombencephalon (37.1%3.7% vs. 45.2%4.3%, p<0.05) of embryos from

diabetic mice in comparison to that of embryos from normal mice (Fig 12E).

6. Maternal diabetes alters the expression of genes involved in neuronal

migration and neurogenesis in cranial neural tubes of mouse embryos

It is well established that the survival, proliferation and differentiation of

neuroepithelial cells of different regions of the brain are controlled by several

developmental control genes during embryogenesis. The microarray analysis

showed that several genes involved in proliferation, migration and differentiation

of neuronal and glial cells in the cranial neural tube were differentially expressed

in embryos of diabetic mice (Table 8). The expression levels of a majority of the

genes which control neuronal migration and neurogenesis including the

microtubule-associated proteins such as doublecortin (Dcx), and doublecortin-like

kinase (Dclk), neurogenic differentiation 1 (Neurod1), neurogenin 2 (Ngn 2),

Notch signaling pathway genes such as Notch gene homolog 1 (Notch 1) and

hairy and enhancer of split 6 (Hes 6), brain lipid binding protein (Blbp),

insulin-like growth factor-2 (Igf-2), paired box gene 6 (pax6), bone morphogenetic

protein 4 (Bmp4), etc were downregulated in cranial neural tubes of embryos from

diabetic pregnancies, indicating an impaired neurogenesis and neuronal migration

in embryos of diabetic pregnancy.

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6.1. Expression pattern of developmental control genes is altered in embryos of

diabetic mice

6.1.1. Paired box gene 6 (Pax6)

Pax6, a paired box transcription factor with key functional roles in the developing

brain, promotes neuronal differentiation via transcriptional regulation of the Ngn2

gene (Scardigli et al., 2003). Microarray analysis showed that expression of Pax6

was downregulated in cranial neural tubes of embryos from diabetic pregnancy

(Table 8). The real time RT-PCR analysis further confirmed the decreased mRNA

expression level of Pax6 in cranial neural tubes of embryos from diabetic mice

compared to that of embryos from control mice (Fig 13). By in situ hybridization

analysis, the mRNA expression of Pax6 was localized to the forebrain including

the telencephalon and diencephalon in embryos of control mice (Fig 14A-C).

However in the forebrains of embryos from diabetic pregnancies, the expression

level of Pax6 appeared to be markedly decreased and its expression domain was

distinctly perturbed (Fig 14D-F).

6.1.2. Neurogenin 2 (Ngn2)

Ngn2, a proneural gene belonging to family of the bHLH factors, is expressed in

the distinct progenitor populations in the nervous system and is involved in the

specification of neuronal phenotype in the developing brain (Parras et al., 2002).

Microarray analysis revealed that expression of Ngn2 was downregulated in

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cranial neural tubes of embryos from diabetic mice (Table 8). The downregulation

of Ngn2 in cranial neural tubes of embryos from diabetic mice compared to that of

embryos from control mice was further confirmed by the real time RT-PCR

analysis (Fig 15). In situ hybridization analysis revealed the mRNA expression

pattern of Ngn2 in the forebrain including the telencephalon and diencephalon,

and rhombencephalon in embryos of control mice (Fig 16A-D). Its expression

level seemed to be markedly decreased and the expression domain was distinctly

perturbed in both telencephalon and diencephalon of embryos from diabetic mice

(Fig 16E-G). However, the expression level of Ngn2 appeared to be unaffected in

the rhombencephalon of embryos from diabetic mice compared with the control

(Fig 16D, H).

6.2. Development of radial glial cell lineages and migration of neurons are

disrupted in the cranial neural tubes of embryos from diabetic mice

6.2.1. Doublecortin (Dcx)

Doublecortin (Dcx), a microtubule-associated protein, is expressed in migrating

neuroblasts and differentiating young neurons throughout the central and

peripheral nervous system during embryonic development (Couillard-Despres et

al., 2001; des, V et al., 1998; Gleeson et al., 1998). Consistent with microarray

results showing down-regulation of Dcx (Table 8), the domain containing Dcx

positive cells was found to be markedly decreased in the forebrain and

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rhombencephalon in cranial neural tubes of embryos derived from diabetic mice

when compared to that of embryos from control mice (Fig 17A-D).

6.2.2. Brain lipid binding protein (Blbp)

During early development, migration of cells in the neural tube occurs along the

radial glial cells, which serve as neuronal progenitors, neuronal migration guides

and astrocyte progenitors (Malatesta et al., 2003; Noctor et al., 2001; Parnavelas

and Nadarajah, 2001). Blbp is expressed in the radial glial cells which are also

essential for maintenance of neuroepithelial cells during early cortical

development (Arai et al., 2005). As observed in microarray analysis (Table 8),

immunofluorescence analysis also revealed that the domains containing Blbp

positive cells in the neuroepithelia of telencephalon, diencephalon and

rhombencephalon were attenuated in embryos of diabetic mice (Fig 17A-D). In

addition, the processes of Blbp-positive radial glial cells extending towards pial

surface appeared to be disrupted in the neuroepithelia of embryos from diabetic

pregnancies (Fig 17E, F). Further, Neurofibromatosis type 1 (Nf-1) which has

been shown to downregulate Blbp (Miller et al., 2003) was upregulated in cranial

neural tubes of embryos from diabetic pregnancies (Table 8), indicating that

downregulation of Blbp is possibly mediated via upregulation of Nf-1 in cranial

neural tubes of embryos from diabetic pregnancy.

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7. Development of choroid plexus is impaired in cranial neural tubes of

embryos from diabetic mice

The choroid plexus (CP) is found in the developing brain at three sites of cerebral

ventricles, the lateral ventricle (LV), the 3rd ventricle (III) and the 4th ventricle (IV).

It is composed of a single layer of epithelial cells around the mesenchymal tissue

containing blood vessels. In the present study, CP was hardly detectable in any of

the ventricles at E11.5 embryos although it was reported to be developed as early

as E10 in the 4th ventricle. However, it was readily evident in the IV of cranial

neural tubes in embryos at E13.5 (Fig 18A, C and E). In addition, the CP was

found to be poorly developed or absent in the IV of E13.5 embryos from diabetic

mice (Fig 18B, D, and F). Quantitative analysis revealed that the number of

epithelial cells was significantly decreased in the CP of embryos from diabetic

mice compared to that of embryos from control mice (Fig 19). The volume of IV

appeared to be markedly reduced in embryos of diabetic mice (Fig 18B, D).

The CP produces the cerebral spinal fluid (CSF) which provides an essential

expanding mechanism that determines the shape of the brain during its

development (Dziegielewska et al., 2001) and several growth factors such as

transthyretin (Ttr) and insulin-like growth factor-2 (Igf-2).

7.1. Transthyretin (Ttr)

Ttr is the major protein synthesized by the CP epithelial cells and is involved in

the transport of thyroxine and retinol from the peripheral circulation to the brain

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(Dickson et al., 1987; Ferguson et al., 1975; Raz et al., 1970). The microarray

analysis showed the decreased expression of Ttr in E11.5 embryos of diabetic

mice, in comparison to that of control mice (Table 5). This result was further

confirmed by the real time RT-PCR which showed downregulation of Ttr in

cranial neural tubes of both E11.5 and E13.5 embryos derived from diabetic mice

(Fig 20).

