Boran Jiang

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.
i
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.
ii
ACKNOWLEDGEMENTS
This thesis is dedicated to

my beloved family
iii
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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TABLE OF CONTENTS
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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TABLE OF CONTENTS
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
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
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
xii
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%).
xiii
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
xiv
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.
xvi
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)
xvii
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
1
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
2
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
3
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%
4
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
5
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
6
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
7
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.
8
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).
9
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
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
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
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
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
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
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
(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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
59
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.
60
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.
61
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)
62
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'
63
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
64
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
65
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
66
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
67
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
68
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
69
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.
70
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 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
71
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
72
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
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 PBS containing 0.1% Tween-20 (PBST):
4% paraformaldehyde (PF)
3, 3 diaminobenzidine tetrahydrochloride (DAB) solution
74
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
75
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
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
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
78
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
79
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.
80
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
81
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
82
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.
83
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)
84
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
85
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
86
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
87
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
88
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
89
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
90
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
92
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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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CHAPTER 3: RESULTS
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).
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CHAPTER 4: DISCUSSION
CHAPTER 4
DISCUSSION
106
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
107
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
108
(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
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
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
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
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
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).
114
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
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
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
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
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
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
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.
122
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148
FIGURES AND FIGURE LEGENDS
FIGURES
AND
FIGURE LEGENDS
149
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
Figure 1
Control Diabetic
dc dc
rc rc
tc tc
e e
ov ov
A
A B
B
151
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
Figure 2
Control Diabetic
tc tc
LV
dc dc
III III
A B
rc
rc
IV
IV
C D
153
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
Figure 3
155
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
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
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
Figure 5
159
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
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
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
Figure 7
Hif-1 mRNA expression
Marker
A
500bp
127bp
100bp
C *
Control Diabetic
163
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
Figure 8
Hif1 protein expression by western blot
A
Hif1 (120kDa)
-actin (42kDa)
Control Diabetic
B
*
Fold change of protein level
Control Diabetic
165
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
Figure 9
Vegf mRNA expression
Marker
A
500bp
138bp
100bp
C **
Control Diabetic
167
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
Figure 10
Vegf protein expression by western blot
A
Vegf (20kDa)
-actin (42kDa)
Control Diabetic
B *
Fold change of protein level
Control Diabetic
169
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
Figure 11
Control Diabetic
tc tc
A B
dc dc
C D
rc rc
E F
171
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
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
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
Figure 13
Pax 6 mRNA expression
Marker
A
500bp
144bp
100bp
C
*
175
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
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
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
Figure 15
Ngn2 mRNA expression
Marker
A
500bp
100bp 124bp
179
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
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
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
Figure 17
Control Diabetic
tc tc
dc dc
A B
Dcx/Blbp/DAPI
rc rc
C D
E F
183
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
Figure 18
CP
CP IV
CP
IV
IV
A B
CP
CP
IV
C D IV
CP
CP IV
IV
E F
185
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
Figure 19
Fold change in number
of epithelial cells
Control Diabetic
187
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
Figure 20
Ttr mRNA expression
Marker
A
500bp
100bp 112bp
*
*
189
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
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
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
Figure 22
Igf-2 mRNA expression
Marker
A
500bp
138bp
100bp
*
*
193
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
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
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
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

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

NM_013820 hexokinase 2 # 2.8 Response to hypoxia, glycolysis

NM_010699 lactate dehydrogenase 1, A chain # 1.5 Response to hypoxia, glycolysis

NM_008826 phosphofructokinase, liver, B-type # 1.8 Response to hypoxia, glycolysis

NM_019703 phosphofructokinase, platelet 1.6 Response to hypoxia, glycolysis

NM_008828 phosphoglycerate kinase 1 # 1.6 Response to hypoxia, glycolysis

(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

NM_009415 triosephosphate isomerase 1 # 1.6 Response to hypoxia, glycolysis

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
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
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

Boran Jiang

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Boran Jiang

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GLOBAL GENE EXPRESSION ANALYSIS OF

CRANIAL NEURAL TUBES IN EMBRYOS OF

NATIONAL UNIVERSITY OF SINGAPORE

A THESIS SUBMITTED FOR THE DEGREE OF

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

well as for his valuable suggestions to my career.

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

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

their secretarial assistance.

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.

endless support for my study.

encouragement and assistance during my study.

This thesis is dedicated to

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

2.1.1. 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

2.2.1.4. Neurogenin 2 (Ngn2) 15

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

4.1. 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

5.1. Specific aims. 35

Chapter 2: Materials and Methods. 37

2. Induction of diabetes mellitus 40