Transcript Primitive Streak Regression

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: [email protected]
The e-mail for my lecture: ppt or any notices
[email protected]
Password: development
杨雪松 Scientific career
1984
1991
生殖内分泌
1997
膜生理,
神经科
学
1999
2007
胰腺外
分泌
细胞生理学
细胞生物学
now
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
Human Embryo development movie
Developmental biology
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Definition: Field of biology that studies how a single cell (the
fertilized egg) gives rise to a fully formed organism.
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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.
Fundamentals questions in developmental biology
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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
Tools for developmental biology study
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Gene knock-out or knock down: mutation, RNAi, antisense
oligos
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Gain-of-function experiment: mRNA injections, transgenics
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Biochemistry and molecular biology: Western blot, PCR…
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Imaging: live, gene and protein expression
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Experimental embryology: cell or tissue ablations, bead
implantation, chick-quail chimeras
Model systems for developmental biology
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Worm (C. elegans)
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Fly (Drosophila melanogaster)
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Fish (zebrafish, medaka)
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Mouse
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Xenopus (laevis, tropicalis)
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Chick (Quail)
Model systems for developmental biology
Chick:
Gallus
domesticus
Fly:
Drosophila
melanogast
er
MODEL SYSTEMS
for the experimental
analysis of development
Mouse: Mus
musculis
Fish:
Danio
rerio
Xenopus
laevis and
tropicalis
C elegans:
Caenorhabdit
is elegans
The early development of chick embryo
The developmental stage of chick embryo
The five fundamental main steps
Gametogenesis
fertilization
cleavage
gastrulation
organogenesis
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.
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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
Cell movement in chick embryo Gastrulation
Hensen’s node
primitive streak
epiblast
hypoblast
migrating cells
endoderm
mesoderm
displacing
hypoblst
When does gastrulation happen ?
List of developmental events in order:
• Fertilization)
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Cleavage
Blastocyst
Grastrulation
Neuralization
Primitive Streak Formation
(elongation to anterior)
The main axes of a developing embryos
Specification of the chick anteriorposterior 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
posterior marginal zone (PMZ)
(Only Vg1 expression PMZ
Supplied by Xuesong Yang’s lab)
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.
The in vitro developmental methods for chick embryos
New Culture
EC culture
Susan C etc. DEVELOPMENTAL DYNAMICS
220:284–289 (2001)
The microscopy for studying cell migration in
gastrulation of chick embryos
inverted
Compound & Dissection
Optical Systems
Two-photon
Confocal
Gene transfection …training required
Just think of ….and keep trying…..
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) Foursomite 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
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
3.
Possibility:
posterior blastoderm drive node
regression and notochord extension
4.
Possibility:
Node actively pull itself posteriorly.
Ray Keller et. Differentiation (2003)
71:171–205
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
EMT – the initial step for mesoderm cell formation
Hensen’s node
primitive streak
epiblast
hypoblast
EMT
migrating cells
endoderm
mesoderm
displacing
hypoblst
(Epithelial-Mesenchymal Transition)
Cadherin-mediated cell adhesion during gastrulation
Epiblast- epithelial cells –
connected by E-Cadherin
EMT – the initial step for mesoderm cell formation
Hensen’s node
primitive streak
epiblast
E-Cadherin
EMT
N-Cadherin
hypoblast
migrating cells
endoderm
mesoderm
displacing
hypoblst
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.
Spatial-temporal regulation of PTEN and PI3K induces
cell polarization in response to a chemo-attractant signal
PI4,5P2
PI3K
PTEN
PI3,4,5P3
Merlot & Firtel, (2003) JCS 116, 3471
Signalling to actin cytoskeleton
PI3kinase/Pten
Dictyostelium
Dock180
PH Gef
Rac/cdc42
Scar/wave
Actin filaments
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
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.
Chemotaxis act as one of
mechanism for directional cell
migration during gastrulation
Questions
• What is gastrulation?
• What is the significance of gastrulation
during embryonic development?