Immunohistochemical analysis of Ttr was carried out in cranial neural tubes

of E11.5 and E13.5 embryos from control and diabetic mice. Ttr appeared to be

weakly expressed in the tela choroidea, the developmental forerunner of the

choroid plexus, in the cranial neural tubes of E11.5 embryos from control mice

(Fig 21A), whereas in E13.5 embryos of control mice, the expression of Ttr was

clearly evident in the epithelial cells of the CP (Fig 21C). However, the

immunostaining of Ttr was markedly reduced in the tela choroidea of E11.5

embryos and CP epithelium of E13.5 embryos from diabetic mice (Fig 21B, D).

7.2. Insulin-like growth factor 2 (Igf-2)

Igf-2, a polypeptide hormone which functions as a mitogen during embryogenesis

and as a morphogen in inducing differentiation of CP epithelial cells during

development (Cavallaro et al., 1993; Eicher et al., 1993). Expression of Igf-2 was

decreased in cranial neural tubes of E11.5 embryos from diabetic mice in

comparison to that of embryos from control mice as shown in the microarray

analysis (Table 5). The real time RT-PCR analysis also confirmed the

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downregulation of Igf-2 in cranial neural tubes of E11.5 and E13.5 embryos

derived from diabetic mice (Fig 22).

As revealed by immunohistochemistry analysis, the Igf-2 protein expression

was detectable in the tela choroidea in cranial neural tubes of E11.5 embryos from

control mice (Fig 23A). Intense immunostaining of Igf-2 was localized to the

epithelial cells of the CP in cranial neural tubes of E13.5 embryos from control

mice (Fig 23C). The immunostaining of Igf-2 was absent or markedly reduced in

the tela choroidea of E11.5 embryos and CP epithelium of E13.5 embryos from

diabetic mice (Fig 23B, D).

7.3. The proliferation index in the choroid plexus and adjacent neuroepithelia

The proliferation index in the CP and the adjacent neuroepithelia around the

ventricles was measured by BrdU labeling in E13.5 embryos of both normal and

diabetic mice. Mitotic cells in the developing CP were hardly detectable in cranial

neural tubes of embryos from both normal and diabetic mice. However, the BrdU

positive cells were detected in the base of the CP and adjacent neuroepithelia

around the ventricles (Fig 24A, C). The percentage of proliferating cells in

relation to total cells in the neuroepithelia around the IV and base of the CP was

significantly decreased (12.9%1.2% vs. 30.7%5.1%, p<0.01) in embryos from

diabetic mice compared to that of embryos from control mice (Fig 24B, D and E).

105
CHAPTER 4: DISCUSSION

CHAPTER 4

DISCUSSION

106
CHAPTER 4: DISCUSSION

It has been widely demonstrated that the maternal diabetes induces malformations

in various organs including neural tubes in embryos of rodents and humans

(Akazawa, 2005; Liao et al., 2004; Mills et al., 1979). Neural tube defects (NTDs)

in embryos of diabetic pregnancy have been shown to be associated with altered

expression of some developmental control genes involved in neurulation (Liao et

al., 2004). More recently, it has been shown that high-glucose concentration alters

the expression of genes that are involved in proliferation and differentiation of

neural stem cells (NSCs), indicating the basis for the CNS malformations in

infants of diabetic mothers (Fu et al., 2006). The present study is the first of its

kind demonstrating the differential gene expression profiling of cranial neural

tubes in embryos of diabetic mice in comparison to that of embryos from control

mice.

During neurulation in mouse embryos, the neural plate closure occurs at

E8.5-9.5 and subsequently the rostral end of the neural tube enlarges in size due to

an increase in the size of ventricles which are filled with CSF secreted by CP

epithelium (Sturrock, 1979). During this expansion process, progenitor cells in the

neuroepithelia around the ventricles undergo proliferation, migration and

differentiation into different types of neurons and glia that form the brain. The

initial neurulation and subsequent brain development require accurate

coordination of the proliferation, differentiation and apoptotic cell death of neural

and glial progenitor cells. These events are mediated by a complex interplay

between genetic and environment factors, which involve the function of inductive

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CHAPTER 4: DISCUSSION

signaling molecules, and thereby activation of transcription factors that control the

position of different cell types in the developing brain (Harris and Juriloff, 1999).

Altered expression of many of those genes caused by environmental factors or

teratogenicity including maternal diabetes appeared to be implicated in neural tube

anomalies.

In the present study, malformations were evident in the telencephalon,

diencephalon, and rhombencephalon as well as the ventricular system of E11.5

embryos from diabetic mice. In addition, several genes involved in intracellular

metabolism, cellular physiological process, morphogenesis, apoptosis,

proliferation and cell differentiation have been found to be differentially expressed

in cranial neural tubes of embryos from diabetic pregnancy. Although the present

study does not reveal the molecular mechanisms that contribute to the neural tube

anomalies, it provides a clue for changes in molecular cues that guide the

development of different functional regions of the brain after the period when the

neural tube closure occurs.

1. Apoptotic cells are increased and mitotic index is decreased in cranial

neural tubes of embryos from diabetic mice.

Apoptosis which is a key feature in the early rapid expansion phase of neural tube

development occurs in the neural tube on E10-E11 in order to halt premature

neurogenesis (Akazawa, 2005). Apoptosis has been shown to be induced markedly

in the developing brain in the absence of several signaling molecules or factors

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CHAPTER 4: DISCUSSION

(Mason et al., 2006; Yu et al., 2002). Recently, downregulation of Pax3 expression

by maternal diabetes has been shown to be associated with increased apoptosis in

the mouse neural tube, resulting in neural tube defects (Fine et al., 1999; Phelan et

al., 1997). The present study also shows a significant increase in the number of

apoptotic cells in neuroepithelia of the telencephalon, diencephalon, and

rhombencephalon in E11.5 embryos of diabetic mice. Increased apoptosis was

further corroborated by microarray results which showed an increased expression

of several proapoptotic genes including p53, Bnip3 and Bid in cranial neural tubes

of embryos from diabetic mice (Shaw et al., 1992; Vande et al., 2000; Wang et al.,

1996). The proapoptotic proteins, such as p53, Bnip3 and Bid, induce programmed

cell death by inhibiting anti-apoptotic proteins, such as Bcl2 and Bcl-XL, which

may protect immature neurons from death (Boyd et al., 1994). Recently, it has

been shown that Pax3 inhibition promotes p53-mediated cell death in the neural

tube, leading to NTD (Pani et al., 2002). Taken together, it appears that maternal

diabetes alters the expression of developmental control genes which subsequently

induce the proapoptotic genes, leading to excessive apoptosis in the cranial neural

tube.

On the other hand, maternal diabetes seems to impair the mitotic index in

cranial neural tubes of mouse embryos. It has been shown that exposure to high

glucose inhibits the cell-cycle progression in neural stem cells, microvessel

endothelial cells and mesangial cells (Abraham et al., 2003; Fu et al., 2006; Wolf

et al., 2001). Further, the placental growth has been shown to be delayed due to

109
CHAPTER 4: DISCUSSION

altered proliferation index in diabetic pregnancies (Weiss et al., 2001). In the

present study, the decreased proliferation index in cranial neural tubes of embryos

from diabetic mice was associated with down-regulation of expression of genes

that are involved in cell proliferation such as Igf2 and Tgfb2. These changes

suggest that the glucotoxicity caused by maternal diabetes alters the cell cycle

progression in the cranial neural tube by regulating the expression of genes

involved in apoptosis and cell proliferation which may subsequently result in

malformations of the developing brain of embryos from diabetic mice.

