The early development of vertebrates: birds and

mammals

杨雪松

医学院 组胚系 & 生科院 再生医学教育部重点实验室

医学院 641 ， Tel ： 85228316 ， E-mail: yang_xuesong@126.com

The e-mail for my lecture: ppt or any notices

Development_2012@126.com

Password: development

杨雪松 Scientific career

哈尔滨医科大学医疗系

哈尔滨医科大学生理学硕士

博士 日本东京医科齿科大学博士

英国

Manchester 大

学 英国

Dundee 大

学

暨南大学医学院

1984 1991 1997 1999 2007 now

生殖内分泌 膜生理 ,神经科学

胰腺外分泌

原肠胚形成期三胚层形成中细胞迁移以及血管发生中的基因调控

细胞生理学

细胞生物学

Human Embryo development movie

• Definition: Field of biology that studies how a single

cell (the fertilized egg) gives rise to a fully formed

organism.

• Enjoyment: why do people get excited about the

topic? Complexity, beauty, imaging, relevance (human

congenital defects, cancer)

Developmental biology

Description: Draw out embryonic development from 1-cell stage, cleavage, blastula, gastrula (germ layer formation), body plan development, neurulation, organogenesis

So most of what you have studied so far has been intracellular biology, focusing on molecules and what happens inside individual cells; one important aspect of biology that has not yet been covered is how cells communicate with each other,

how they form multicellular organisms, how cells form different tissues, how these different tissues form organs that in turn interact with each other.

Developmental biology

- Axis determination: AP, DV, LR. how do you ‘break’ the symmetry of the egg? Sperm entry, localized determinants

- Cell differentiation - Cell proliferation - Cell migration: mainly in gastrulation - Cell polarization (symmetric vs asymmetric cell division), - Cell shape (giving cells different morphologies, e.g. neurons, EMT), - Cell death- Morphogenesis (how cells come together to form tissues- Organogenesis- Germ cell development: fertilization- Stem cells: multipotency, ability to self-renew, so ‘trendy’ - Regeneration

Fundamentals questions in developmental biology

• Gene knock-out or knock down: mutation, RNAi, antisense oligos

- Gain-of-function experiment: mRNA injections, transgenics - Biochemistry and molecular biology: Western blot, PCR…- Imaging: live, gene and protein expression- Experimental embryology: cell or tissue ablations, bead

implantation, chick-quail chimeras

Tools for developmental biology study

• Worm (C. elegans)• Fly (Drosophila melanogaster)• Fish (zebrafish, medaka)• Mouse• Xenopus (laevis, tropicalis)• Chick (Quail)

Model systems for developmental biology

MODEL SYSTEMS for the experimental

analysis of development

Fly: Drosophila melanogaster

Mouse: Mus musculis

Chick: Gallus domest

icus

C elegans: Caenorhabdit

is elegans

Xenopus laevis and tropi

calis

Fish: Danio rerio

Model systems for developmental biology

The early development of chick embryo

The developmental stage of chick embryo

Gametogenesis

fertilization

cleavage

gastrulation

organogenesis

The five fundamental main steps

Modulation of cell migration in chick embryo

gastrulation

Is cell migration the only event happen during gastrulation?

Answer is No

• Proliferation: divide 2-3 time during gastrulation

• Apoptosis

However, comparing those… no doubt that cell migration is major event, at least in chick gastrulation

The advantage of chick embryo

1. Good model for human being, short life-span

2. Very easy to accessible and be manipulated.

3. Combination with Classic and molecular biological

technologies.

• transplantation• cell labelling• Gene overexpression• Gene knock-down• Immunocytochemistry• chemical treatment• in situ hybridization

What is gastrulation?

Gastrulation – the stage of animal development

where a ball of cells consisting of a single,

undeveloped layer is converted into an embryo

consisting of three separate tissue layers :

• Ectoderm – outer layer• Endoderm – inner layer surrounding the gut• Mesoderm – middle tissue layer

The first structure – primitive streak

Hensen’s nodeprimitive streak

epiblast

hypoblast

endodermdisplacing hypoblst

migrating cells

mesoderm

Cell movement in chick embryo Gastrulation

When does gastrulation happen ?

List of developmental events in order:

• Fertilization)• Cleavage • Blastocyst• Grastrulation• Neuralization

Primitive Streak Formation (elongation to anterior)

The main axes of a developing embryos

Specification of the chick anterior-posterior axis by gravity

Rotation in the shell gland (A) results in the lighter components of the yolk pushing up one side of the blastoderm (B). That more elevated region becomes the posterior of the embryo (C).

