Download Gastrulation, Vertebrates

2016.10.11.
Ernst Haeckel (1834-1919)
Karl Ernst von Baer (1792-1876)
GASTRULATION –
formation of germ layers II.
„It is not birth, marriage, or geath, but gastrulation,
which is truly the most important time in yor life”
Lewis Wolpert (1983)
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DETAILS AND BACKGROUND 3.
Zebrafish

The blastoderm consists of a superficial enveloping layer (EVL) and
deep cells (blastomeres), which will give rise to all embryonic
tissues (YSL: yolk cell)

Epiboly begins when the flat yolk cell domes into the blastoderm
and more deeply located blastomeres intercalate radially into
more superficial layers → blastoderm becomes thinner and
expands toward the vegetal pole

At 50% epiboly gastrulation begins (synchronized ingression)

Marginal zone: mesendodermal precursors (prospective mesoand endodermal cells intermingle) move via the blastopore
beneath the prospective ectoderm

Upon internalization, the mesodermal progenitors migrate away
from the blastopore toward the animal pole via directed
migration and convergent extension (planar intercalation)
(chorda dorsalis); endodermal precursors also spread toward the
animal pole via a random walk
YSL
Zebrafish Gastrulation: Cell Movements, Signals, and Mechanisms
IntRevCytol2007 DOI: 10.1016/S0074-7696(07)61004-3
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APICAL CONSTRICTION
?
DETERMINATION OF CELL MIGRATION TYPE
 migration ← PCP pathway
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 migration type depends on cell position in the BMP gradient: BMP
signaling inhibit Wnt/PCP pathway components and cell adhesion
(see positions of extension ↔ mediolateral intercalation in the right
picture)
Figure 11. Rho mediates actin remodeling during
gastrulation in zebrafish. Rho acts through its
downstream effector, Rho associated kinase (ROCK),
which presumably phosphorylates regulatory myosin
light chains (MRLCs) and enforces actomyosin
contractility. In conjunction, zDia2 or other formins
may be activated by Rho and/or Cdc42 to
accelerate
actin-nucleation
through
the
cooperation with profilin I at the front edge of
migrating cells for the control of cellular migration
during gastrulation in the zebrafish.
doi:10.1371/journal.pone.0003439.g011
Annu. Rev. Cell Dev. Biol. 2012.
Epiboly – mesendoderm specification
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CLUE: b-CATENIN IN NUCLEUS

1st: in early development, under maternal gene control and
defines the dorsal organizer by nuclear labelling restricted
to a few cells → axis formation

2nd (after epiboly has started):
 translocation expands to encompass the entire marginal zone
 nuclear b-catenin → ntl transcription (no tail protein; zebrafish T;
bra in zebrafish)
 Wnt anatogins dickkopf does not pervent
 b-catenin nuclear localization and
 does not inhibit ntl expression
 b-catenin nuclear localization is Wnt independent!!!
(a) Zebrafish embryos at sphere stage (4 hpf). (b) Domestage zebrafish embryos at the onset of epiboly (4.3
hpf). (c) b-cat labelling, margin cells (left) and dorsal
pole (right, white arrows: nuclear b-cat) at sphere. (d) bcat labelling around the margin (left and right) at dome
(ubiquitous nuclear b-cat). (e) b-cat labelling at animal
pole (upper pole) at sphere stage. (f) b-cat labelling at
animal pole (upper pole) at dome stage. Scale bar, 20
mm (white bars).
Bra: Brachyury – its early expression leads mesoderm induction in Xenopus
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MECHANICAL FORCE – β-catenin as a mechanoeffector
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Gastrulation is a profoundly physical event: cells deform, get pulled and are compressed,
generating hinges, folds and borders

marginal b-catenin nuclear translocation coincides with the
first epiboly movements in zebrafish

marginal zone is, however, not fixed: it advances as
gastrulation proceeds, and its cellular composition changes as
cells transit through the margin before rolling inside the
embryo (a insert)