2. Expression of genes involved in neuronal migration and differentiation is

altered in cranial neural tubes of embryos from diabetic mice

The neural tube originates from a single layer of neuroepithelial cells (Bayer and

Altman, 1991; Panchision and McKay, 2002). These progenitor cells proliferate

and then differentiate sequentially into distinct neurons and glia that form

functional networks in the CNS. Throughout the neurodevelopment, a distinct

pattern of gene expression specific for neurogenesis and gliogenesis is evident in

the progenitor cells. Regulation of cell fate specification in the developing brain is

complex and influenced by both cellular intrinsic factors and extracellular

environmental cues. Maternal diabetes appears to alter the expression pattern of

genes that are involved in differentiation and specification of different types of

neurons and glia.

Development of mammalian brain substantially depends on migration and

110
CHAPTER 4: DISCUSSION

axon projection of newborn neurons. Doublecortin (Dcx) encoding

microtubule-associated protein is expressed in the migrating neurons (Francis et

al., 1999; Friocourt et al., 2003; Gleeson et al., 1999). Mutations in the human

DCX gene are associated with abnormal neuronal migration, epilepsy,

lissencephaly and mental retardation (LoTurco and Bai, 2006). This gene together

with doublecortin-like kinase (Dclk), a gene that shares high homology with Dcx,

function in the formation of axonal projections across the midline and in the

neuronal migration (Bai et al., 2003; Koizumi et al., 2006). Dclk has been further

shown to control mitotic division by regulating spindle formation and also to

determine the fate of neural progenitors during neurogenesis (Shu et al., 2006). In

the present study, downregulation of both Dcx and Dclk mRNA expression and

decreased number of Dcx immuno-positive cells indicate the disruption of

neuronal proliferation and migration as well as axonal wiring in cranial neural

tubes of embryos from diabetic mice.

Migration of cells from the ventricular zone in the cranial neural tube occurs

along the radial glial cells during early stage and through the meshwork of

astrocyte processes after the formation of astrocytes postnatally (Thomas et al.,

1996). Radial glial cells which are identifiable with a molecular marker, Blbp, a

member of the fatty acid-binding protein (FABP) family, serve as neuronal

progenitors and neuronal migration guides as well as astrocyte progenitors (Hatten,

2002; Hunter and Hatten, 1995; Malatesta et al., 2003; Noctor et al., 2001;

Parnavelas and Nadarajah, 2001). The differentiation of radial glial cells is

111
CHAPTER 4: DISCUSSION

regulated by Notch1 signaling through the transcriptional activation of Blbp

(Patten et al., 2006). Further, Blbp is known to be a downstream gene of Pax6

transcription factor which promotes neuronal differentiation via transcriptional

regulation of the Ngn2 (Arai et al., 2005). The maternal diabetes-induced

downregulation of Notch 1 receptor and Pax6 as well as its downstream genes

Blbp and Ngn2 in the cranial neural tube indicates the impaired differentiation of

radial glial cell lineages, neuronal migration and neurogenesis in cranial neural

tubes of embryos from diabetic mice. However, further study is required to

determine whether altered expression of these genes contributed to neural tube

malformations or the change was a mere consequence of neural tube

malformations caused by the glucotoxicity of maternal diabetes.

3. Expression of genes that are involved in glucose metabolism and that

respond to hypoxia is altered in cranial neural tubes of embryos from

diabetic mice

Abnormal metabolic environment caused by maternal diabetes appears to

contribute to neural tube malformations in mouse embryos. Several studies have

shown that a deficiency of myoinositol, abnormalities of arachidonic acid and

prostaglandin metabolism, excessive production of ROS, activation of

diacylglycerol-protein kinase C and induced-hypoxic as well as oxidative stress

mediate the toxic effect of maternal diabetes on development of various organs

including the neural tube (Baker et al., 1990; Chang et al., 2003; Goldman et al.,

112
CHAPTER 4: DISCUSSION

1985; Hashimoto et al., 1990; Hiramatsu et al., 2002; Li et al., 2005). The present

study further revealed an altered expression of genes involving hypoxia and

glycolysis in cranial neural tubes of embryos from diabetic pregnancy.

Physiologically optimal oxygenation in all cells is essential to the survival of

embryos (Michiels, 2004). Limitation of oxygen availability to the cells and

tissues contributes to the several pathological conditions. However, the cells are

capable of triggering an adaptive response to hypoxic condition by increasing the

efficiency of energy producing pathways, mainly through increased anaerobic

glycolysis activity, and on the other hand by decreasing energy-consuming

processes (Boutilier, 2001). Recently it has been shown that maternal diabetes

induces excessive physiological hypoxia in embryos and many of the maternal

diabetes-induced changes in various tissues during development are the results of

hypoxia-induced oxidative stress (Li et al., 2005). In the present study, the maternal

diabetes-induced physiological hypoxia appeared to have triggered an adaptive

response in the cranial neural tube by activating the expression of several genes

encoding enzymes involved in aerobic and anaerobic glycolysis pathways (such as

phosphoglycerate kinase 1, pyruvate kinase, phosphofructokinases, aldolase 1A,

enolase 1, 2, 3, lactate dehydrogenase and hexokinases) that help to increase the

energy production by decreasing hypoxic state in the neural tissue of embryos from

diabetic mice.

However, the adaptive response seems to be insufficient to reverse the

hypoxic state of the neural tissue in embryos from diabetic mice as there is a

113
CHAPTER 4: DISCUSSION

continuously increased glucose transport from maternal circulation and increased

oxygen consumption which subsequently leads to depletion of oxygen availability

in the neural tissue. Hence, it is suggested that contribution of hypoxia-induced

oxidative stress to the cranial neural tube malformations in embryos of diabetic

mice can not be excluded.

Further, the transcriptional response to excessive hypoxia is mediated largely

by the action of hypoxia-inducible factor-1 (HIF-1) which is upregulated under

hypoxia and rapidly degraded in the presence of oxygen (Wang et al., 1995).

HIF-1 which has been implicated in a range of brain pathologies can be harmful

or beneficial, depending on the circumstances (Bergeron et al., 1999; Gidday et al.,

1999). In the present study, the upregulation of Hif-1 expression appears to be

harmful to the development of cranial neural tubes in embryos of diabetic

pregnancy as it has been shown to mediate the hypoxia-induced apoptosis by

promoting the transcription of Bnip3, a pro-apoptotic member of the Bcl family

of cell death factors and to contribute to the cell growth arrest by upregulating the

expression of cyclin-dependent kinase inhibitor 1A (P21) (Bruick, 2000; Feldser

et al., 1999; Goda et al., 2003). Taken together, the altered expression of genes

that respond to hypoxia appears to be associated with cranial neural tube

malformations in embryos from diabetic mice. Further, hypoxia is an important

factor contributing to neural tube malformations since hypoxia-induced oxidative

stress has been shown to impair the neuronal migration and maturation (Li et al.,

2005; Zechel et al., 2005).

