Gastrulation III establishment of body axes

1
Gastrulation III - establishment of body axes

• Anterior-Posterior Axis Formation in Drosophila.
• Bicoid, the molecular interpretation of a
  gradient
• Anterior-Posterior Axis Formation in Frog and
  Mouse.
• The 3 Signal Model
• Formation of the primitive streak
• Anterior Visceral Endoderm

2
Gastrulation III - establishment of body axes

• Anterior-Posterior Axis Formation in Drosophila.
• Gradients and Morphogens
• Bicoid, the molecular interpretation of a
  gradient
• Anterior-Posterior Axis Formation in Frog and
  Mouse.
• The 3 Signal Model
• Formation of the primitive streak
• Anterior Visceral Endoderm

3
Drosophila malenogaster
4
Fly Anatomy
Egg
First Instar Larva
Anterior
Posterior
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The Axis is Established Through a Graded
Reduction in Pattern
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Drosophila malenogaster
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The Axis is Established Under Maternal Control
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Maternal Effect Mutations

• Mutations that do NOT result in damage to the
  mother but have effects on her offspring that can
  not be rescued by wildtype sperm.

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Mutations Affecting the Anterior-Posterior Axis
Anterior-Posterior Axis Formation is Independent
of Dorsal-Ventral Axis Formation.
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Bicoid (Bcd) Controls Drosophila A-P Patterning
Bcd Hb DAPI
Gregor et al., Cell 2007
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Evidence the Bcd is the Key Regulatory Gene

• 1. Strong Bcd alleles lead to a complete loss of
  head structures.
• 2. Bcd mutants can be completely rescued by
  injection of wild-type anterior cytoplasm.
• 3. Size of head directly related to Bcd gene
  dosage.

12
Bcd is Required for the Formation of Anterior
Structures
Phenocopies Removal of Anterior Cytoplasm
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Evidence the Bcd is the Key Regulatory Gene

• 1. Strong Bcd alleles lead to a complete loss of
  head structures.
• 2. Bcd mutants can be completely rescued by
  injection of wild-type anterior cytoplasm.
• 3. Size of head directly related to Bcd gene
  dosage.

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Transplantation of Wildtype Anterior Cytoplasm
Can Rescue Bcd Mutants
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Transfer of Bcd RNA is Sufficient to Induce
Anterior Structures
Bcd May Act as a Morphogen
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Evidence the Bcd is the Key Regulatory Gene

• 1. Strong Bcd alleles lead to a complete loss of
  head structures.
• 2. Bcd mutants can be completely rescued by
  injection of wild-type anterior cytoplasm.
• 3. Size of head directly related to Bcd gene
  dosage.

17
Hypothesis Bcd establishes the
anterior-posterior axis through the establishment
of a morphogen gradient.
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Morphogen
An inducing factor that can evoke more than one
cell state from the responding tissue.
19
Gradient

• The asymmetric distribution of a protein or
  protein activity in a tissue.

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Hypothesis Bcd establishes the
anterior-posterior axis through the establishment
of a morphogen gradient.
Prediction Bcd must be a cytoplasmic determinant
localized to the anterior pole.
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Establish A-P axis through localization of Bcd
RNA and protein in anterior pole
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Establish A-P axis through localization of Bcd
RNA and protein in anterior pole
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Posterior pole must be cleared of hunchback by
nanos.
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Posterior Posterior pole must be cleared of
hunchback
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Main Function of nanos is to Block Hunchback
From The Making of a Fly, Lawrence
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The Axis is Established Through a Graded
Reduction in Pattern
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How does the embryo interpret the Bcd gradient?

• The Conversion of Maternal Information to Zygotic
  readout.

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The Maternal Systems That Establish A-P Position
in the Drosophila Egg Activate Exclusively
Zygotic Transcription Factors
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Drosophila malenogaster
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Quantitative Relationship Between the Number of
Bcd and the Pattern of the Embryo
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Bcd can Activate and Repress Target Genes at
Defined Thresholds
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The Axis is Established Through a Graded
Reduction in Pattern
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Gastrulation III - establishment of body axes

• Anterior-Posterior Axis Formation in Drosophila.
• Gradients and Morphogenesis
• Bicoid
• Anterior-Posterior Axis Formation in Mouse.
• The 3 Signal Model
• Formation of the primitive streak
• Anterior Visceral Endoderm

34
Xenopus laevis and Xenopus tropicalis
Like fly early development in Xenopus under
maternal control
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The Organizer Forms on the Dorsal Side of the
Embryo
Animal
Ectoderm
Dorsal
Ventral
Mesoderm
Endoderm
Vegetal
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Spemann and Mangolds Organizer Grafts
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What Determines Where the Organizer Will Form?
Animal
Ectoderm
Dorsal
Ventral
Mesoderm
Endoderm
Vegetal

• i.e. is the information in the ectoderm or in the
  endoderm?

