Patterning a Target Field

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Patterning a Target Field
2
Morphogen Model
Target Field
Organizing Center
Wolpert L. (1969), J. of Theoretical Biology, 25,
1-47
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The Vertebrate Limb
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Limb Polarity
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Organizing Centers in the Vertebrate Limb
Proximal-Distal
AER
(removal)
Dorsal-Ventral
mesenchyme (proximal limb) ectoderm (autopod)
(rotation)
Anterior-Posterior
ZPA
(transplantation)
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ZPA Model
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Notochord patterning and neural development
D
V
D
V
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Notochord and floor plate induction of ventral
cell identities
D
p0
p1
p2
pMN
p3
FP
V
(Jessell and coworkers)
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Morphogens and ventral neural tube patterning
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A8
A6
A7
A5
A4
A3
A2
A1
T3
T1
T2
T1
T2
T3
A8
A1
A7
A2
A6
A3
A4
A5
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Shh expression
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Production of an active Shh signal(N-Shhp)
N-Shhp
secretory pathway
Cholesterol
C
N
signal
catalytic
Shh
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Hedgehog Signaling
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Generating Knockout Mutant Mice
x
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Homologous recombination

• Studies in yeast, and several other simple
  organisms showed possible to mutate gene by
  homologous recombination

Gene A
X
X
NEO
NEO
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n
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Cre/Flp-mediated recombination
Gene X
CRE or FLP
LoxP or FRT sites
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Does Reducing N-Shhp Activity Lead
to Preferential Loss of Posterior Digits -
Morphogen Model?
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Reducing N-Shhp Levels (Signaling Activity) Leads
to Loss of the Most Posterior Digit
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Evidence for direct long-range action of Shh
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Notochord and floor plate induction of ventral
cell identities
D
p0
p1
p2
pMN
p3
FP
V
(Jessell and coworkers)
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Hedgehog Signaling
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Generation of chimeras to study requirement for
Smo activity
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Use of Chimeras to study direct requirement for
Shh signaling

• Mutation of Smo in some, but not all neural cells
• Cell autonomous block to Shh signaling
• Direct signaling cell fates lost
• Indirect signaling cell fates present

Smo -/-
wt
wt
wt
wt
V
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Smo is required forp3, pMN and p2 specification
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Notochord and floor plate induction of ventral
cell identities
D
p0
p1
p2
pMN
p3
FP
V
(Jessell and coworkers)
29
Notochord patterning and neural development
D
V
D
V
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Shh induces different ventral cell identities at
distinct concentration thresholds
D
Shh
p0
0.05
nM
p1
0.25
nM
p2
0.50
nM
pMN
2.00
nM
p3
3.00
nM
FP
4.00
nMgt
V
(Jessell and coworkers)
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Hedgehog Signaling
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Patched (Ptc) Hh receptor

• Negative regulator of Hh signaling pathway
• - ligand-independent inhibits Smo
• - ligand-dependent sequesters Hh
• Transcriptional target of Hh signaling
• Negative feedback regulation to limit the range
  of Hh signaling

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Hedgehog interacting protein (Hip1)

• Identified in screen for Shh binding proteins
• Membrane protein anchored by C-terminal
  hydrophobic domain
• Binds all mammalian Hedgehogs
• Binding affinity comparable to Ptc1 (low nM)
• Overexpression/loss-of-function studies
  antagonist of Hh signaling
• Transcriptional target of Hh signaling

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Mt-Ptc1 Ptc1-/-Hip1-/-
Mt-Ptc1 Ptc1-/-
Mt-Ptc1 Ptc1-/-Hip1/-
Hip1/-
Wt
HNF3b
Shh
Olig2 Nkx2.2
Pax6 Nkx6.1
Irx3 Dbx1
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Conclusions

• Shh long-range organizer.
• Distinct roles in different systems
• Response in target field dependent on
  concentration and feedback control

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FUTURE
gtRelease (Dispatched), movement (GFP),
transduction of signal (Gli-code derepression
and activation) gtTarget genes (microarrays,
bioinformatics)
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