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CHAPTER 4: DISCUSSION

4. Development of choroid plexus is impaired in the cranial neural tubes of

embryos from diabetic mice

During neurodevelopment, choroid plexus (CP) develops in the 4th, lateral and

3rd ventricles of the cranial neural tube and functions as the dorsal signaling

centre (Dziegielewska et al., 2001; Yamamoto et al., 1996). The CP epithelial

cells produce cerebrospinal fluid (CSF) which provides an essential expanding

mechanism that determines the shape of the brain. CSF also transfers nutrients,

proteins and other molecules required for neuroepithelial cell survival as well as

proliferation and neurogenesis during brain development (Gato et al., 2005; Miyan

et al., 2003; Parada et al., 2005). In the present study, the development of CP and

its epithelial cells was found to be impaired in embryos (E11.5-13.5) of diabetic

pregnancy. In addition, the number of proliferating cells in the adjacent

ventricular zone that contains ependymal cells which are considered to be the

origin of CP epithelial cells (Matsumoto et al., 2003) and in the base of the CP,

was significantly decreased in embryos of diabetic pregnancy, indicating that the

impaired development of CP is associated with reduced proliferation of CP

epithelial progenitor cells caused by maternal diabetes. Further, the malformation

of CP observed in embryos of diabetic mice appears to be partly contributed by

the downregulation of insulin-like growth factor 2 (Igf-2), a polypeptide hormone

which functions as a mitogen during embryogenesis (Eicher et al., 1993; Lee et al.,

1990) and as a morphogen in inducing differentiation of CP epithelial cells during

brain development (Cavallaro et al., 1993). The abnormal development of CP also

115
CHAPTER 4: DISCUSSION

indicates the decrease in CSF production, which may be associated with impaired

proliferation of neuroepithelial cells, neurogenesis and subsequently cranial neural

tube dysmorphogenesis in embryos of diabetic mice.

CP epithelial cells have been shown to synthesize and release several

macromolecules into the CSF. Transthyretin (Ttr) which is involved in the

transport of thyroxine and retinol (a metabolite of vitamin A) from the peripheral

circulation to the brain (Dickson et al., 1987; Ferguson et al., 1975; Raz et al.,

1970), is the major protein synthesized by the CP epithelial cells. Thyroxine and

retinol regulate neuronal differentiation in the developing brain (Kilby, 2003;

Maden, 2002). Moreover, congenital hypothyroidism (causing thyroid hormone

deficiency in the circulation) leads to devastating effects upon brain development

resulting in cognitive deficits in infants (Oerbeck et al., 2007; Santalucia et al.,

2006). Maternal diabetes has also been shown to affect the level of fetal brain

thyroxine, T3 and T4 (Calvo et al., 1997) and subsequently intellectual

development and behavior of the brain in infants (Rizzo et al., 1991). Taken

together, in the present study, a significant reduction of mRNA and protein

expression of Ttr in cranial neural tubes of E11.5-E13.5 embryos from diabetic

pregnancy could reduce the availability of thyroxine and retinol in the neural

tissue, thereby resulting in abnormal brain development which may subsequently

contribute to intellectual impairment of the offspring as reported in human (Rizzo

et al., 1991).

In conclusion, maternal diabetes induces metabolic derangements in the

116
CHAPTER 4: DISCUSSION

cranial neural tube by altering the expression of genes involved in cellular

metabolism such as glycolysis and in physiological hypoxia. The metabolic

changes are associated with altered expression of genes involved in apoptosis,

proliferation, migration and differentiation of neurons. Moreover, malformation of

the CP and the ventricular systems together with reduced production of Ttr, Igf-2

and other components of CSF appear to influence the neuroepithelial survival,

proliferation and neurogenesis. It is possible that these changes may impede

further development of functional domains of the brain (including the

telencephalon derivatives, i.e., cerebral cortex; diencephalon derivatives, i.e.,

hypothalamus; and rhombencephalon derivatives, i.e., pons, medulla oblongata,

etc.) and subsequently contribute to intellectual impairment in the offspring of

diabetic mothers. However, an extensive follow-up study is required to understand

the molecular mechanisms and consequence of cranial neural tube malformations

observed in embryos of diabetic mice.

117
CHAPTER 5: CONCLUSION

CHAPTER 5

CONCLUSION

118
CHAPTER 5: CONCLUSION

A high frequency of birth defects has been widely reported in infants born to

diabetic mothers (Hanson et al., 1990; Mills, 1982). Experimental analysis in

animal models also revealed that there is an increased risk for fetal malformations

and spontaneous abortions in diabetic pregnancy (Chang et al., 2003; Sakamaki et

al., 1999). Although most neonatal problems have declined, diabetes-associated

anomalies remain a major health problem having significant social and financial

implications. Gestational diabetes develops in 2-12% of total pregnancies,

depending on ethnic background. In Asia, it accounts for 4-6% of total

pregnancies. About 15% of the fetuses from diabetic pregnancy display congenital

malformations in various organ systems including the nervous system (Eriksson,

1995; Kucera, 1971). The association between maternal diabetes and the incidence

of congenital malformations including neural tube defects has been well

established. The basis of these malformations is quite variable and may include

chromosomal aberrations, mutation of genes, environmental factors, interactions

between hereditary tendencies and genetic factors, and idiopathic causes (Kalter

and Warkany, 1983).

High blood sugar levels in the fetus at the time of conception and in the first

few weeks of development are associated with higher rates of congenital anomalies

(Temple et al., 2002). Despite apparent declines in pregnancy-related complications

among women with diabetes mellitus, recent studies continue to show increased

rates of perinatal mortality and congenital anomalies in these women compared to

the non-diabetes mellitus population (Casson et al., 1997; Cundy et al., 2000;

119
CHAPTER 5: CONCLUSION

Hawthorne et al., 1997; Towner et al., 1995). It has been shown that congenital

anomalies can be almost completely avoided with careful blood sugar control prior

to conception and during organ development in the first few weeks of pregnancy

(Fuhrmann et al., 1984; Kitzmiller et al., 1991; Steel et al., 1990). Moreover, the

frequency of embryonic malformations in diabetic pregnancy has been reported to

be markedly decreased by dietary supplements of anti-oxidants, such as vitamin C

or E and butylated hydroxytoluene (Chang et al., 2003; Eriksson and Borg, 1991;

Eriksson and Siman, 1996; Siman and Eriksson, 1997), indicating that oxidative

stress is involved in the etiology of embryonic dysmorphogenesis.

In recent years, considerable efforts have been made to investigate the etiology

of birth defects among infants of diabetic mothers. It has been shown that

diabetes-induced embryonic dysmorphogenesis is accompanied by some metabolic

abnormalities including elevated superoxide dismutase activity, reduced levels of

myoinositol and arachidonic acid, and inhibition of the pentose phosphate shunt

pathway (Cederberg et al., 2000; Eriksson, 1991; Eriksson and Borg, 1993;

Goldman et al., 1985; Hashimoto et al., 1990). Further, several experimental studies

have shown that neural tube anomalies in embryos of diabetic mice are associated

with altered expression of genes involved in the neural tube development (Chang et

al., 2003; Li et al., 2005; Liao et al., 2004; Loeken, 2005; Loeken, 2006). More

recently, it has been shown that high-glucose concentrations alter the expression of

genes that are involved in proliferation and differentiation of neural stem cells

(NSCs), indicating the basis for the CNS malformations in infants of diabetic

120
CHAPTER 5: CONCLUSION

mothers (Fu et al., 2006). The present study is the first of its kind demonstrating the

differential gene expression profiling of cranial neural tubes in embryos of diabetic

mice in comparison to that of embryos from control mice.

The data presented in this thesis clearly demonstrate that maternal diabetes

induces metabolic derangements in the cranial neural tube by altering the

expression of genes involved in cellular metabolism such as glycolysis and in

physiological hypoxia. The metabolic changes are associated with altered

expression of genes involved in apoptosis, proliferation, migration and

differentiation of neuronal cell types. Moreover, malformation of the CP and the

ventricular systems together with reduced production of Ttr, Igf-2 and other

components of the CSF appear to influence the neuroepithelial survival,

proliferation and neurogenesis. It is possible that these changes may impede

further development of functional domains of the brain and subsequently

contribute to intellectual impairment in the offspring of diabetic mothers.

However, an extensive follow-up study is required to understand the molecular

mechanisms and consequence of cranial neural tube malformations observed in

embryos of diabetic mice.

