Download Gastrulation 1

2016.10.11.
Ernst Haeckel (1834-1919)
Karl Ernst von Baer (1792-1876)
GASTRULATION –
formation of germ layers I.
„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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 Definitions
 Types of morphogenetical transformations
 background: apical constriction, planar polarity
 Details with background
 C. elegans
 Drosophila
 Zebrafish
 Xenopus
 Amniote
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DEFINITIONS
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 GASTRULATION is a fundamental phase of animal embryogenesis during
which
 the germ layers of an embryo are specified, formed and
 the body plan of the mature organism is established
 Complex and coordinated movements on a massive scale allow cells to
establish great complexity from a very simple starting form.
 A GERM LAYER is a group of cells in an embryo that interact with each
other as the embryo develops and contribute to the formation of all organs
and tissues
 germ layers are epithelial like cell layers without specialized cellular
junctions and polarity
 Diploblastic organisms have only the two primary germ layers
 Triploblastic animals have three germ layers
Gastrulation must be exquisitely regulated to ensure that specific cells move to the
correct position at the appropriate developmental time
MORPHOLOGICAL TISSUE TRANSFORMATION
Annu. Rev. Cell Dev. Biol. 2012.28:687-717
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GASTRULATION = 4 evolutionarily conserved morphogenetic movements
 Internalization/ emboly: mesodermal and endodermal cells become internalized
beneath the outer layer;
 Epiboly: epibolic movements spread and thin germ layers;
 Convergence movements narrow germ layers dorsoventrally;
 Extension movements elongate germ layers antero-posteriorly
cell shape changes,
• directed migration,
• planar and radial intercalations,
• cell divisions,
• EMT (epithelial-mesenchymal transition),
Cell behaviours ← actomyosin cytoskeleton
•
guided by
• differential cell adhesion,
• chemotaxis, chemokinesis
• planar polarity.
Coordination of gastrulation movements with embryonic polarity involves regulation by
anteroposterior and dorsoventral patterning systems
Chemotaxis: directional cell movement of cells towards concentration gradients of solubilized attractants
Chemokinesis: increased nondirectional activity of cells due to presence of a chemical substance (random cell movement)
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Internalization / emboly
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 Gateway = blastopore
 Epithelial to mesenchymal transition (EMT) ← cell adhesion molecules are
downregulated, while intermediate filament network is formed and microtubule
network is rearranged
0. INICIATION: APICAL CONSTRICTION („csúcsi összehúzódás”)
 actomyosin mediated contraction ← apical enrichment of activated (phosphorylated) nonmuscle myosin II
Functions and examples of apical
constriction. (A-C) Apical constriction
functions in various contexts
including: (A) tissue folding and tube
formation, seen in examples of
gastrulation and vertebrate
neurulation; (B) ingression of
individual cells and epithelial-tomesenchymal (EMT) transitions, as
occur in other examples of
gastrulation and in tissue
homeostasis; and (C) healing and
sealing of embryonic tissues in
response to wound healing. The cell
and tissue movements (green
arrows) that occur as specific cells
undergo constriction of their apical
sides (orange) are indicated in each
context. Wound healing can involve
apical constriction of an underlying
layer of cells, or of a ring of cells
(dashed line; just two such cells of
the ring are drawn) at the periphery
of a wound.
homophilic cell
adhesion molecule
E-cadherin
Development 2014 141: 1987-1998; doi: 10.1242/dev.102228
Developmental cue
G protein-coupled receptor
(a unit: G12/13) signaling
recruiting to the apical membrane
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Regulation and positioning of
contractility - mechanotransduction
PDZ-RhoGEF
(guanin nucleotide exchange factor)
RhoA
(small GTPase)
Mechanisms of apical
constriction. Key
components involved in
apical constriction include Factin (red) and myosin
(orange), which form
contractile networks. Actinmyosin networks can be
organized into contractile
bundles/fibers or can be
organized into a more
loosely organized twodimensional network that
underlies the plasma
membrane, called the
apical cortex. Shrinkage of
the apical cortex (green
arrows) is driven by actinmyosin contractions. Apical
adherens junctions (AJs,
gray) link cells, allowing
apical actin-myosin
contractions to drive tissue
shape changes. In this
example, only the apical
actin cortex is shown.
RhoA*-GTP
Dia formin
Model of protein localization in apical
constriction. F-actin is present in an apical
meshwork and in cables at the level of the AJ.
Apical-basal-oriented microtubules (brown)
transport argo myosin ( green), RhoGEF2 (blue),
actin (orange), and endocytic vesicles ( purple).
F-ACTIN
ASSEMBLY
Type II MYOSIN ACTIVATION
APICAL
CONSTRICTION
ROCK
myosin-phosphatase
(Rho-associated
coiled-coil kinase)
myosin-P
Rho family small GTPases: RhoA, Rac, and Cdc42
Dia: Diaphanous
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I.
