Characterization of the regulation of Msx2 expression during limb development: Studies using transgenic mice and polydactylous chicken mutants, and cultured chicken limb cells.

摘要

The chicken Msx2 gene is a homologue of the Drosophila msh gene. During embryonic development, Msx2 is dynamically expressed in regions of active epithelial-mesenchymml interactions, such as cranial neural crest, the mammary gland and limb bud. In the limb bud, Msx2 is expressed in both the apical ectodermal ridge (AER) and several regions of mesenchyme. The expression domains change as the limb develops. To characterize the regulation of Msx2 during limb development, three different approaches were used to analyze the nature of Msx2 regulation.; Sumoy et al. (1995), Wang (1997), and Pan et al. (2002), have identified different regions important for expression in the AER. In this thesis, I show that a 55bp fragment made by combining parts of two previously identified regulatory regions is capable of directing AER specific expression of a LacZ reporter in transgenic mice. There are two TAAT sites in this 55bp fragment. They are essential and interchangeable and the relative orientations of these two sites are important for enhancer activity.; I have showed that a recombinant micromass culture system can, at least in part, recapitulate the stage specific induction of Msx2 expression in the limb distal mesenchyme by the limb distal epithelium. By using this system, a 928bp fragment was identified which contains elements important for responding to the AER inductive signal(s).; In the chicken polydactylous mutants talpid2 and diplopodia5, Msx2 transcript is absent in the limb mesenchyme but expression in the AER is unaffected. I have demonstrated that the loss of Msx2 expression in the mutant limb mesenchyme in both talpid2 and diplopodia 5 is likely due to the presence of excessive inhibition of the Shh signaling pathway, caused by defects of in the intracellular components in the pathway. Furthermore, I have shown that Gli1 is mis-expressed in anterior mesenchyme of talpid2 limb and Gli3 is mis-expressed in the Zone of Polarizing Activity of diplopodia5 limbs. This is the first report of a difference between these two similar mutants at the molecular level.