Discoidal meroblastic cleavage in a chick egg

(A-D) Four stages viewed from the animal pole (the future dorsal side of the embryo). (E) An early-cleavage embryo viewed from the side

Formation of the two-layered blastoderm of the chick embryo

(A, B) Primary hypoblast cells delaminate individually to form islands of cells beneath the epiblast. (C) Secondary hypoblast cells from the posterior margin (Koller's sickle and the posterior marginal cells behind it) migrate beneath the epiblast and incorporate the polyinvagination islands. (D) This sagittal section of an embryo near the posterior margin shows an upper layer consisting of a central epiblast that trails into the cells of Koller's sickle (ks) and the posterior marginal zone (mz).

Streak formation in chick

Regulation of the chick blastoderm

The ability to initiate a primitive streak is found throughout the marginal zone. When the blastoderm is divided into four parts, each part can initiate gastrulation and give rise to an embryo.

posterior marginal zone (PMZ)

(Only Vg1 expression PMZSupplied by Xuesong Yang’s lab)

Susan C etc. DEVELOPMENTAL DYNAMICS 220:284–289 (2001)

EC culture New Culture

The in vitro developmental methods for chick embryos

Optical Systems

Two-photon Confocal

Compound & Dissection inverted

The microscopy for studying cell migration in gastrulation of chick embryos

Just think of ….and keep trying…..

Gene transfection …training required

Cell migration in gastrulation

Cell movement pattern:

1. Epiblast cells movement during primitive streak formation.

2. Mesoderm formation during gastrulation.

3. Vasculogenesis

Cell movement mechanism:

1. Cell-cell adhesive molecule.

2. Cell polarity.

3. chemotaxis.

The correlation between morphogen and cells in pattern formation

Cell migration in gastrulation

Cell movement pattern:

1. Epiblast cells movement during primitive streak formation.

2. Mesoderm formation during gastrulation.

3. Vasculogenesis

Cell movement mechanism:

1. Cell-cell adhesive molecule.

2. Cell polarity.

3. chemotaxis.

The genes involved in streak formation in chick

Convergent extension in Keller explants

The Wnt planar polarity pathway in convergent extension

Cell movement patterns in gastrulation

The role of Wnt11 signalling

Streak formation as a result of intercalation and/or oriented cell division

R Keller et al, 2003

Cell movements of the primitive streak of the chick embryo

(A-C) Dorsal view of the formation and elongation of the primitive streak. The blastoderm is seen at (A) 3–4 hours, (B) 7–8 hours, and (C) 15–16 hours after fertilization. The early movements of the migrating epiblast cells are shown by arrows. (D-F) Formation of notochord and mesodermal somites as the primitive streak regresses, shown at (F) 19–22 hours, (E) 23–24 hours, and (F) the four-somite stage. Fate maps of the chick epiblast are shown for two stages, the definitive primitive streak stage (C) and neurulation (F). The endoderm has already ingressed beneath the epiblast, and convergent extension is seen in the midline.

Migration of endodermal and mesodermal cells through the primitive streak

(A) Scanning electron micrograph shows epiblast cells passing into the blastocoel and extending their apical ends to become bottle cells. (B) Stereogram of a gastrulating chick embryo, showing the relationship of the primitive streak, the migrating cells, and the two original layers of the blastoderm. The lower layer becomes a mosaic of hypoblast and endodermal cells; the hypoblast cells eventually sort out to form a layer beneath the endoderm and contribute to the yolk sac.

哈尔滨医科大学 – 难忘的大学及研究生时代

Advertising for old school and hometown…

哈尔滨– 难忘的大学及硕士研究生时代

Advertising for old school and hometown…

Primitive Streak

Regression (shorten to

posterior)

Chick gastrulation roughly from 24 to 28 hours

(A) The primitive streak at full extension (24 hours). The head process (anterior notochord) can be seen extending from Hensen's node. (B) Two-somite stage (25 hours). Pharyngeal endoderm is seen anteriorly, while the anterior notochord pushes up the head process beneath it. The primitive streak is regressing. (C) Four-somite stage (27 hours). (D) At 28 hours, the primitive streak has regressed to the caudal portion of the embryo. (E) Regression of the primitive streak, leaving the notochord in its wake. Various points of the streak (represented by letters) were followed after it achieved its maximum length. Time represents hours after achieving maximum length (the reference line, about 18 hours after incubation).

