Basic the principle of

Developmental
Biology
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University 1
Email: bhthuy@hcmiu.edu.vn
 The questions of Developmental Biology ?

Developmental Biology deals with a multitude of

questions arising from the fact that a single cell,
a fertilized egg cell, can give rise to a complex
multicellular organism.

1. The question of differentiation

2. The question of morphogenesis
3. The question of growth
4. The question of reproduction
5. The question of evolution

2
This subject will introduce you to two levels
of studying developing embryos:
1. Descriptive Embryology and comparative anatomy
of the embryo; describing major events during
embryonic development.
(descriptions of embryos and how they develop)
2. Molecular and cellular aspects, developmental
genetics:
(examining how genes influence cell behavior
and bring about changes in the embryo and how
this is all co-ordinated in building an organism)

3
 The questions of Developmental Biology ?
Fertilized egg Placenta
Trophectoderm
(TE) cells

1 Cell
Inner cell
(Zygot Blastocyst Mass
e)
(ICM)
Fetus

6,000,000,000 cells
(230 different
cell types)

4
Ectodermal derivatives

5
Mesodermal derivatives

6
Endodermal derivatives

7
The germline produces gamates

8
The similarities and differences among different
vertebrate embryos

They each begin with a basically similar

structure, although they acquire this structure at different
ages and sizes. As they develop, they become less like each
other. 9
Morphological Changes During Human
Maturation

10
The Cell Cycle:
Meiosis & Mitosis
Bui Hong Thuy, Ph.D.
Associated Professor
School of
Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
 The Cell Cycle:
Meiosis &
Mitosis

12
 Metaphase Cell cycle (Mitosis)
(M phase)

• Chromosome Daughter
condensation cells
• Nuclear membrane
breakdown
• Chromosome • Chromosome
segregation decondensation
• Reformation of
nuclear membrane

Centrosome
Chromosome
s 13
 Cell cycle
M PHASE
4. ANAPHASE
3. METAPHASE
5. TELOPHASE
2. PROMETAPHASE
6. CYTOKINESIS
1. PROPHASE CELL
CYCL
E

INTERPHASE 14
The cell cycle : Mitosis
G2 of INTERPHASE PROPHASE PROMETAPHASE

15
The cell cycle : Mitosis (cont.)
TELOPHASE
METAPHASE ANAPHASE AND
CYTOKINESIS

16
The cell cycle : Meiosis

17
The cell cycle : Meiosis (cont.)

During another round of cell division, the sister

chromatids finally separate; four haploid daughter
cells result, containing single chromosomes.

18
The centrosome cycle
Centrosome Mitotic Centrosome
maturation commitm separation
ent
Chromosom NEBD
e Bipolar spindle
condensation formation
Centriole
engageme
nt
M

S
DNA
replication

Centrosome
duplication
Centriole 19
disengagem
Components of the Cell-Cycle
control system
Bui Hong Thuy,
Ph.D.
Associated Professor
School of
Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
20
 What drives entry into M phase
MATURATION ACTIVATION
Oocyte Egg Embryo

1st polar body 1st pb

2nd pb
Hormonal
trigger Meiosis I Meiosis II Cleavage

Fertilizatio
n
Oocyte Metaphase Oocyte Fertilized egg
Arrested of meiosis arrested in in interphase
in I M phase of
G2 phase meiosis II
of meiosis (M II)
I

What controls cell cycle transitions ?