marginal zone undergoes local dilation of the tissue ← yolk
cell contractile force pulling margin cells in epiboly (cell
mechanics is rooted in cytoskeletal tension and architecture,
and tuned by the physical resistance of surrounding cells or
the extracellular matrix)

applying cytoskeleton targeting drugs (arrest epiboly) →
expression of the ntl gene was not activated in the ring of cells
at the embryonic margin (b)

injection the mechanically defective embryos with magnetic
particles → margin cells to be mechnically towed by an
electromagnet ring positioned around the embryo! (c)
↓
DOI: 10.1038/ncomms3821
Mechanics in the embryo, Nature 2013
rescued cell deformation, epiboly movements and
mesoderm gene expression!
(rescue occurred even in neighbouring cells that did not receive
the magnetic particles, suggesting that mechanical forces are
transmitted through cell–cell junctions)
IX. Gergely (1227-1241; 94 éves!)
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 1239. „Si vera sunt (Ha ezek igazak)” kezdetű bulla
az eretnekség pestis, ami ellen csak tűzzel lehet védekezni
Az eretnekség és a boszorkányság egy és ugyanaz
Hecket (Egyiptom) → kopt őskeresztények
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DETAILS AND BACKGROUND 4. Xenopus
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EPIBOLY - GASTRULATION

Pigmented animal cells overgrow the yolk cells

gastrulation movement iniciated just below the equator, in the
marginal zone, where the animal and vegetal hemispheres meet

blastopore
 the slitlike blastopore bands gradually and forms a circle →
encircles the yolk plug
 It has one dorsal, two lateral and one ventral lips, where
prospective endodermal and mesodermal cells involute
 blastocoel shrinks while the newly formed archenteron extends
blastocoel=„empty” cavity
inside the embryo
Initiation of the involution
APICAL CONSTRICTION

beginning of Xenopus laevis
gastrulation is marked by the
apical constriction of bottle
cells in the dorsal marginal
zone, which bends the tissue
and creates a crevice at the
blastopore lip
Summary and model of the cytoskeletal mechanisms of Xenopus bottle cell formation. DMZ cells at stage 9 (left) are cuboidal. In unperturbed bottle cells, F-actin (fuchsia) and
myosin (orange) are apically localized and intact microtubules (blue) emanate from the apical side, and bottle cells undergo apical constriction and apicobasal elongation
while blastopore depth increases as one result of cell shape changes (top row). When F-actin dynamics are inhibited (second row), F-actin does not accumulate apically while
pMLC localization is undisturbed (data not shown). Bottle cells do not apically constrict without F-actin, nor do they invaginate to increase blastopore depth. In the presence of
myosin inhibitors (third row), F-actin localization still occurs, while pMLC localizes apically in blebbistatin but is reduced in ML-7 treatment. All aspects of bottle cell formation and
blastopore depth are disturbed. Nocodazole treatment (bottom row) does not affect F-actin or Pmlc localization. Bottle cells without intact microtubules undergo apicobasal
elongation normally, but do not apically constrict efficiently, nor do they exhibit significant blastopore depths compared to untreated embryos. Plus signs mean the protein is
functional (left columns) or that the event occurs normally (right columns). Minus sign indicates the activity or structure of the protein has been perturbed with inhibitors. Down
arrows signify a reduction in cell shape change or decrease in blastopore depth. Question marks indicate unknown results, as those experiments or analyses were not performed.
MT, microtubules. (doi:10.1016/j.ydbio.2007.08.010)
MECHANICAL FORCE – β-catenin as a mechanoeffector (see Zebrafish)
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DETAILS AND BACKGROUND 4. Amniote - Chicken
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THE EMBRIONIC DISC AND THE PRIMITIVE STREAK
 segregation: discoidal meroblastic cleavage →
blastoderm: bilaminar embrionic disc:
 Its upper layer is the epiblast, and
Gilbert – Chapter 8
 Its lower layer is the hypoblast
 margin of the disc is opaque (area opaca), while the
 centre is pellucid / transparent (area pellucida)
 between the area opaca and pellucida there is a thin ring
of cells, called the marginal zone
 epiblast and hypoblast is joined together at the marginal
zone, and
 The space between them forms a blastocoel
 embryo develops mainly from the epiblast
 The major characteristic of avian and mammalian
gastrulation is the PRIMITIVE STREAK
 it taking shape as a local thickening at the posterior edge
of the area pellucida, called Koller’s sickle and the
thickening epiblast above it – the latter formes the posterior
marginal zone
PROGRESSION OF THE STREAK
MORPHOLOGY OF THE STREAK
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Gilbert – Chapter 8
Gilbert – Chapter 8

