MCDB 4650 Class 28
Patterning of the Limb,
continued
Answers to in class questions
Shh is expressed in the posterior region of each limb bud
Shh expression
16.18
Which family of
molecules is most
directly responsible
for the expression
of Shh only in the
posterior region of
each limb bud ?
a. Hox
b. FGF
c. BMP
d. Wnt
In a limb with duplicated digits (produced by an ectopic
ZPA), why are the extra digits produced in mirror image?
a. Shh diffuses toward the center,
so the higher concentration at
the center induces two Digit 2s
Original
Extra ZPA
ZPA
b. High Shh concentration induces
Digit 4, so there are Digit 4s
closest to each ZPA
c. The cells on either end are
prespecified to make digit 2, 3,
or 4
d. Digit 4 requires Shh; the other
digits form in the absence of
Shh.
Gain of function
mutation in Shh “Hemimelic Extra Toes”
Expression of Shh in
anterior and posterior
of limb leads to extra
digits developing
What kind of a mutation in
Shh would produce this
phenotype? A mutation in an:
a.  exon
b.  regulatory region
How were these mice made?
So what is Shh role in digit #?
Determines how many
condensation units will form in
lateral plate mesoderm to begin
making the bones of the digits
Remove interdigital spaces
2
anterior
3
4
1
posterior
B= Interdigital space (ID) 2 removed
C= ID 3 removed
What does this experiment tell you?
a. The interdigital space is not important for cell identity
b. The interdigital space to the posterior regulates a digit s identity
c. The interdigital space to the anterior regulates a digit s identity
NORMALLY, the posterior ID space is important. In its absence the digit
receives information from the anterior ID space.
Review of the progress zone/pre-pattern model of limb development
Children born with limb defects as a result of thalidomide exposure
Thalidomide: causes:
1. oxidative stress, leading to an upregulation of BMP signaling and an
increase in apoptosis
2. inhibit transcription of FGF
Discuss with your table the two models of proximal-distal limb development:
Which one could account for these abnormalities? Why?
• 
Thalidomide causes primarily a truncation of the limb (although sometimes limbs
are missing altogether, and sometimes there are digits abnormalities as well).
The most common phenotype is that the middle structures of the limb are
missing-- ie, there are distal elements fused to more proximal elements.The
most obvious explanation for the phenotype is that there is an overall shortening
of the limb, which is reasonable if FGF is inhibited, and if there is more cell
death due to BMPs. Because the middle structures are often more affected, one
could postulate that the middle element is more dependent on BMP and FGF
levels than the more distal or proximal elements.
• 
The evidence best supports the pre-pattern model, because distal structures are
not always completely lost. The only way you can still have distal structures if
you’re losing the proliferative capacity of the limb bud, is if that lack of
proliferation prevents the prepatterned limb from spreading out, thus each
section of the limb is still made, but is smaller than it should be. Concept Map of Limb patterning
Anterior/Posterior
Proximal/Distal
Digit identity
Interdigital mesenchyme
ZPA (zone of polarizing activity)
AER
Shh
FGF8
To add after we discuss last axis:
FGF10
Dorsal/Ventral
BMPs
Wnt 7a
En
Hox genes
Tbx genes
Sample limb concept map
Make a prediction:
How does Wnt7a interact with
the molecules that help pattern
the other axes (P-D, A-P)?
a. 
b. 
c. 
d. 
Wnt
Wnt
Wnt
Wnt
7a
7a
7a
7a
activates Shh expression, represses FGF expression
represses Shh expression, activates FGF expression
activates expression of Shh and FGF
represses expression of Shh and FGF