21
 Maturation promoting factor (MPF):
An activity that drives oocytes into M phase
Inject cytoplasm from Spindle detected
A. M phase oocyte At cell surface

Mature oocyte Interphase oocyte Oocyte is

(Metaphase II) (GV oocyte) driven
into M phase
B. Nucleus Nucleus Nucleus

Interphase oocyte Interphase oocyte Oocyte remain

(GV oocyte) (GV oocyte) in GV 22
 What is MPF: discovery of cyclins

mitosis mitosis interphase

Relative concentration
MPF

Cyclin A

Cyclin B
Ribonuc
leotide
reductas
e
23
 MPF is a heterodimer of cyclins and Cdks

Synthesized
Cyclin Degraded
Cyclins

Cyclin-dependent
Kinase (Cdk1/Cdc2)

Maturation Promoting Factor

(MPF)
• The most important of these Cdks regulators are protein known as Cyclin.
• Cyclin undergo a cycle of synthesis and degradation in cell cycle.
• However, Cdk levels are constant in the cell cycles. 24
 The core of the cell-cycle control system
Trigger mitosis machinery

M-Cdk (Cyclin act in Metaphase phase)

M-cyclin Cyclin
degradation

Cdk

Cyclin
S-cyclin
degradation

S-Cdk (Cyclin
act in Synthesis phase)
Trigger DNA replication machinery 25
Activating MPF: Cyclin & Phosphorylation

Cdk-Activating
kinase
Inhibitory Phosphatase
M-cyclin phosphatase
CAK
P
P P
Activating
Wee1 phosphatase
Cdk1/Cdc2 Inactive MPF Active MPF
Cdk-
Inhibitory
kinase

Cdc2 has 2 phosphorylation sites: Inhibitory Tyrosine 14/15

Activating Threonine 160

26
Summary
The central components of the cell-cycle control
system are cyclin-dependent protein kinases (Cdks),
whose activity depends on association with
regulatory subunits called cyclins.

Oscillations in the activities of various cyclin-Cdk

complexes leads to the initiation of various cell-cycle
events.

The activities of cyclin-Cdk complexes are influenced

by several mechanisms, including phosphorylation of
the Cdk subunit, the binding of special inhibitory
protein, and proteolysis of cyclins.
27
Control of Meiosis in
Oocyte
Bui Hong Thuy, Ph.D.
Associated Professor
School of Biotechnology,
International University
Email: bhthuy@hcmiu.edu.vn
 MPF activity increases at every M phase

Interphase arrest Maturation Fertilization Embryo development

MPF activity

G arrest Meiosis I Meiosis II First Second

2 Metaphase arrest embryonic embryonic
mitosis mitosis

29
MPF and MAP kinase
Mitogen Activated
Maturation Promoting Factor Mitogen Activated
Protein Kinase
MPF (Cdc2 kinase) Protein Kinase
MAPK (MAP kinase)
P
P MAPK (MAP kinase)
p34 cdc2 P
MAPK
cyclin B

Cdc2 kinase
M II MAP
GV MI
kinase

Chromosome condensation
Germinal vesicle breakdown
Spindle formation
30
 DNA
Structure of the double
helix (2-nm
Nucleosome diameter)
Histone
“Beads on
a string

Nucleosome
(10-nm diameter)

Tight helical fiber

(30-nm diameter) Supercoil
(200-nm
diameter
)

700-nm
Metaphase chromosome 31
 Epigenetic modifications
In biology, the term epigenetics refers to changes in
phenotype (appearance) or gene expression caused by
mechanisms other than changes in the underlying DNA
sequence, hence the name epi- (Greek: over; above) -genetics.

NH3+

-K9 Ac Me

Nucleosome P S10-
DNA -K14 Ac

-K18 Ac

H2B H2A
-K27 Me
P S28-
H4H3
DNA H3
Octameric
histone core Histone H3 32
 Activities of Cdc2 kinase (MPF) and histone H3
kinase during oocyte maturation

GV I GV II - IV Diakinesis Metaphase I Ana-Telo I Metaphase II

Cdc2 kinase

Histone
H3 kinase
0 3 6 9 12 15 18 21 24 27 h

Biology of Reproduction. 2004

Involvement of histone H3 (Ser10) phosphorylation in chromosome
condensation without Cdc2 kinase and mitogen-activated protein
kinase activation in pig oocytes.
33
Bui
Expression of Histone H3 phosphorylation at Serine
10 (P-H3-S10) and Serine 28 (P-H3-S28) during oocyte
maturation
0h 12 h 18 h 27 h 31 h 33 h 42 h
A B C D E F G

