Body axes in mammals

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Lecture 4

• Body axes in mammals
• Germ layer specification in Xenopus

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The early embryo forms two populations of cells
16-cell stage
Inner cells make ICM Outer cells make
trophectoderm (TE) Does relative position
determine their fate?
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Early sources of polarizing information--1
Polar bodies (animal pole)

• animal-vegetal axis at right angles to
  embryonic-abembryonic axis

Fig 3.13
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Early sources of polarizing information--2
blastomere that inherits sperm entry point will
divide first
blastomere that divides first will give rise to
ICM
mark one blastomere with dye (NOT by injection!)
Piotrowska, K. and Zernicka-Goetz, M. (2001).
Role for sperm in spatial patterning of the early
mouse embryo. Nature 409 , 517-521.
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but pattern can be re-specified if positions
scrambled
Fig 3.12
Conclusion at 8-cell stage cells are not
determined to become ICM or TE
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When do cells become determined to ICM or TE?
homotopic
heterotopic
16-cell stage

• Ziomek and Johnson 1982
• all-outer or all-inner aggregates develop
  normally
• By 32-cell stage, no regulation

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determination, again
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more terms

• potency range of fates a cell can generate
• totipotency--all fates
• pluripotency--many fates
• multipotency
• during development
• cells become more determined
• potency becomes restricted to final fate

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Regulation in mouse embryos

• Combine two 8-cell embryos into one 16-cell
  embryo
• aggregation chimera (with 4 parents)
• Cell division slows so that blastocyst is normal
  size
• Very rare examples of human chimeras (XX-- XY)
  formed by fusion?

Fig 3.24
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Making transgenic mice requires embryonic stem
cell chimeras

• ES cells derived from ICM, retain totipotency in
  vitro
• Manipulate DNA in culture (gene knockouts,
  insertions)
• Inject mutated ES cells into host blastocyst
• injection chimera
• ES cells can contribute to germline

Box 3C
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Regulation and body axes

• Early embryonic pattern may use asymmetries of
  oocyte, sperm entry fate mapping implies some
  consistency
• But regulation means cells are not determined to
  particular germ layers or body regions (up to
  E4.5)
• Patterns can self-organize

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When do body axes of embryo develop?
First molecular differences along body axis
First overt sign of body axes
Fig 2.22
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The first molecular markers of antero-posterior
polarity

• Distal VE cells express Hex
• Move to one side, future anterior
• Proximal ectoderm cells express Brachyury
• Move towards future posterior

Fig 3.14
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How is symmetry of egg cylinder broken?

• Signals from uterine wall? (but cant be in
  mammala that implant after gastrulation)
• Gravity?
• Stochastic?
• Mammalian epiblast 600 cells
• Chick blastodisc 60,000 cells
• If twinning is less likely, may not need external
  cues.

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twinning

• monozygotic (MZ) versus dizygotic (DZ)
• natural clone
• MZ twins rare in mammals (except armadillo)
• 1 in 400 live human births
• 5x more common after IVF
• most twin fetuses die and are resorbed
• prone to congenital abnormalities
• 10 are mirror-image (left-right asymmetry)

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3 kinds of monozygotic twin
dichorionic diamniotic 30
monochorionic diamniotic 70
monochorionic monoamniotic 1

• time of splitting deduced from structure of
  extra-embryonic membranes
• Chorion diverges earlier, from trophectoderm
• Amnion derived later, from extra-embryonic part
  of ICM
• illustrates regulation in morula, blastocyst,
  epiblast stages

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Summary

• Oocyte has animal-vegetal axis that correlates
  with axis at blastocyst stage
• chimeras show that oocyte polarity can be
  overridden
• First pattern is specification of inner and
  outer cells as ICM and TE.
• Earliest markers of body axis are asymmetries
  within extra-embryonic tissue, of unknown origin
• Signals set up the anteroposterior axis of the
  epiblast

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Review axis formation

• What is symmetry-breaking step?
• How does embryo use this to set up body axes?
• Frog sperm ? cortical rotation ? Nieuwkoop
  center ? Spemann organizer
• Chick blastodisc tilt ? PMZ ? primitive streak
• Nieuwkoop center and PMZ are both dorsal-midline
  signaling centers that initiate gastrulation
• Mouse asymmetric signals from extra-embryonic
  endoderm in anterior and posterior

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Origin and specification of germ layers

• Three early embryonic tissues endoderm,
  mesoderm, ectoderm
• How are they made different?
• How does their topology arise (gastrulation)
• How are they patterned w.r.t the body axis
  asymmetries?

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Fate mapping tells us where things come from in
normal development
Mark cell at early stageand look later to see
what it made
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Fate map of frog blastula

• Composite, based on many cell marking experiments
• Normal cell mixing limits resolution
• Fate map does not tell us if cells specified or
  determined to these fates--need to do isolation
  or transplant experiments

Fig 3.18
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Tissues formed from explants in culture
Fig 3.25

• animal cells make epidermis, but not neurons
• marginal cells make only most dorsal and ventral
  fates
• Vegetal explants make large yolky cells, sort
  of endoderm?

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compare fate and specification
cf Fig 3.28
fate map--describes normal development, based on
cell marking
specification map--describes cell behavior in
isolation (in vitro)
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How are germ layers specified?

• are determinants localized along An-Vg axis?
• VegT -- behaves as localized endodermal
  determinant
• Determinants for ectoderm not known
• Mesoderm is different

Ec
Ec
Ec
Ec
Ec
Ec
Ec
Ec
M M M M M M M
En En En En En En
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Mesoderm induction in vitro
Fig 3.25

• Nieuwkoop (1960s)
• Dale and Slack (1980s, using fluorescent labels)
• Analyze properties of induction in vitro

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Does induction require cell-cell contact?

• Place micropore filter between vegetal and animal
  explants
• Mesoderm induction occurs normally

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What is the range of the inductive signal?
Animal cap
4 cells 80 mm
mesoderm
Vegetal base

• Block cell movement, division in animal cap
  (cytochalasin--inhibits actin)
• Long-range diffusion or relay of short-range
  signals?

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Competence

• Property of receiving cells
• Animal cap cells competent 4-11 hours after
  fertilization
• Induction requires at least 2 hours contact
• 5 hours gives full induction of mesoderm
  specific genes (e.g. Brachyury)

Scale bar about 500 mm
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Can single cells be induced?

• No need a critical mass of gt100 cells
• The community effect
• May be mediated by embryonic FGF (eFGF)
  signaling between induced cells

Fig 3.26
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Timing of mesoderm gene expression set by
internal clock, not time of induction

• Blocking protein synthesis has no effect on
  timing of competence or response to induction

Fig 3.27
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Tentative model

• Mesoderm fate requires an inductive signal from
  vegetal cells to animal cells
• Signal is diffusible, spreads over several cell
  diameters
• Caveat explant experiments are artificial (in
  vitro, not in vivo)
• Vegetal cells normally contact marginal zone (MZ)
  cells by the time MZ can be dissected its
  already specified
• Data show inductive signals sufficient, but are
  they necessary?
• Need to find the signals and inhibit them in vivo.