The controlling mechanism of directional limb outgrowth and limb patterning at cellular and molecular levels
This study focuses on two key questions in limb development: limb patterning and directional limb outgrowth. In the first part of this work, I studied the role of Msx2 gene in limb patterning. Msx2 cDNA was transferred into a mouse posterior limb bud (MPLB) cell line, MPLB2.1, which maintains ZPA signaling. Msx2 is shown to be able to increase the frequency and to improve the quality of supernumerary digit formation, probably by influencing the pre-existing ZPA signal in MPLB2.1 cells. Msx2 alters the surface properties of the cells, and Msx2-expressing MPLB-2.1 cells acquire an ability to recognize each other and to aggregate, suggesting that Msx2 might affect ZPA signaling via influencing cell-cell interaction. In the second part of this work, I studied the effects of the AER, FGF-2 and FGF-4 on the expression of Sonic hedeghog (Shh). FGF-2 and FGF-4 can maintain Shh expression after the AER removal. FGF-2 and FGF-4 also regulate the spatial expression of Shh domain in the presence of the AER. The alteration of Shh domain by FGFs is found to be due to the movements of Shh-expressing cells. Furthermore, FGF-2 and FGF-4 cause the reduction of the length of limb skeletal elements. FGF-2 also causes the loss of the digits. These results suggest that cell movements are important for normal limb formation. To further characterize how FGFs influence cell movements, limb bud cells are labeled with DiI. FGF-4 is shown to stimulate posterior cells within Shh domain to migrate, while FGF-2 does not induce a migratory response in a similar assay. In addition, anterior cells only weakly respond to FGF-4-stimulated cell migration. On the other hand, the AER can stimulate cells to migrate distally and FGF-4 can replace the AER in stimulating cell migration. Prevention of normal distalward cell migration causes the reduction of length of limb skeletal elements, indicating that cell migration stimulated by FGF-4 is involved in normal limb development. Taken together, I provided the first evidence showing the in vivo limb cell migration stimulated by FGF-4. I propose that both cell migration and cell proliferation are required for normal limb formation