A’ B’ C’ D’ E’ F’ G’

GVA’ I GVB’’ II - IV D’’Meta I E’’Ana-I F’’Telo I G’’Meta II

C’’Diakinesis

P-H3-S10

P-H3-S28 34
GVI GVII GVIII D MI AI TI M
Effects of protein phosphatase 1/2A (PP1/PP2A)
inhibitors on the chromosome condensation
It is suggested in somatic cells that the balance of histone
H3 kinase and PP1 acting on Ser10 of histone H3
regulates chromosome condensation.
The inhibitor of the protein serine/ threonine phosphatases
PP1 and PP2A, such as okadaic acid and calyculin A, induce
rapid chromosome condensation in oocytes.
Okadaic acid
Calyculin A
S10 S10 P

PP1/PP2A

H3 H3
? Chromosome
Cdc condensation
2 kinase ?
MAP kinase?
Histone H3 kinase? 35
Materials and methods

• 2.5 M OA
• 50 nM CL-A
• 50 nM CL-A + 0.1 mM U0126

Immunostaining

0.5, 1, 2, 3, 4 and 6 h Kinase assay

Culture medium:
TCM 199 + 10% FCS + 0.1 mg/ml
OCs Na pyruvate + 0.08 mg/ml kanamycin
+ 0.1 IU/ml hMG
PP1/PP2A inhibitors : Okadaic acid (OA) and Calyculin A (CL-A)
MAP kinase inhibitor : U0126

Hong-Thuy Bui et al. Biology of Reproduction, 2004 36

Effects of calyculin A on histone H3 phosphorylation,
nuclear membrane and chromosome morphology
0.5 h 1h 2h 3h 4h 6h

GV GV GV’ ED LD MI
Nuclear membrane: Anti-lamin A/C antibody-Alexa 350 Chromosomes:
Propidium iodide
Phosphorylated histone H3 : Anti-phospho-histone H3 (Ser10) -Alexa 488
Hong-Thuy Bui et al. Biology of Reproduction, 2004 37
Effect of calyculin A on histone H3
phosphorylation
and germinal vesicle breakdown

%
100

80

60

40

20
0 0.5 1 2 3 4 6 0.5 1 2 3 4 6 h
Calyculin A (-) Calyculin A (+)
Germinal vesicle breakdown Histone H3 phosphorylation

Hong-Thuy Bui et al. Biology of Reproduction, 2004 38

Effect of calyculin A on the activation of Cdc2
kinase, MAP kinase and histone H3 kinase

Cdc2

MAP
Cdc2

H3
0 0.5 1 2 3 4 6 M I 0 0.5 1 2 3 4 6 0.5 1 2 3 4 6 M I h
CL-A (-) CL-A (+) CL-A + U0126
Maturation promoting factor (Cdc2 kinase) : Cdc2
MAP kinase : MAP;
Histone H3 kinase: H3
39
Hong-Thuy Bui et al. Biology of Reproduction,
 Conclusion

PP1/PP2A inhibitors induce chromosome

condensation associated with histone H3
phosphorylation in pig oocytes.

Chromosome condensation is
correlated with histone H3 kinase activity,
but not with Cdc2 kinase and MAP kinase
activities.