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TYPE: INVAGINATION („betüremlés, betűrődés”)
 Apical constriction → tissue folding and tube formation
 ventral midline epithelial cells creates a furrow where mesoderm folds
inward
http://dx.doi.org/10.1016/j.bpj.2012.07.018
II. TYPE: INVOLUTION („legörbülés”)
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 The prospective mesoderm and part of endoderm form a cohesive tissue
above the prospective blastopore
 apical constriction of so-called bottle cells marking the nascent blastopore
in the dorsal gastrula region, where the Spemann-Mangold organizer (SMO)
resides
 through that blastopore, which will expand laterally in the course of
gastrulation, the mesoderm progenitors roll as a coherent tissue
blastopore
 only when inside the gastrula do the mesodermal cells break away from the
involuted tissue mass to migrate on the internal side of the uninvoluted
tissue (blastocoel roof)
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III. TYPE: INGRESSION („beözönlés”)
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 EMT precedes internalization: progenitors undergo EMT to break away from
the epithelium and
 move as individuals deep into the embryo, where they continue to migrate
as individual cells
 blastopore = primitive streak
 Examples: Nematode and Amniotes: chicken and mouse
crossection
Functions and examples of
apical constriction. (B)
ingression of individual cells
and epithelial-tomesenchymal transitions
(EMT)
IV. TYPE VARIATIONS OF THE I-III TYPES
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 zebrafish gastrulation: prospective mesoderm and endoderm cells
(mesendoderm) of mesenchymal character move through the blastopore
largely as individuals, but in a synchronized manner
embryonic shield
germ ring = blastopore
50% epiboly
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EPIBOLY
 morphogenetic process that results in isotropic spreading of tissue, usually
associated with its thinning. It is achieved by
 radial intercalation of cells from deeper to more superficial layers
 intercalations are random (not polarized) with respect to embryonic axes,
they result in isotropic expansion of tissues around the nascent embryo
 classic examples: fish and frog
 cell shape changes
 directed migration of cells away from a tightly packed and thick cell mass at
the embryo equator results in its thinning and spreading toward the vegetal
pole
 example: zebrafish
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CONVERGENCE AND EXTENSION
 evolutionarily conserved process that elongates the nascent germ layers
from head to tail and narrows them from back to belly
 organogenesis: elongation of various tubular organs
I. CONVERGENT EXTENSION
•
planar / medio-lateral intercalation: simultaneous AP elongation and
mediolateral (ML) narrowing
 mediolaterally elongated cells that move between their anterior and
posterior cell neighbors
 example: Xenopus
Rearrangement of cells during convergent extension
of the mesoderm in Xenopus embryos. (A) The
dorsal region of the IMZD (which forms the
notochord) was taken from an embryo labeled with
fluorescienated dextran particles and placed into an
unlabeled embryo. (B) Tracings of individual cells
followed with video recorder during the formation of
the notochord in vitro. (After Keller et al., 1985;
Keller, 1986.)
Convergent extension of the mesoderm appears to be
autonomous, because the movements of the cells
occur even if the cells are removed from the rest of the
embryo (Keller, 1986)
http://10e.devbio.com/ chapter 8
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 polarized radial intercalation:
 cells in multilayered tissue intercalate from one layer into another,
preferentially separating their anterior and posterior neighbors
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 zebrafish gastrulation
 polarized cell divisions
 the cell division plane is polarized such that the daughters are aligned with
the AP axis
 directed cell migration
 migration trajectories of cells in the lateral mesoderm point dorsally, such
that this population converges toward the dorsal midline
 trajectories of cells closer to the animal pole (anterior) are biased anteriorly,
and
 those closer to the vegetal pole (posterior) are biased posteriorly
 undirected cell migration (random walk)
 endodermal precursors: ingress beneath the ectoderm during zebrafish
gastrulation via the circumferential blastoderm margin (blastopore) and
migrate on the surface of the yolk cell in an undirected fashion, thus
extending the nascent cell population in animal (anterior) and later also in
vegetal (posterior) direction
 extension without convergence
Collections
zebrafish
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zebrafish
Amniote
BACKGROUND:
PLANAR POLARITY
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Planar cell polarity (PCP)
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 refers to the coordinated alignment of cell polarity across the tissue plane
 Keys:
 asymmetric partitioning of cortical PCP components AND
 intercellular communication to coordinate polarity between
neighboring cells
 Contributors:
 protein transport, endocytosis, and intercellular interactions
 Establishment of PCP involves
 (1) a global orienting cue
 (2) asymmetric segregation of dedicated polarity proteins, and
 (3) translation of polarity information into polarized outputs.
www.jcb.org/cgi/doi/10.1083/jcb.201408039
The core system
 it was first describe in Drosophila
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 core PCP pathway is composed of the
 multipass transmembrane proteins Frizzled (Fz), Van Gogh (Vang; also known as Strabismus/Stbm),
and cadherin Flamingo (Fmi; also known as Starry night/Stan), and
 cytosolic components Dishevelled (Dsh), Prickle (Pk), and Diego (Dgo)
 the asymmetric segregation of Fz–Dsh–Fmi and Vang–Pk–Fmi complexes to opposite sides of the cell
relies on their mutual exclusion intracellularly and their preferential binding between neighboring cells
 develops progressively from initially uniform distributions → result of feedback amplification of an initial
directional bias
Feedback interactions between core PCP components. A Fz–Fmi complex
interacts preferentially with a Vang–Fmi complex between cells, whereas proximal
and distal complexes antagonize one another within the cell.