depression forms within the streak: primitive groove - it serves
as an opening through which migrating cells pass into the
depp layer of the embryo
PRIMITIVE GROOVE IS HOMOLOGOUS TO THE AMPHIBIA
BLASTOPORE – OR MAY BE NOT? see later
•
•

there is a local thickening at the anterior end of the streak:
Hensen’ node / primitive knot

the centre of the node contains a funnel-shaped depression:
primitive pit
mediolateral intercalation and
convergent extension → progression
Gilbert – Chapter 8
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EMT → INGRESSION
EMT is a multi-step process beginning with well-polarized
and adhesive epithelial cells and producing nonpolarized cells embedded in the extracellular matrix
(ECM).
a) specification of a group of cells destined to undergo
EMT (1),
b) loss of intercellular adhesion mediated by cadherins
at adherens junctions (2,3)
c) cytoskeletal reorganization to actively drive cell
delamination (4) and
d) degradation of the basement membrane (5)
e) Ingression (5).
Remark: EMT marker: Snai(l)2
2008 Nature Cell Biology 10: 757-759
EMT comprises up to five steps: 1) EMT specification. Different signalling
pathways (for example, HGF, TGF-β) specify the cells that will undergo
EMT; 2, 3) Downregulation of adhesion and loss of polarity markers;
4) Regulation of forces allowing cell ingression, such as apical constriction;
5) BM disassembly. The red bars represent adherens junctions and the
green bars indicate integrins and sites of adhesion to the ECM.
New concept: BM disassembly is the starting point of EMT.
STREAK FORMATION SEEMS TO BE UNCOUPLED FROM MESODERM FORMATION!
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Mesoderm formation
QUESTIONS
1) evolutionary origin of the streak
2) its relationship to the blastopore
 Mesoderm induction is regulated by evolutionarily conserved molecular
mechanisms, yet morphogenetic events during its formation vary in
different vertebrate clades
 in lower vertebrates (anamniotes), mesoderm cells are induced radially at
the blastopore - this circumblastoporal mode of mesoderm formation was
lost during early amniote evolution (B, Anamniotes)
 reptiles dont have a streak!
 in birds and mammals, mesoderm cells are generated exclusively from
the primitive streak (B, Amniotes)
 Ectopic activation of the FGF pathway generated a ring of Brachyurypositive (blue) cells in the marginal zone! (C)
(B) Schematic comparison of mesoderm formation (blue) in
anamniotes and amniotes. an, animal; veg, vegetal. a.o.,
area opaca; a.p., area pellucida. (C) FGF4-induced
mesoderm rings shown schematically (left) and stained for
Brachyury (right). Top, control injected; middle and bottom,
FGF4 injected. White line indicates section level shown in
supplementary material Fig. S3A. doi:10.1242/dev.094318
 whether a streak forms or not, mesoderm cells can be induced radially
(‘circumblastoporally’) in the marginal zone
 This ectopically formed mesoderm cells undergo migration and initiate
terminal differentiation
 to generate mesoderm in an ancestral ‘circumblastoporal’ mode, is
conserved among amniotes (including reptiles)
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Correct positioning of blastopore /
primitive streak ↔ body axis formation
next time
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