40
 Chromatin remodeling
Histone H3 phosphorylation: NH3+
Regulated chromosome condensation during -K9 Ac Me
pig oocytes maturation P S10-
(Bui H-T et al., Biology of Reproduction 2004) -K14 Ac

Histone H3 acetylation: -K18 Ac

- Chromatin remodeling
-K27 Me
- Transcriptional P S28-
activity
Histone H3 methylation: H3
- Transcriptional repression
- Epigenetic memory Histone
H3
Reproduction. 2007
Regulation of chromatin and chromosome morphology by
histone H3 modifications in pig oocytes
Bui HT, Van Thuan N, Kishigami S, Wakayama S, Hikichi T, Ohta H, Mizutani E, Yamaoka E,
Wakayama T, Miyano S. 41
 Oocyte development

Follicular antrum
Follicular
Zonna pellucida
Priomary oocyte cells
Theca
externa

Theca
interna

Granulose
cells
A. Primordial follicle Cumulus
B. Maturing follicle cells
C. Graafian follicle
(Full growth follicle)

Arrest at the prophase of meiosis I

42
 Growing phase

Filamentous Stringy Germinal

chromatin chromatin vesicle (GV
(FC) (SC) I)
Maturation

GV I GV II - IV Diakinesis Metaphase I Ana-Telo I Metaphase II

Fertilization (activation)

Metaphase II Ana-Telo II Pronucleus

formation 43
Regulation of Cdc2 kinase and Histone H3 kinase
during oocyte maturation
100

80
Histone H3 kinase
60

40
Cdc2 kinase
20

0
FC SC GVI GVII-IV D MI AI –TI MII AII-TII PN

Growth Maturation Activation

Cdc2 kinase

Histone
H3 kinase 44
GV D M I AI-TI M II G
Expression of Histone H3 acetylation at Lysine 14
(Ac-H3-K14) during oocyte maturation
DNA
Merge Ac-H3-K14 GVI GVIII D MI AI TI MII

Lamin A/C  tubulin

Anaphase I Telophase I 45
Expression of Histone H3 methylation at Lysine 9
(Ac-H3-K14) during oocyte maturation
DNA
Merge Me-H3-K9 GVI GVII-IV D MI AI TI MII

Lamin A/C  tubulin

GVI MI MII 46
Expression of histone H3 phosphorylation at Serine 10
(P-H3-S10) after oocyte activation
MII AII TII PN
2nd pb

1st pb
DNA

1st pb 1 st pb 1st pb
P-H3-S10
Merge

0h 2h 4h 6h 47
Expression of histone H3 phosphorylation at Serine 28
(P-H3-S28) after oocyte activation
MII AII TII PN
1st pb 2nd pb
1st pb
DNA

1st pb 1st pb
P-H3-S28
Merge

48
 Regulation of Chromosome Morphology by
Histone Modifications in Pig Oocytes

NH3 NH3 Me NH3 Me NH3 Me NH3 NH3 NH3 Me NH3 NH3 Me NH3 Me
9 Ac 9 Ac 9 Ac 9 Ac 9 Me 9 Me 9Ac 9 Me 9 Ac 9 Ac
1 10 10 10 P 1 P 1 P 10 P 1 P 10 10
0 14 Ac 14 Ac 14 Ac 14 Ac 0 14 Ac 0 14 14 Ac 0 14 14 Ac 14 Ac

18 Ac 18 Ac 18 Ac 18 Ac 18 Ac 18 18 18 18 18 Ac
28 28 28 P 28 P 28 P 28 P 28 P 28 P 28 P 28

2 nd FC GVI GVII-IV D MI AI –TI MII AII-TII FPN

Follicl
e Growth Maturation Activation

FC SC GVI GVII-IV D MI AI –TI MII AII-TII FPN

49
Summary

Chromosome condensation/decondensation in pig

oocytes is regulated by the acetylation/deacetylation
and phosphorylation/dephosphorylation of histone
H3.

Histone H3-K9 methylation is established in parallel

with oocyte growth. Although histone acetylation
and phosphorylation were reversible, the stable
methylation of H3-K9 was maintained throughout
the course of oocyte maturation and activation.

