gastrulation

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Lecture 16

• gastrulation
• neurulation
• organogenesis

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Gastrulation in Xenopus overview
Fig 8.20
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check out Jeff Hardins dynamics of development
tutorial http//worms.zoology.wisc.edu/embryo_main
/embryology_main.html for movies of frog
gastrulation, etc
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5 regions
Animal cap 3 cells thick, undergoes epiboly
superficial involuting marginal zone (sIMZ)
outer layer of MZ, form archenteron roof
deep involuting marginal zone (dIMZ) inner layer
of MZ, forms mesoderm
bottle cells invaginate at blastopore make
sides of archenteron
vegetal base undergoes vegetal rotation, makes
floor of archenteron
for details see Keller et al 2003
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gastrulation questions

• what cells move where?
• descriptive embryology
• which cells generate force?
• experimental embryology
• biomechanical models
• what is molecular basis of force generation?
• genetics and biochemistry

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Movie 1

• gastrulation.org

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1. invagination
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role of the bottle cells

• display apical constriction, basal expansion
• invagination forms the blastopore (starts
  dorsally)
• remove bottle cells surgically gastrulation
  proceeds but less reliable, involution erratic
  (Keller 1981)
• bottle cells bend epithelial sheet, may also
  attach endoderm to involuting mesoderm
• so--what drives involution?

Fig 8.21
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2. involution
bottle cells at leading edge of endoderm
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role of the involuting marginal zone (IMZ)

• Keller is driving force from deep or superficial
  IMZ?
• replace superficial cells with animal
  cap--gastrulation fine
• replace superficial PLUS deep cells with animal
  cap--no movement
• conclusion deep IMZ cells drive involution,
  everything else hitches a ride

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does dIMZ involution require blastocoel roof?

• Holtfreter
• remove part of animal cap--hole in blastocoel
  roof
• MZ involution continues, so blastocoel roof not
  essential?
• blastocoel roof ECM contains fibronectin
• blocking fibronectin function blocks involution
• probably both processes operate

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3. convergent extension

• extension along the dorsal midline
• occurs in MZ explants--autonomous

Fig 8.24 Brachyury
Fig 8.22
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convergent extension

• Keller et al 1985
• transplant labeled IMZ cells into unlabeled host
• see cells intercalate
• probably a major force producing movement in
  gastrulation

Fig 8.25
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two types of intercalation

• timelapse movies of cell movement in explants
• radial intercalation makes IMZ thinner, allows
  expansion
• mediolateral intercalation causes extension along
  dorsal midline

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cellular basis of intercalation

• cells become polarized (Wnt signaling involved)
• one end becomes motile, other end immotile
• motile ends exert traction--pull cells over each
  other
• IMZ becomes a stiffened rod

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Intercalation movements
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4. epiboly

• spreading of animal cap
• driven by
• cell division
• cells become more squamous (in outer layers)
• radial intercalation (in deep layers) may drive
  epiboly

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3 epiboly movements

• Epiboly of the entire embryo--zebrafish
• Epiboly of the epidermis
• Drosophila dorsal closure
• C. elegans ventral closure

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result of gastrulation movements
Fig 8.23
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summary of frog gastrulation

• bottle cells initiate invagination at blastopore
• deep IMZ cells autonomously undergo involution at
  blastopore lip, migrate over inner blastocoel
  roof
• deep IMZ cells undergo convergent extension,
  strongest on dorsal side generates hoop stress
  that forces blastopore closure
• superficial IMZ pulled along passively by deep
  IMZ
• vegetal base undergoes rotation (Winklbauer
  Schurfield 1999) that may also generate force
• epiboly of animal cap covers embryo, closes
  blastopore leaving the yolk plug

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molecular basis of gastrulation?

• Wnt signaling
• polarizes cells in IMZ, necessary for convergent
  extension
• organizer proteins
• probably regulate other genes involved in cell
  movements?
• e.g. 2 protocadherins one expressed only in
  axial mesoderm, one only in paraxial mesoderm
• some functional data

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gastrulation in chick, quick.

• epiblast undergoes gastrulation
• cells move towards axis (force generated by
  convergence and extension, also oriented cell
  division)
• primitive streak cells ingress (EMT)
• ingressing cells form deep layer (endoderm,
  displaces hypoblast) and middle layer (mesoderm)

Fig 2.13
Molecules --Scatter factor (HGF) secreted
protein, promotes ingression --downregulates
E-cadherin --local degradation of basal lamina
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neurulation

• formation of a tube from a flat sheet of cells
• 4 phases
• specification of neural plate convergence and
  extension to form single cell layer
• palisading to form region of columnar epithelium
  (placode)
• bending and invagination
• tube closure

Fig 8.29
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driving forces in neural folding

• apical constriction (change in cell shape)
• crawling (change in cell motility)

