Functional analysis of Pcl2 in vertebrate left-right asymmetry
Description
Left-right (L-R) asymmetry is a vital feature of vertebrate embryogenesis. A failure in the normal development of L-R axis is often associated with laterality defects. The genetic pathways regulating L-R asymmetry are beginning to be elucidated. In an effort to identify differentially expressed genes in L-R axis development, a chick Polycomblike 2 ( cPcl2) gene was identified. cPcl2 resides genetically downstream from Activin-betaB and Bmp4, and displays asymmetric expression pattern in the right side of Hensen's node, complementary to Sonic hedgehog (Shh). Moreover cPcl2 encodes a transcription repressor with the repression domain mapped to the PHD domain. Repression of cPcl2 expression in early chick embryos results in randomized heart looping direction, which is accompanied by the ectopic expression of Shh in the right side of the node and Shh downstream genes in the right lateral plate mesoderm (LPM), while overexpression of cPcl2 represses Shh expression in the node. The repression of Shh by cPcl2 was also supported by overexpression studies in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of cPcl2 in the developing mouse limb inhibits Shh expression in the ZPA. The mechanism of Shh repression by cPcl2 was studied further. cPcl2 was found to repress Shh promoter, but not TK directed reporter activity in vitro. In vitro pull-down assays demonstrated a direct interaction of the cPcl2 PHD finger with EZH2, a component of the EED/EZH2 repressive complex. Taken together, these data suggest cPcl2 might play a critical role in regulating L-R axis patterning in the chick by silencing Shh in the right side of the node To study the function of Pcl2 during L-R axis development in mice, the mPcl2 mutant mice were generated. The heart looping direction and the position of other internal organs in the mPcl2 mutant mice were normal, indicating that mPcl2 seems to be dispensable for L-R axis development in mouse. These results are consistent with the idea that the mechanism for initial symmetry breaking might be divergent among vertebrate species