50
 The basic events during
oocyte growth,
maturation
and fertilization
Bui Hong Thuy, Ph.D.
Associated Professor
School of Biotechnology, International
University
Email: bhthuy@hcmiu.edu.vn
 Oocyte growth and maturation in the ovary
Fully grown
oocyte (GV)
GTH (FSH, LH)
Growing Oocyte
oocytes
Maturation
Antral follicle

Secondary follicles

Primary follicles

Primordial Mature oocyte

follicles
(M II)
Oocyte Growth Non-
growing
52
oocytes
Fertilization: beginning of new organism
-Mouse

53
Development of a human embryo from fertilization to
implantation
 The basic embryo development
* The question of differentiation: A single cell (a
zygote), give rise to hundreds of different cells types: muscle
cells, neurons, blood cells fat cells….and so on. This
generation of cellular diversity is called differentiation.

Zygote Blastocyst Neuron Blood cells Liver cell

The first differentiation

differentiation

Preimplantation Postimplantation
development development 55
 Reproduction. 2007
Regulation of chromatin and chromosome morphology by
histone H3 modifications in pig oocytes
Bui HT, Van Thuan N, Kishigami S, Wakayama S, Hikichi T, Ohta H, Mizutani E, Yamaoka E,
Wakayama T, Miyano S.

SC GV I Growing phase
FC

DNA
2nd follicle Filamentous
chromatin (FC)

Ac-H3-K14
Stringy
chromatin (SC)
Merge

Germinal
vesicle (GV 56
 Oocyte Maturation

GV I GV II - IV Diakinesis Metaphase I Ana-Telo I Metaphase II

A B C D E F G H

Fully grown
oocyte (GV)

Mature oocyte
(M II)

Hong-Thuy 57

Bui et al. Reproduction 2007

 Fertilization (activation)

Metaphase II Ana-Telo II Pronucleus

formation
MII AII TII FPN
A B C D 2nd pb
1st pb
DNA

1st pb 1st pb 1st pb

A’ B’ C’ D’
P-H3-S10

A’’ B’’ C’’ D’’

Merge

Hong-Thuy Bui et al. Reproduction 2007 58

 The early development of embryos
* Preimplantation development

Pronuclei
Zygote (1-cell embryo) 2-cell embryos 4-cell embryos

8-cell embryos Morula (compaction) Blastocyst

The smaller cells in embryo called blastomeres (embryonic

cells)
The early development of embryos
* Preimplantation development

Early blastocyst Expanded blastocyst

Hatching blastocyst Hatched blastocyst

60
The first cell division in zygote (the first mitotic division)
a n b n

n
n

Sperm head n

c d
Blue:
n
Male chromosomes
Green:
n Female chromosomes
Red:
The first mitotic metaphase Microtubules
61
The first cell division in zygote (the first mitotic division)
e 2nd pb f 2 n d p b 1 st p b

The first mitotic metaphase The first mitotic anaphase

g 2nd pb h 2nd pb

Blue:
Male chromosomes
Green:
Female chromosomes
The first mitotic Red:
The first mitotic telophase late telophase Microtubules 62
Differences between male and female
pronuclei in zygote

Nucleolus

Nucleoplasm

Cytoplasm

63
Distribution of DNA replication in one-cell ICSI embryos
5h 6h 7h 8h 9h
♀ ♀ ♀ ♀ ♀

♂ ♂ ♂ ♂ ♂

Expression of Histone acetylation and Transcription

in male and female pronuclei
DNA Ac-H4-K12 Transcription

64
 Expression of Histone methylation (Me-H3-K9) in
male and female chromosome
DNA Me-H3-K9 Merge
1-cell

♂ ♀
2-cell

♂ ♀

♂
♀
65
 Differences between male and female
pronuclei
• Male pronucleus (PN) has a
larger volume & higher levels of chromosome
decondensation than female PN.
• Male PN promote DNA replication faster than
female PN.
• Male PN has a higher levels of histone acetylation
than female PN.
• Male PN has a higher level of transcription and
concentrations of transcription factors than female PN.
• Female PN has a higher levels of DNA and
histone
methylation than male PN.