The predisposing effect of intrauterine exposure to a diabetic environment

has major public health implications in the present context of the growing diabetes

epidemic. Since the mechanisms of fetal malformations in diabetic pregnancies

appear to be complex, it is suggested that intensive glycemic control in the

preconception period and throughout pregnancy may contribute to a decrease in

121
CHAPTER 5: CONCLUSION

the prevalence of diabetes-induced malformations in the fetus. Based on present

findings together with previous reports, it is also suggested that supplementation of

molecules that are found to be deficient in embryos under hyperglycemic conditions

and antioxidants to alleviate the adverse effects of oxidative stress would prevent

embryonic malformations.

Although the present study does not reveal the molecular mechanisms that

contribute to the neural tube anomalies, it provides a clue for changes in molecular

cues that guide the development of different functional regions of the brain. This

direction of study is of clinical importance as it could open up new vistas of

research to develop safer modes of therapeutic strategies and multi-nutrient dietary

supplements to eliminate embryonic abnormalities induced by maternal diabetes.

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148
FIGURES AND FIGURE LEGENDS

FIGURES

AND

FIGURE LEGENDS

149
FIGURES AND FIGURE LEGENDS

Fig.1. Embryos (E11.5) obtained from control (A) and diabetic (B) mice. The

embryo from a diabetic mouse exhibits exencephaly phenotype extending from

the telencephalon to rhombencephalon (arrows). The line in Fig A and B shows

the approximate region of cranial neural tube (containing forebrain, midbrain and

hindbrain) that was used for RNA extraction in this study. tc, telencephalon; dc,

diencephalon; rc, rhombencephalon; e, eye; ov, otic vesicle. Scale bar: 1mm

150
FIGURES AND FIGURE LEGENDS

Figure 1

Control Diabetic

dc dc
rc rc
tc tc
e e
ov ov

A
A B
B

151
FIGURES AND FIGURE LEGENDS

Fig.2. Transverse sections (stained with Hematoxylin & Eosin) showing

morphological changes in the telencephalon, diencephalon and rhombencephalon

of embryos (E11.5) obtained from control (A, C) and diabetic (B, D) mice. The

neuroepithelia of forebrain including telencephalon and diencephalon and the

rhombencephalon in embryos of diabetic mice appear to be distorted and fused.

The size and volume of the 3rd and 4th ventricles (III and IV) are markedly reduced

in cranial neural tubes of embryos from diabetic mice in comparison to that of

embryos from normal mice. LV, lateral ventricle; III, third ventricle; IV, fourth

ventricle; tel, telencephalon; dc, diencephalon; rb, rhombencephalon. Scale bar:

200 m.

152
FIGURES AND FIGURE LEGENDS

Figure 2

Control Diabetic

tc tc

LV

dc dc
III III
A B
rc

rc
IV
IV

C D

153
FIGURES AND FIGURE LEGENDS

Fig.3. Cluster analysis showing changes in gene expression profiles of the cranial

neural tube in embryos from diabetic mice. The data analysis revealed the

differential expression of 1613 genes between embryos of control and diabetic

mice. Agglomerative average-linkage hierarchical clustering of the six

independent samples was obtained for selected groups of genes using GeneSpring

7.3 software. Each colored box represents the normalized expression level of a

given gene in each sample and is colored according to the fold change.

154
FIGURES AND FIGURE LEGENDS

Figure 3

155
FIGURES AND FIGURE LEGENDS

Fig.4. Functional categorization of genes that are differentially expressed in

cranial neural tubes of embryos (E11.5) from diabetic mice in comparison to that

of control mice. About 390 genes displaying greater than 2-fold changes in

expression level are grouped into 9 categories as follow: (1) metabolism (27.7%);

(2) cellular physiological process (20.3%); (3) cell communication (12.1%); (4)

morphogenesis (6.9%); (5) response to stimulus (3.8%); (6) cell death (2.3%); (7)

cell differentiation (2.1%); (8) small subsets of genes (5.4%) that are implicated in

regulation of cellular process, regulation of gene expression, epigenetic,

extracellular structure organization, biogenesis and cell adhesion; and (9)

unclassified genes with no functional annotation.

156
FIGURES AND FIGURE LEGENDS

Figure 4

Metabolism
19.4 %
27.7% Cellular physiological process
Cell communication

5.4 % Morphogenesis
Response to stimulus
2.1 %
Death
2.3 %
3.8 %
Cell differentiation
Other functions
6.9 %
20.3 % Unclassified

12.1 %

157
FIGURES AND FIGURE LEGENDS

Fig.5. Microarray analysis shows that expression of genes that encode key enzymes

involved in glycolysis pathway is upregulated in cranial neural tubes of embryos

from diabetic mice. In the flow chart of glycolysis pathway, substrates and products

are in rectangle, enzymes are in circle, genes are in italic format and fold changes of

gene expression are in parenthesis. Abbreviations used: 1,3-BPG,

1,3-bisphosphoglycerate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate;

ALD, aldolase; ENO, enolase; F-1,6-BP, fructose 1,6-bisphosphate; F-6-P, fructose

6-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G-6-P, glucose

6-phosphate; GPI, glucose phosphate isomerase; GADP, glyceraldehyde

3-phosphate; DHAP, dihydroxyacetone phosphate; HK, hexokinase; LAC, lactate;

LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PFK,

phosphofructokinase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate

mutase; PK, pyruvate kinase; PYR, pyruvate; TPI, triosephosphate isomerase; aldoa,

aldolase A; pkm2, pyruvate kinase, muscle; pfkl, phosphofructokinase, liver; pfkp,

phosphofructokinase, platelet

158
FIGURES AND FIGURE LEGENDS

Figure 5

159
FIGURES AND FIGURE LEGENDS

Fig.6. The real time RT-PCR analysis of mRNA expression of some genes which

are involved in glycolysis in cranial neural tubes of embryos (E11.5) from control

and diabetic mice. The mRNA expression levels (E) hk1, hk2, pfkl, pfkp pkm2, and

ldh are increased in cranial neural tubes of embryos from diabetic mice compared

with that of embryos from control mice. Panels A and C show the size of PCR

products loaded on agarose gels. Panels B and D show the melting curves which

depict the specificity of the PCR products amplified. The data are presented as

mean SE (n=4), *p<0.05.

160
FIGURES AND FIGURE LEGENDS

Figure 6
A B C D

Hk1 Hk1 Hk2 Hk2


Marker Marker

500bp 500bp

100bp 112bp 100bp 117bp

Pfkl Pfkl Pfkp Pfkp

Marker Marker

500bp
500bp
100bp 106bp
100bp 115bp

Pkm2 Pkm2 Ldh Ldh


Marker
Marker

500bp 500bp

100bp 134bp 100bp 135bp

E
*

* *
* * *

161
FIGURES AND FIGURE LEGENDS

Fig.7. Hif-1 gene (127bp) product (A) is amplified in the cranial neural tube of

E11.5 mouse embryos. Melting curve (B) indicates the specificity of the product.

The quantitative real time RT-PCR analysis (C) shows that expression level of

Hif-1 is significantly increased in the cranial neural tubes of embryos derived

from diabetic mice in comparison to that of embryos from control mice (mean

S.E, n=4, *p<0.05).