(The cell biology of planar cell polarity, JCB 2014
(www.jcb.org/cgi/doi/10.1083/jcb.201408039))
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WHAT MEDIATES THESE INTERCELLULAR ASYMMETRIC INTERACTIONS?
1)
Vang and Fz interact directly → cell positioning
2)
Fmi
17 → cell positioning
2)
4)
 is essential for the junctional recruitment of Fz and Vang
 Fmi homodimers appear to be functionally asymmetric
 exist in two forms depending on whether it is paired with Fz or Vang
1)
 unpaired Fmi is in a configuration that has higher affinity for Fmi–Fz than Fmi–Vang
AMPLIFICATION OF ASYMMETRY
3)
through clustering by PCP components
 clusters are stably associated with the plasma membrane (have limited lateral mobility) within the membrane
and are resistant to endocytosis
 precise mechanism: ?
 direct transport
 transcytosis in proxamal-to-distal direction: subapical, non-centrosomal MTs: plus ends oriented with a
slight distal bias (another, „feeding” system)
 sorting from the trans-Golgi-network (in vertebrates)
4)
Repulsive interactions between Vang- and Fz-containing complexes:
 Pk and Dgo interact with the same domain on Dsh in a mutually exclusive manner
 positive feedback: stabilization of asymmetry by clustering
The setup
Downstream effectors
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If PCP is the cell’s compass, it is also the steering wheel, directing downstream, polarized cell
behaviors in response to global directional cues.
 PCP can polarize a wide range of cell behaviors, which suggests that it can intersect with
numerous downstream effectors; drives
 CE (A)
 asymmetric cell division (B)
 positioning of centrosome
and kinocilia (C)
Polarized cell behaviors controlled by PCP. (A) PCP drives convergent extension (CE). CE in vertebrates is
driven by mediolateral intercalation, which narrows the mediolateral axis while simultaneously lengthening the
A-P axis. Mediolateral intercalation is accompanied by cell polarization and elongation and the formation of
mediolateral protrusions, all of which require core PCP function. Pk localizes anteriorly (Ciruna et al., 2006;
Yin et al., 2008), whereas Dsh localizes posteriorly (Yin et al., 2008). In addition, PCP proteins recruit myosin
to A-P cell borders, leading to actomyosin contractility and junctional shrinking. (B) Asymmetric cell division.
Drosophila sensory organ precursors (SOPs) divide asymmetrically along the epithelial plane, giving rise to
distinct anterior and posterior daughters. Spindle alignment along the A-P axis is PCP dependent. Dsh
interacts with Mud/NuMA and the dynein complex posteriorly while Vang links Pins/LGN-Mud/NuMA-dynein on
the anterior. This links astral MTs to the A-P cortex, bringing the spindle into register with the A-P axis. (C)
Positioning of the kinocilium in the inner ear. The placement of kinocilium in sensory hair cells of the inner
ear determines the position of V-shaped stereocilia bundles. G i and mPins/LGN localize on the abneural
side on the hair cell, where they are required for abneural positioning the Mtbased kinocilium. The collective
alignment of kinocilia and stereocilia bundles across the epithelium requires the core PCP component Vangl2.
Vangl2 (light green) localizes to the abneural side of supporting cells. Whether Fz (dark blue) associates on
the opposite face is not yet clear (Ezan et al., 2013).
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 In VERTEBRATES: WNT/PCP PATHWAY
 Wnts are clearly important regulators of PCP (but in different ways, such in Drosophila)
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The Wnt
 additional components (ligands and membrane components)
 able to control the myosin contractility through Rac, RhoA and Rho kinase (ROK)
 it alters the E-cadherin membrane stabilitiy → endocytosis (see APICAL CONSTRICTION!!)
 E-cadherin may be indirectly linked to the actin cytoskeleton through b-catenin!
 b-catenin Ga12/13 competition!
Vertebrate
canonical Wnt
signaling pathway
(A). Vertebrate
noncanonical Wnt
pathways,
Wnt/PCP (B), and
Wnt/Ca 2+ (C).
Diversin=Diablo, G
(green): G-proteins
Wnt canonical pathway
→ gene transcription
BUT
Wnt noncanonical
pathways →
actin cytoskeleton
DOI: 10.1016/S0074-7696(07)61004-3
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