Fig 8.30
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forces from inside and outside

• hinge points abrupt changes in shape
• medial hinge point may be induced by notochord
• an isolated neural plate will fold up inside-out
• surrounding ectoderm forces invagination inside
  embryo

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role of cadherins
Fig 8.32

• neural plate cells switch from E to N cadherin
• N-cadherin gain of function experiment

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organogenesis
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3 kinds of organ

• internal organs
• fluid handling function
• branching morphogenesis
• sensory organs
• sensory neurons in epithelium
• organ identity
• Limbs (next lecture)
• mechanical function
• pattern formation

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internal organs
fly tracheal system

• fluid handling functions
• maximize surface area, minimize volume
• branched epithelial tubes in mesenchymal matrix
• lung, kidney, pancreas, glands, ...
• models tracheal system in Drosophila, mammalian
  kidney

adult kidney collecting ducts
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drosophila tracheal system

• form from 20 groups of 80 cells each
• adult has 10,000 branches
• form by changes in cell shape/migration, not
  proliferation

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types of branch

• 1 and 2 branches
• contain multiple cells
• invariant pattern
• terminal (3) branches
• tubes form within single cells
• variable pattern

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3 kinds of epithelial tubes
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tubulogenesis
(cf blastulation)
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development of tracheal system
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what specifies branching pattern?

• 1 branches FGF signaling
• FGF (Bnl) expression is chemoattractant
• trachea express FGFR (Btl)

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what specifies tracheal branching?

• 2 branches FGF signaling
• cells at tip of 1 branch stimulated to form 2
  branches
• cells further away inhibited (sprouty)

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terminal branching is controlled by oxygen need
more branches, more tortuosity
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hypoxia activates local FGF expression

• self-limiting by negative feedback

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the mammalian kidney

• many steps can be reconstituted in organ culture
• develops from intermediate mesoderm
• epithelial tube, the nephric duct (aka Wolffian
  duct)
• adjacent nephrogenic mesenchyme

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primitive versus definitive kidneys

• pronephros
• mesonephros
• metanephros
• becomes definitive kidney in mammals

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three kidneys
ureteric bud induces metanephric mesenchyme to
condense into nephrons
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branching of ureteric bud
15 rounds of branching 300,000-1 million
branches both collateral branching and
bifurcation of branch tips
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development of tubules
Fig 10.45. Note dual origin of tubule
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reciprocal signaling
ureteric bud
LIF
LIFR
metanephric mesenchyme
Ret
GDNF
FGF2, BMP7
(permissive)

• LIF signaling induces tubule formation
• GDNF induces branching of ureteric bud
• FGF2/BMP7 signals prevent cell death in mesenchyme

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eye development

• compare eyes in vertebrate and Drosophila
• organ identity and the role of Pax-6

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eye development

• 3 tissues
• CNS --retina
• epidermis--lens cornea
• neural crest--ocular mesenchyme--other bits
• multiple reciprocal interactions
• all express the same transcription factor PAX6

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eye development-1

• optic vesicle invaginates to form optic cup
• sends BMPs to induce adjacent ectoderm to form
  lens placode

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eye development-2

• lens placode
• invaginates and pinches off to form future lens
• signals back to optic vesicle (FGFs) to induce
  retinal differentiation
• signals to newly overlying ectoderm to make
  cornea

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lens development

• lens epithelium differentiate into lens fiber
  cells
• lens cells do not die--oldest cells in your body
• express crystallin proteins (90 of mass of
  lens)--molecular chaperones

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Human eye disorders

• Aniridia
• iris remnants (arrowheads)
• cataracts (arrow
• Ectopia pupillae
• pupil is not central
• Peters anomaly
• central opacity of cornea
• all due to different mutations in PAX6

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PAX6 in mice

• 2 copies of PAX6
• wild type

1 copy of PAX6 Small-eye (cf Aniridia)
0 copies of PAX6 lethal
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PAX6 turns on in many eye precursors
corneal epidermis
some in mesenchyme?
invaginating lens vesicle
optic cup
does PAX6 determine eye organ identity?

• mouse eye E10.5 stained with anti-PAX6

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Drosophila eye structure
each compound eye is made of 800 simple eyes
(ommatidia)
develop from epithelium of eye imaginal disc
Fig 11.21
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Drosophila eye development
develop from epithelium of eye imaginal disc
Fig 11.22
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eyeless mutants
...are due to mutations in the Drosophila PAX6
gene!
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PAX6 sufficient to initiate eyes
ectopic expression of PAX6 can induce ectopic eye
tissue
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its not that simple
PAX6 is expressed in other tissues as well as
eyes ectopic expression of several other genes
can induce ectopic eyes PAX6 does not act
alone...network of eye transcripton factors
that turn each other on Some aspects of the
network conserved fly to mouse