162
FIGURES AND FIGURE LEGENDS

Figure 7

Hif-1 mRNA expression

Marker
A

500bp

127bp
100bp

C *

Control Diabetic
163
FIGURES AND FIGURE LEGENDS

Fig.8. Western blot analysis (A) shows the expression of Hif1 protein (120kDa)

and -actin (42kDa) in the cranial neural tubes of E11.5 embryos from control and

diabetic mice. The quantity of Hif1 protein (B) is found to be significantly

increased in cranial neural tubes of embryos from diabetic mice compared to that

of embryos from control mice (mean S.E, n = 3, *p < 0.05)

164
FIGURES AND FIGURE LEGENDS

Figure 8

Hif1 protein expression by western blot

A
Hif1 (120kDa)

-actin (42kDa)

Control Diabetic

B
*
Fold change of protein level

Control Diabetic

165
FIGURES AND FIGURE LEGENDS

Fig.9. Vegf gene (138bp) product (A) is amplified in the cranial neural tube of

E11.5 mouse embryos. Melting curve (B) indicates the specificity of the product.

The quantitative real time RT-PCR (C) shows that expression level of Vegf is

significantly increased in cranial neural tubes of embryos derived from diabetic

mice in comparison to that of embryos from control mice (mean SE, n=4,

**p<0.01).

166
FIGURES AND FIGURE LEGENDS

Figure 9
Vegf mRNA expression

Marker
A

500bp

138bp
100bp

C **

Control Diabetic
167
FIGURES AND FIGURE LEGENDS

Fig.10. Western blot (A) shows the protein expression of Vegf (20kDa) and

-actin (42kDa) in cranial neural tubes of E11.5 embryos from control and

diabetic mice. The Vegf protein expression level (B) is found to be significantly

increased in cranial neural tubes of embryos from diabetic mice compared to that

of embryos from control mice (mean SE, n = 3, *p < 0.05)

168
FIGURES AND FIGURE LEGENDS

Figure 10

Vegf protein expression by western blot

A
Vegf (20kDa)

-actin (42kDa)

Control Diabetic

B *
Fold change of protein level

Control Diabetic

169
FIGURES AND FIGURE LEGENDS

Fig.11. Apoptosis is induced in cranial neural tubes of embryos (E11.5) from

diabetic mice as revealed by TUNEL assay. Apoptotic cells (arrows in B, D, and F)

are markedly increased in the neuroepithelia of telencephalon (B), diencephalons

(D) and rhombencephalon (F). However, apoptotic cells are hardly detectable in

neuroepithela (A, C, E) of embryos from control mice. tc, telencephalon; dc,

diencephalon; rc, rhombencephalon. Scale bar: 50 m

170
FIGURES AND FIGURE LEGENDS

Figure 11

Control Diabetic

tc tc

A B

dc dc

C D

rc rc

E F

171
FIGURES AND FIGURE LEGENDS

Fig.12. Transverse sections showing BrdU-labelled cells (red) in cranial neural

tubes of embryos (E11.5) from control and diabetic mice. Sections are

counterstained with DAPI (blue), a nuclear marker. The BrdU-labelled cells are

evenly distributed (arrows) in the entire forebrain (A) and rhombencephalon (C)

of control embryos. In embryos of diabetic mice, the proliferating cells (arrows)

appear to be decreased in neuroepithelia of the forebrain (B) and

rhombencephalon (D). Quantitative analysis (E) shows that the percentage of

BrdU-labelled cells in the neuroepithelia of telencephalon, diencephalon and

rhombencephalon was significantly decreased in embryos from diabetic mice

compared with that of embryos from control mice. tc, telencephalon; dc,

diencephalon; rc, rhombencephalon; LV, lateral ventricle; III, third ventricle; IV,

fourth ventricle. Scale bar (A-D): 200 m. Data are presented as mean values of

% of BrdU positive cells S.E (n=3). *p<0.05; **p<0.01.

172
FIGURES AND FIGURE LEGENDS

Figure 12

Control Diabetic
tc tc

LV
LV

dc
dc III
BrdU/DAPI

III

A B

rc IV rc IV

C D

Control
% of BrdU labeled cells

Diabetic

** *
*

E
tc dc rc

173
FIGURES AND FIGURE LEGENDS

Fig.13. Pax6 gene (144bp) product is amplified by the real time RT-PCR (A).

Melting curve (B) indicates the specificity of the product. The quantitative real

time RT-PCR analysis (C) shows that expression level of Pax6 is significantly

decreased in the cranial neural tube of E11.5 embryos from diabetic mice in

comparison to that of embryos from control mice. The data are presented as mean

S.E (n=4), *p<0.05.

174
FIGURES AND FIGURE LEGENDS

Figure 13

Pax 6 mRNA expression

Marker
A

500bp

144bp
100bp

C
*

175
FIGURES AND FIGURE LEGENDS

Fig.14. In situ hybridization analysis of pax 6 mRNA expression in cranial neural

tubes of embryos (E11.5) from control and diabetic mice. Expression of pax 6 in

embryos of control mice (A-C) is localized to the telencephalon and diencephalon

(arrow-heads). No expression is detectable in rhombencephalon. In embryos of

diabetic mice, the expression level of pax 6 (D-F) is markedly reduced and the

domain is distorted. Arrows in D show the cranial neural tube closure defects in

embryos of diabetic mice. Red lines depict the approximate planes of transverse

sections. Letters on the line indicate the locations of the sections shown in B, C,

E,and F. tc, telencephalon; dc, diencephalon; rc, rhombencephalon. Scale bar:

1mm (A, D); 200 m (B,C,E,F).

176
FIGURES AND FIGURE LEGENDS

Figure 14

Control Diabetic

rc rc
dc dc
tc C
e tc F
B e
E

A D
telencephalon

B E
diencephalon

C F

177
FIGURES AND FIGURE LEGENDS

Fig.15. Ngn2 gene (124bp) product (A) is amplified by the real time RT-PCR.

Melting curve (B) indicates the specificity of the product. The quantitative real

time RT-PCR (C) shows that expression level of Ngn2 is significantly decreased

in the cranial neural tube of E11.5 embryos derived from diabetic mice in

comparison to that of embryos from control mice. The data are presented as mean

S.E (n=4), *p<0.05.

178
FIGURES AND FIGURE LEGENDS

Figure 15

Ngn2 mRNA expression

Marker
A

500bp

100bp 124bp

179
FIGURES AND FIGURE LEGENDS

Fig.16. In situ hybridization analysis of Ngn2 mRNA expression in cranial neural

tubes of embryos (E11.5) from control and diabetic mice. Expression domain of

Ngn2 is restricted to the telencephalon, diencephalon and rhombencephalon

(arrow-heads) in embryos of control mice (A-D). However, its expression domain

is disrupted and the expression level appears to be decreased in the telencephalon

and diencephalon of embryos from diabetic mice (E-G). However, the expression

level of Ngn2 appears to be unchanged in the rhombencephalon of embryos from

diabetic mice (H) compared with the control (D). Arrows in panel E show the

cranial neural tube closure defect in embryos of diabetic mice. Red lines in panels

A & E indicate the approximate planes of transverse sections. Letters on the lines

depict the locations of sections shown in B-D and F-H. tc, telencephalon; dc,

diencephalon; rc, rhombencephalon. Scale bar: 1mm (A, E); 200 m (B-D, F-H).

