Download Vertebrate limbs From 2D to 3D 40 hrs. 60 hrs. 80 hrs. 100 hrs

Vertebrate limbs
Mouse forelimb
1
2
3
4
Anterior
Proximal
2
Dorsal
Posterior
Chicken forelimb
:Wing
4
3
Distal
Ventral
5
Vertebrate limb
patterning:
Retrospection
and
Introspection
From 2D to 3D
40 hrs.
60 hrs.
80 hrs.
100 hrs.
Limb bud in
3.5 days old
chick embryo
Day 3
Day 3.5
Day 3
Day 3.5
The lateral plate mesoderm and overlying ectoderm is specified to form the limb buds.
Day 3
The lateral plate mesoderm proliferates in the region where the limb buds develop.
Day 3.5
The body wall folds over to close ventrally.
Day 4
Vertebrate limbs
From 2D to 3D
40 hrs.
60 hrs.
80 hrs.
100 hrs.
Induction of limb bud
Specification of limb bud
Specification of limb bud
RE-Specification of limb bud
Nature. 1999 Apr 29;398(6730):814-8
Anterior
Dorsal
Posterior
Distal
Proximal
Ventral
D-V axis
Dorsal
Ventral
{ Wnt-7a
r-fng
Lateral plate
mesoderm
D
V
Lmx-1
Dorsal
AER
en-1
Lessons from D/V axis formation
• Ectoderm specifies mesoderm
• Ventral fate seems default
• AER positioning and Dorsalization of mesoderm
• Both dictated by en-1
• But are disconnected
{ Wnt-7a
r-fng
en-1
r-fng
Wnt-7a
Lmx-1
Dorsal
Lmx-1
AER
AER
formation
Dorsal
pattern
en-1
Apical Ectodermal Ridge
A signaling center
P/D axis and the AER
Time of
AER removal
Early
Intermediate
Late
Normal
Resultant wing
Progress zone model
Lewis Wolpert, 1973
Schematic of X-irradiation of stage 19-21 limb buds
No irradiation or low-dose
Intermediate dose
High dose
Cell death and decreased cell proliferation are significant
factors in phenotypes produced by AER removal.
AER removed
At stage 19
FGF at T0
FGF at T12
AER removed
At stage 23
FGF at T0
FGF at T12
Nature. 2002 Aug 1;418(6897):539-44, Dudley AT, Ros MA, Tabin CJ.
Descendents of cells in the early limb bud contribute to only
one proximodistal segment.
100-200 μM
200-300 μM
U
200-300 μM
100-200 μM
U
100-200 μM
Nature. 2002 Aug 1;418(6897):539-44, Dudley AT, Ros MA, Tabin CJ.
Progressive specification model
Dudley and Tabin, 2002
Progress zone Vs Progressive specification model
Genes Dev. 2007 21: 1433-1442
FGFs are the signals from AER
FGFs: Necessary and Sufficient for AER function
A/P axis and ZPA
Normal wing bud
Normal wing
2
4
ZPA
3
A/P axis and ZPA
Ectopic ZPA
transplanted wing bud
Ectopic ZPA
transplanted wing
3
4
2
2
4
Retinoic acid bead implantation can also achieve this
3
ZPA and Sonic Hedgehog
Shh is expressed at the ZPA
Expression of
Shh mRNA
Riddle and Tabin, 1993
ZPA and Sonic Hedgehog
Shh protein can substitute ZPA
Ectopic Shh bead
transplanted wing bud
Ectopic Shh bead
transplanted wing
Shh protein
soaked bead
3
4
2
2
4
3
Is Shh the real morphogen
Is BMP2 the real morphogen acting downstream of Shh?
Shh protein domain
Shh mRNA domain
BMP2 mRNA domain
BMP4 does not cause patterning defect
defect
Combined loss of BMP2 and BMP7
Albeit combined loss of BMP2 and BMP4 causes severe skeletal defect
Forelimb
WT
Hindlimb
Bmp2c/c, Bmp7-/-, Bmp2c/c, Bmp4c/c,
Prx1 cre
Prx1 cre
If not secondary morphogen then what?
Expansion-based temporal gradient of Shh
If not secondary morphogen then what?
Expansion-based temporal gradient of Shh
Supporting evidences :
1. Loss of Shh spares digit 1 => Shh independent
2. Delayed marking marks only digit 4 and 5
3. Non-diffusible Shh mutant only loses digits 2 and 3
4. Agrees with bead implantation studies
Digit identity –
Graded signal interpretation
Randall D. Dahn and John F. Fallon*
21 JULY 2000 VOL 438 289 SCIENCE
Posterior ID wins
Randall D. Dahn and John F. Fallon*
21 JULY 2000 VOL 438 289 SCIENCE
Stapled
Not stapled
Randall D. Dahn and John F. Fallon*
21 JULY 2000 VOL 438 289 SCIENCE
Inter-digital mesenchyme specifies DIGIT identity
Posterior ID prevails
Modulation of ID BMP levels causes homeotic digital transformation
BMP4 does not cause patterning defect
defect
Combined loss of BMP2 and BMP7
Albeit combined loss of BMP2 and BMP4 causes severe skeletal defect
Forelimb
WT
Hindlimb
Bmp2c/c, Bmp7-/-, Bmp2c/c, Bmp4c/c,
Prx1 cre
Prx1 cre
Competence Vs permissibility
Restriction of Shh expression domain
1. Retinoic acid can induce ectopic ZPA
2. Hoxb8 is expressed immediately following RA application
ZPA cells or even flank cells (prior to Shh expression) if
transplanted in the anterior margin can induce Shh. Retinoic
acid bead implanted in the anterior promote Shh expression and
ZPA activity.
Why should not Shh be expressed in the anterior margin?
Anterior necrotic zone OR Gli3 mediated repression?
RA ----- Hoxb8--- Shh
Gli3
Interaction between axes
Competence to express Shh
Retinoic acid bead implantation
Non-AER D/V border : Another signaling center
:: Regulates Tbx2 expression
Non-AER D/V border is required
to maintain Tbx2 expression
Non-AER D/V ectoderm can induce ectopic Tbx2
expression
Tbx2 is in red, anti-quail antibody QCPN
is in green - Non-AER D/V ectoderm
can induce ectopic Tbx2 expression
Non-AER D/V border : Another signaling center
:: Regulates Tbx2 expression => Upregulates Shh expression
Shh expression upon
non-AER D/V transplantation
- Restricted to the posterior
margin only
Why is Tbx2 and Shh excluded from under AER?
Rather Shh expression expands underneath the AER
Dorsal
ectoderm
Wnt-7a, r-fng
BMP
FGF
Ventral
ectoderm
en-1
Gremlin
Dorsal
ectoderm
Wnt-7a, r-fng
BMP
FGF
Ventral
ectoderm
en-1
Gremlin
Way forward?
• Reductionist approach
• Build mini circuits
• Construct predictive model
• Experimental validation and revisiting the model