Formation of Hensen's node from Koller's sickle

(A) Diagram of the posterior end of an early (pre-streak) embryo, showing the cells labeled with fluorescent dyes in the photographs. (B) Just before gastrulation, cells in the anterior end of Koller's sickle (the epiblast and middle layer) were labeled with green dye. Cells of the posterior portion of Koller's sickle were labeled with red dye. As the cells migrate, the anterior cells formed Hensen's node and its notochord derivatives.

Induction of a new embryo by transplantation of Hensen's node

(A) A Hensen's node from a duck embryo is transplanted into the epiblast of a chick embryo. (B) A secondary embryo is induced (as is evident by the neural tube) from host tissues at the graft site.

Nobel prize was presented to the discover of Organizer

The change in cell shape in the neural plate during chick neurulation

The change in cell shape in the neural plate during chick neurulation

Ray Keller et. Differentiation (2003) 71:171–205

Hensen’s node regress & notochord extension

1. Possibility: addition extra cells by Hensen’s node, notochord and notochord

plate cell division, mediolateral cells move into notochord

2. Possibility: Convergence and extension of

notochord maybe active process, which push Hensen’s node back posteriorly

4. Possibility: Node actively pull itself posteriorly.

3. Possibility: posterior blastoderm drive node regression and notochord extension

Mesoderm cell fate map

What is mesoderm formation apart from gene expression?

• How does the streak form?

• Which signals control movement?

• What is mechanism of movement?

• Role of cell division

• Chemoattractants/repellents (FGF/VEGF?)

• Adhesion molecules

Hensen’s nodeprimitive streak

epiblast

hypoblast

endodermdisplacing hypoblst

migrating cells

mesoderm

EMT – the initial step for mesoderm cell formation

EMTEMT

(Epithelial-Mesenchymal Transition)

Cadherin-mediated cell adhesion during gastrulation

Epiblast- epithelial cells – Epiblast- epithelial cells – connected by E-Cadherinconnected by E-Cadherin

Hensen’s nodeprimitive streak

epiblast

hypoblast

endodermdisplacing hypoblst

migrating cells

mesoderm

EMT – the initial step for mesoderm cell formation

EMTEMT

E-CadherinE-Cadherin

N-CadherinN-Cadherin

日本东京医科齿科大学 – 难忘的日本留学 Ph.D时代

Six years there!!!!!

日本东京医科齿科大学 – 难忘的日本留学 Ph.D时代

Cell migration in gastrulation

Cell movement pattern:

1. Epiblast cells movement during primitive streak formation.

2. Mesoderm formation during gastrulation.

3. Vasculogenesis

Cell movement mechanism:

1. Cell-cell adhesive molecule.

2. Cell polarity.

3. chemotaxis.

Merlot & Firtel, (2003) JCS 116, 3471

Spatial-temporal regulation of PTEN and PI3K induces cell polarization in response to a chemo-attractant signal

PI4,5P2 PI3,4,5P3

PTEN

PI3K

Signalling to actin cytoskeleton

PH Gef

Rac/cdc42

Scar/wave

Actin filaments

PI3kinase/Pten

Dock180Dictyostelium Growth

factor

Small GTPase – cell polarity

Cell migration in gastrulation

Cell movement pattern:

1. Epiblast cells movement during primitive streak formation.

2. Mesoderm formation during gastrulation.

3. Vasculogenesis

Cell movement mechanism:

1. Cell-cell adhesive molecule.

2. Cell polarity.

3. chemotaxis.

Mesoderm cell migration

pattern during

gastrulation

The fate map of primitive streak cells

Somite formation in chick

The xxx eggs make me crazy everyday!!!!

Could fully understand the student’s complain…

英国 Manchester 大学 – 难忘的英国 Post-Doc 时代

英国 Dundee 大学 – 工作近十年的城市 - 家？

Many hypothesis for the mechanism of cell migration

Migratory cell contact inhibition

Supplementary Figure 1: Contact Inhibition of

Locomotion controls Neural Crest (NC) directional

migration in vivo a. When two NC cells collide they change their respective direction of migration. We show here that this process is dependent on the PCP pathway (symbolised here with yellow tees). b. In the embryo, NC cells migrate with high directionality (green arrow) in streams of limited width (represented as a white area). We showed that cell-cell contact is essential for this directionality. c. Cell polarization is inhibited by cell contact. This inhibition is controlled by the same mechanism that the one shown in panel a (yellow tees). d. Only leading cells are polarised as they have a free edge. Cells can only migrate in this direction and thusdirectionality is achieved.

Migratory cell contact inhibition

Chemotaxis act as one of

mechanism for directional

cell migration during

gastrulation

• What is gastrulation?

• What is the significance of

gastrulation during embryonic

development?

Questions