180
FIGURES AND FIGURE LEGENDS

Figure 16

Control Diabetic

rc
hb
hb hb rc
dc D dc H

tc dc dcG
tel C eC tc
e tel F F
e
B
B e
E

A D
E
telencephalon

B F
diencephalon

C G
rhombencephal

hin

dbr

ain
D H

181
FIGURES AND FIGURE LEGENDS

Fig.17. Immunofluorescence staining of Dcx (green) and Blbp (red) in transverse

sections through telencephalon, diencephalon and rhombencephalon of embryos

(E11.5) from control (A, C, E) and diabetic (B, D, F) mice. Sections are

counterstained with DAPI (blue). The number of Dcx positive cells (arrows) in the

telencephalon, diencephalon and rhombencephalon of embryos from diabetic mice

(B, D) appears to be decreased, compared to that of controls (A, C). In addition, a

decreased number of Blbp-positive cells (arrow-heads) is clearly seen in the

telencephalon, diencephalon and rhombencephalon of embryos from diabetic mice

(B, D) in comparison with that of embryos from control mice (A, C).

Blbp-positive radial glial cells (arrow-heads) exhibit elongated processes (arrows)

which extend to the pial surface (E). However, processes of these cells (arrows)

appear to be disrupted in the neuroepithelia of embryos from diabetic mice (F).

Panels E & F are magnified regions of boxes shown in panels C & D, respectively.

tc, telencephalon; dc, diencephalon; rc, rhombencephalon. Scale bar: 200m

(A-D); 50m (E, F)

182
FIGURES AND FIGURE LEGENDS

Figure 17

Control Diabetic

tc tc

dc dc

A B
Dcx/Blbp/DAPI

rc rc

C D

E F

183
FIGURES AND FIGURE LEGENDS

Fig.18. Transverse sections (stained with Hematoxylin & Eosin) display

developing choroid plexus (CP) in the 4th ventricle (IV) of cranial neural tubes in

embryos (E13.5) of control (A, C, E) and diabetic (B, D, F) mice. The

development of CP in embryos of diabetic mice appears to be markedly impaired.

In addition, the volume of the 4th ventricle is greatly reduced in the embryo of

diabetic mice. Panels E and F are enlarged regions that are outlined in panels C &

D, respectively. Scale bar: (A-B): 400m; (C-D): 200m: (E-F): 25m

184
FIGURES AND FIGURE LEGENDS

Figure 18

CP
CP IV
CP
IV
IV

A B

CP
CP
IV
C D IV

CP
CP IV

IV

E F

185
FIGURES AND FIGURE LEGENDS

Fig.19. Quantitative analysis reveals that the number of epithelial cells in the

choroid plexus is significantly decreased in embryos of diabetic mice compared

with that of embryos from control mice. The data are presented as mean S.E

(n=3), *p<0.05.

186
FIGURES AND FIGURE LEGENDS

Figure 19
Fold change in number

of epithelial cells

Control Diabetic

187
FIGURES AND FIGURE LEGENDS

Fig.20. Ttr gene product (112bp) is amplified in cranial neural tubes of mouse

embryos by the real time RT-PCR (A). Melting curve indicates the specificity of

the product (B). The quantitative real time RT-PCR (C) shows that expression

level of Ttr is significantly decreased in cranial neural tubes of E11.5 and E13.5

embryos derived from diabetic mice in comparison to that of embryos from

control mice. The data are presented as mean S.E (n=4), *p<0.05.

188
FIGURES AND FIGURE LEGENDS

Figure 20
Ttr mRNA expression

Marker
A

500bp
100bp 112bp

*
*

189
FIGURES AND FIGURE LEGENDS

Fig.21. Immunostaining of Ttr protein is detected in the tela choroidea (arrow in

A) and choroid plexus epithelium (arrows in C) in cranial neural tubes of E11.5

and E13.5 embryos respectively. The staining appears to be markedly reduced in

the tela choroidea (arrow in B) of embryos (E11.5) from diabetic mice and the

choroid plexus epithelium (arrows in D) of embryos (E13.5) from diabetic mice

compared with that of embryos from control mice (A and C). CP, choroid plexus;

IV, fourth ventricle; LV, lateral ventricle; TC, tela choroidea. Scale bar: 50m

190
FIGURES AND FIGURE LEGENDS

Figure 21

Control Diabetic
E11.5 E11.5

LV

TC TC LV
A B
Ttr

E13.5
IV E13.5

CP

IV
CP

C D

191
FIGURES AND FIGURE LEGENDS

Fig.22. Igf-2 gene product (138bp) is amplified in cranial neural tubes of mouse

embryos by the real time RT-PCR (A). Melting curve indicates the specificity of

the product (B). The quantitative real time RT-PCR (C) shows that expression

level of Igf-2 is significantly decreased in cranial neural tubes of E11.5 and E13.5

embryos derived from diabetic mice in comparison to that of embryos from

control mice. The data are presented as mean S.E (n=4), *p<0.05.

192
FIGURES AND FIGURE LEGENDS

Figure 22

Igf-2 mRNA expression

Marker
A

500bp

138bp
100bp

*
*

193
FIGURES AND FIGURE LEGENDS

Fig.23. Immunostaining of Igf-2 protein is detected in the tela choroidea (arrow in

A) and choroid plexus epithelium (arrows in C) in cranial neural tubes of E11.5

and E13.5 embryos, respectively. The immunostaining of Igf-2 is hardly

detectable in the tela choroidea of E11.5 embryos from diabetic mice (B) and

appears to be markedly reduced in the choroid plexus epithelium (arrows in D) of

E13.5 embryos from diabetic mice (D) compared with that of embryos from

control mice (A, C). CP, choroid plexus; IV, fourth ventricle; LV, lateral ventricle;

TC, tela choroidea. Scale bar: (A-D): 50m

194
FIGURES AND FIGURE LEGENDS

Figure 23

Control Diabetic
E11.5 E11.5

LV

LV
TC
TC
Igf-2

A B
E13.5 E13.5

IV CP
CP
IV
C D

195
FIGURES AND FIGURE LEGENDS

Fig.24. Cell proliferation index by BrdU incorporation in the base of the choroid

plexus (CP) and adjacent neuroepithelia around the 4th ventricle (IV) in embryos

(E13.5) from control and diabetic mice (A-E). Immunostaining shows that the

number of BrdU labeled cells (arrows) is markedly decreased in the base of the

CP and adjacent neuroepithelia in embryos from diabetic mice (B, D) compared

with that of embryos from control mice (A, C). Panels C and D are enlarged

portions that are outlined in panels A & B, respectively. Quantitative analysis (E)

reveals that the percentage of BrdU-labeled cells is significantly decreased in the

base of the CP and adjacent neuroepithelia in embryos of diabetic mice compared

with that of embryos from control mice. The data are presented as mean values

of % of BrdU labeled cells S.E (n=3), **p<0.01. Scale bar: (A, B): 50um; (C,

D): 20um.

196
FIGURES AND FIGURE LEGENDS

Figure 24

Control Diabetic

CP

IV
CP

IV

A B
BrdU

CP
CP
IV IV

C D
% of BrdU labeled cells

**

E
Control Diabetic

197
TABLES

TABLES

198
TABLES

Table 5: Microarray results showing altered expression of genes in cranial neural

tubes of embryos from diabetic mice are validated by the real time RT-PCR

Fold Changes
Accession No Gene Name Real time
Microarray
RT-PCR

NM_013697 transthyretin -5.9 -6.8

NM_010514 insulin-like growth factor 2 -1.8 -3.7

NM_010025 doublecortin -1.9 -1.8

NM_007523 BCL2-antagonist/killer 1 1.7 1.4

NM_011249 retinoblastoma-like 1 3.6 1.6

BCL2/adenovirus E1B 19kDa-interacting protein


NM_009760 4.5 1.4
1, NIP3

NM_016669 crystallin, mu 2.1 2.2

NM_026998 sorting nexin 6 3.4 5.0

199
TABLES

Table 6: Changes in expression of genes that are involved in glycolysis and that

respond to hypoxia

Accession Fold
Gene Name Gene Function
No. Change
NM_007438 aldolase 1, A isoform # 2.1 Response to hypoxia, glycolysis
and glucose metabolism

