Chick Pcl2 regulates the left-right asymmetry by repressing Shh expression in Hensen's node

Asymmetric expression of sonic hedgehog (Shh) in the left side of Hensen's node, a crucial step for specifying the left-right (LR) axis in the chick embryo, is established by the repression of Shh expression in the right side of the node. The transcriptional regulator that mediates this repression has not been identified. We report the isolation and characterization of a novel chick Polycomblike 2 gene, chick Pcl2, which encodes a transcription repressor and displays an asymmetric expression, downstream from Activin-betaB and Bmp4, in the right side of Hensen's node in the developing embryo. In vitro mapping studies define the transcription repression activity to the PHD finger domain of the chick Pcl2 protein. Repression of chick Pcl2 expression in the early embryo 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 chick Pcl2 represses Shh expression in the node. The repression of Shh by chick Pcl2 was also supported by studies in which chick Pcl2 was overexpressed in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of chick Pcl2 in the developing mouse limb inhibits Shh expression in the ZPA. In vitro pull-down assays demonstrated a direct interaction of the chick Pcl2 PHD finger with EZH2, a component of the ESC/E(Z) repressive complex. Taken together with the fact that chick Pcl2 was found to directly repress Shh promoter activity in vitro, our results demonstrate a crucial role for chick Pcl2 in regulating LR axis patterning in the chick by silencing Shh in the right side of the node.