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Dorsal Side Of Xenopus is Determined by Sperm
Entry Point
Grey Crescent
Signaling Center in the Vegetal Region of the
embryo.
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Axis Formation is Sensitive to UV
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Not All Endoderm is Created Equal
Ectoderm
Mesoderm
Endoderm
Ventral
Dorsal
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Organizer Grafts UV Tissue Recombination
Studies Must be Localized Signal in Either the
Ectoderm or Endoderm
Animal
Ectoderm
Ventral
Dorsal
Mesoderm
Endoderm
Vegetal
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Nieukwoop Center Induces Secondary Axis Without
Contributing to the Duplicated Axis
i.e. Induction signaling cells instruct their
neighbors to change their fate but does not
itself participate in the differentiation.
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Nieuwkoop Center and the Canonical Wnt Pathway
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Wnt Signaling
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Nieuwkoop Center and the Canonical Wnt Pathway

• Wnts can mimic Nieuwkoop Center i.e. inject
  ligands into ventral endoderm get secondary axis
  formation.
• Dominant negative GSK3, ß-catenin or plakoglobin
  injections into ventral endoderm get secondary
  axis formation.
• Maternal depletion of maternal ß-catenin mRNA
  leads to a ventralizied embryo.

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Two Signal Emanate From the Endoderm

• A general mesoderm inducing signal
• A signal from the Nieukwoop Center which induces
  the organizer

Animal
Ectoderm
Ventral
Dorsal
Mesoderm
Endoderm
Vegetal
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Specification ?Fate

• Hypothesis Must exist region a third signal that
  modifies mesodermal cell types.

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Nature of Signals from Organizer
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The 3-Signal Model(Smith and Slack)
Animal
Ectoderm
Ventral
Dorsal
Mesoderm
Endoderm
Vegetal
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3-Signal Model Circa 2008
Wnt
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Mus Musculus domesticus
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Xenopus vs Mouse
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Node verses Organizer

• Transplantation of either to distal regions of
  the embryo can lead to axis (a-p and d-v)
  formation.
• Both ultimately will give rise to notochord and
  gut endoderm
• Both the organizer and the node express many of
  the same genes.
• The organizer but NOT the node will induce
  anterior structures i.e. head
• The organizer is intimately linked with the
  blastopore lip spatially and temporally the
  organizer is not linked with the primitive streak
  spatially and temporally

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Node verses Organizer

• Transplantation of either to distal regions of
  the embryo can lead to axis (a-p and d-v)
  formation.
• Both ultimately will give rise to notochord and
  gut endoderm
• Both the organizer and the node express many of
  the same genes.
• The organizer but NOT the node will induce
  anterior structures i.e. head
• The organizer is intimately linked with the
  blastopore lip spatially and temporally the
  organizer is not linked with the primitive streak
  spatially and temporally

Conclusion Must be other source of organizing
signals responsible for anterior structures in
the mouse.
55

• Thursdays Class ?

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The AVE is Required for Proper Anterior Patterning
57
AVE Patterns the Anterior Portion of the Embryo
Embryonic Ecotoderm
AVE
Visceral Endoderm
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Formation of the AVE
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Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

60
Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

61
Restriction of Gene expression Patterns in AVE
Precedes Streak Formation
AVE itself can be subdivided molecularly into
head verses heart inducing tissue
62
Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

63
Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

64
Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

65
Does gene expression in the AVE correlate with
anterior organizing function?

• Evidence
• Transplant mouse node get a duplicated axis but
  secondary axis lacks a head.
• Genes found to be expressed exclusively within
  the AVE prior to primitive streak formation.
• Remove AVE headless embryos results or add AVE to
  epiblast tissue not normally giving rise to
  anterior structures induces expression of
  anterior genes.
• Mutation in genes expressed in AVE (e.g. Hesx1,
  Lim1, Otx2) associated with anterior truncations.
• Cripto -/- mice lack an organizer but retain some
  anterior patterning.
• Removal of nodal only from the VE results in
  anterior truncations.

66
Removal of nodal only from the VE results in
anterior truncations

• Basis of experiments
• 1. Decedents of tetraploid blastocysts only give
  rise to extraembryonic tissue.
• 2. ES cells injected into blastocysts give
  little to no contribution to the visceral
  endoderm and trophectoderm.

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Chimeric Studies to Test the Requirement for
nodal in the AVE
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AVE Patterns the Anterior Portion of the Embryo
ES Cell Descendents
Embryonic Ectoderm
AVE
Visceral Endoderm
Host Descendents
69
AVE Patterns the Anterior Portion of the Embryo
Wildtype ES Cell Descendents
Embryonic Ectoderm
AVE
Visceral Endoderm
Nodal -/- Blastocyts Descendents
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AVE Patterns the Anterior Portion of the Embryo
Nodal -/- ES Cell Descendents
Embryonic Ectoderm
AVE
Visceral Endoderm
Wildtype Blastocyts Descendents
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Nodal and Anterior Patterning
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Removal of the TGF-ß family member nodal only
from the VE results in anterior truncations
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Chimeric Studies to Test the Requirement for Otx2
in the AVE
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AVE Patterns the Anterior Portion of the Embryo
Wildtype ES Cell Descendents
Embryonic Ecotderm
AVE
Visceral Endoderm
Otx2 -/- Blastocyts Descendents
75
AVE Patterns the Anterior Portion of the Embryo
Otx2 -/- ES Cell Descendents
Embryonic Ectoderm
AVE
Visceral Endoderm
Wildtype Blastocyts Descendents
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Otx2 and Anterior Patterning
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Otx2 is Required Both in the AVE and in the
Underlying Ectoderm
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AVE Patterns the Anterior Portion of the Embryo
Embryonic Ectoderm
AVE
Visceral Endoderm
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Current Model for Anterior Patterning
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(No Transcript)
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Evidence for a Role for the AVE in Anterior
Patterning