NM_023119 enolase 1, alpha non-neuron # 1.4 Response to hypoxia, glycolysis


and glucose metabolism

NM_013509 enolase 2, gamma neuronal 3.9 Glycolysis and glucose


metabolism

NM_007933 enolase 3, beta muscle 1.6 Glycolysis and glucose


metabolism

NM_008155 glucose phosphate isomerase 1 2.0 Glycolysis and glucose


metabolism

NM_010438 hexokinase 1 # 1.5 Response to hypoxia, glycolysis


and glucose metabolism

NM_013820 hexokinase 2 # 2.8 Response to hypoxia, glycolysis


and glucose metabolism

NM_010699 lactate dehydrogenase 1, A chain # 1.5 Response to hypoxia, glycolysis


and glucose metabolism

NM_008826 phosphofructokinase, liver, B-type # 1.8 Response to hypoxia, glycolysis


and glucose metabolism

NM_019703 phosphofructokinase, platelet 1.6 Response to hypoxia, glycolysis


and glucose metabolism

NM_008828 phosphoglycerate kinase 1 # 1.6 Response to hypoxia, glycolysis


and glucose metabolism

(To be continued)

200
TABLES

NM_023418 phosphoglycerate mutase 1 1.7 Glycolysis and glucose


metabolism

NM_011099 pyruvate kinase, muscle # 1.4 Response to hypoxia, glycolysis


and glucose metabolism

NM_009415 triosephosphate isomerase 1 # 1.6 Response to hypoxia, glycolysis


and glucose metabolism

NM_009760 BCL2/adenovirus E1B 4.5 Response to hypoxia and apoptosis


19kDa-interacting protein 1, NIP3 #

NM_008343 Igf binding protein 3 # 1.5 Response to hypoxia, cell


proliferation and apoptosis

NM_010431 hypoxia inducible factor 1, alpha 1.5 Response to hypoxia, positive


subunit regulation of vascular endothelial
growth factor receptor signaling
pathway
NM_009505 vascular endothelial growth factor A 2.1 Response to hypoxia, cell
# proliferation and angiogenesis

NM_011400 solute carrier family 2 (facilitated 2.9 Response to hypoxia and glucose
glucose transporter), member 1 transport

NM_030696 solute carrier family 16 3.5 Response to hypoxia and


(monocarboxylic acid transporters), monocarboxylic acid transport
member 3
NM_007669 cyclin-dependent kinase inhibitor 1.7 Response to hypoxia and negative
1A (P21) # regulation of cell proliferation

NM_011030 procollagen proline, 2-oxoglutarate 2.9 Response to hypoxia and protein


4-dioxygenase (proline metabolism
4-hydroxylase) alpha 1 polypeptide
#
NM_011498 basic helix-loop-helix domain 4.9 Response to hypoxia and
containing, class B2 regulation of transcription

# Genes that are transcriptionally regulated by HIF-1 (For details, please refer to Semenza, 2001)

201
TABLES

Table 7: Changes in expression of genes that are involved in apoptosis

Accession Fold
Gene Name Gene Function
No. Change
NM_009760 BCL2/adenovirus E1B 19kDa 4.5 apoptosis
interacting protein 1, NIP3

NM_009761 BCL2/adenovirus E1B 19kDa 1.5 apoptosis


interacting protein 3-like

NM_007523 BCL2-antagonist/killer 1 1.7 apoptosis and caspase activation


via cytochrome c

NM_007544 BH3 interacting domain death 1.6 apoptosis


agonist

NM_028133 EGL nine homolog 3 2.8 apoptosis and protein metabolism

NM_010370 granzyme A 6.0 apoptosis and cytolysis

NM_008655 growth arrest and DNA damage 1.9 apoptosis and activation of
inducible 45 beta MAPKK

NM_030152 nucleolar protein 3 (apoptosis -1.5 apoptosis and nuclear mRNA


repressor with CARD domain) splicing, via spliceosome

NM_009383 Tial1 cytotoxic granule-associated 1.8 apoptosis


RNA binding protein-like 1

NM_011640 transformation related protein 53 2.3 apoptosis and DNA damage


response, signal transduction by
p53 class mediator
NM_010177 tumor necrosis factor (ligand) 2.0 apoptosis
superfamily, member 6

202
TABLES

Table 8: Changes in expression of genes that regulate cell proliferation,

differentiation, migration and neurogenesis during brain development

Accession Fold
Gene Name Gene Function
No. Change
NM_009655 activated leukocyte cell adhesion -2.2 cell adhesion
molecule

NM_010875 neural cell adhesion molecule 1 -2.1 cell adhesion

NM_130448 protocadherin 18 -1.6 cell adhesion

NM_008714 Notch gene homolog 1 -1.6 cell differentiation

NM_008716 Notch gene homolog 3 1.6 cell differentiation

NM_008782 paired box gene 5 2.3 cell differentiation

NM_021881 quaking -1.8 cell differentiation

NM_008927 mitogen activated protein kinase 1.5 cell differentiation and map kinase
kinase 1 pathway

NM_009170 sonic hedgehog 1.5 brain morphogenesis

NM_007554 bone morphogenetic protein 4 -1.5 cell fate commitment

NM_021272 brain lipid binding protein -1.8 cell migration

NM_009409 topoisomerase (DNA) II beta -1.6 cell migration and forebrain


development

(To be continued)

203
TABLES

NM_010514 insulin-like growth factor 2 -1.8 cell proliferation

NM_009367 transforming growth factor, beta 2 -2.3 cell proliferation

NM_008634 microtubule-associated protein 1 B -2.4 dendrite morphogenesis

NM_133719 meteorin, glial cell differentiation 1.5 glia cell differentiation


regulator

NM_001033 zinc finger homeobox 1b -1.5 neural crest cell migration


635

NM_019978 double cortin and -1.9 neurogenesis


calcium/calmodulin dependent
protein kinase like 1
NM_010025 doublecortin -1.9 neurogenesis

NM_146100 internexin neuronal intermediate -2.2 neurogenesis


filament protein, alpha

NM_010894 neurogenic differentiation 1 -1.9 neurogenesis

NM_009718 neurogenin 2 -1.5 neurogenesis

NM_013627 paired box gene 6 -2.1 neurogenesis

NM_178804 slit homolog 2 -1.5 neurogenesis

NM_019479 hairy and enhancer of split 6 -1.5 neurogenesis

NM_010512 insulin-like growth factor 1 -1.8 neurogenesis, anti-apoptosis and


glial cell differentiation

(To be continued)

204
TABLES

NM_010897 neurofibromatosis 1 1.6 regulation of glial cell


differentiation

NM_010133 engrailed 1 1.5 regulation of transcription

NM_010134 engrailed 2 1.6 regulation of transcription

NM_010835 homeo box, msh-like 1 -1.6 regulation of transcription

NM_008687 nuclear factor I/B -1.7 regulation of transcription

NM_009697 nuclear receptor subfamily 2, 1.5 regulation of transcription


group F, member 2

NM_013634 peroxisome proliferator activated -2.6 regulation of transcription


receptor binding protein

NM_009189 sine oculis-related homeobox 1 -1.8 regulation of transcription


homolog

NM_172688 mitogen activated protein kinase -1.7 transforming growth factor beta
kinase kinase 7 receptor signaling pathway

NM_009370 transforming growth factor, beta -1.5 transforming growth factor beta
receptor I receptor signaling pathway

205