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
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