Summary Chick Pcl 2 regulates the left-right asymmetry by repressing Shh expression in Hensen ’ s node

The left-right (LR) asymmetry of an embryo, which is characterized by asymmetric structures and/or asymmetric placement of internal organs, is a vital feature of vertebrate embryogenesis. The rightward looping of the developing heart represents the first morphological indication of LR asymmetry. A failure in the normal development of the LR axis is associated with laterality defects, including isomerism, heterotaxia and situs inversus. Recent studies demonstrate that the initial positional information that specifies the LR axis originates in/around the node, a disc-shaped structure at the anterior end of the primitive streak in developing chick and mouse embryos. In the chick, the asymmetric expression of Activin-βB and ActRIIA in the right side of Hensen’s node induces the expression of Bmp4, via chick Mid1, which then induces right-sided Fgf8 expression (Levin et al., 1995; Levin et al., 1997; Monsoro-Burg and Le Douarin, 2001; Granata and Quaderi, 2003). The asymmetrically expressed BMP4 inhibits Shhexpression in the right side of the node, thereby restricting Shhexpression to the left side of the node (MonsoroBurg and Le Douarin, 2001). This asymmetric pattern of gene expression in the node is translated into asymmetric gene expression in the lateral plate mesoderm (LPM), which further regulates the LR axis development. The asymmetrically expressed Shh is responsible for the asymmetric expression of Nodal in the left LPM. Caronte (Car), a member of Cerberus/Dangene family, was shown to mediate Nodal induction in the left LPM by antagonizing symmetrically expressed BMP activity (Rodriguez-Esteban et al., 1999; Yokouchi et al., 1999; Zhu et al., 1999). However, it has recently been demonstrated that BMPs are also involved in the activation of Nodalin the left LPM through the activation of CFC, a member of the EGF-CFC family, and ActRIIA (Schlange et al., 2001; Schlange et al., 2002; Piedra and Ros, 2002). Meanwhile, Nodal expression around the node is also independently regulated by the Wnt/β-catenin signaling pathway, a protein kinase A-dependent pathway and Notch signaling pathway (Garcia-Castro et al., 2000; Kawakami and Nakanishi, 2001; Rodriguez-Esteban et al., 2001; Raya et al., 2004). Studies in other vertebrate species, including the mouse and Xenopus, indicate that the asymmetrical expression of Nodalin the left LPM is conserved across species and is crucial for establishing initial LR asymmetry (Collignon et al., 1996; Hyatt et al., 1996; Lowe et al., 1996; Sampath et al., 1997). Downstream from this left side signaling cascade reside Nkx3.2 and Pitx2 (Campione et al., 1999; Logan and Tabin, 1998; Piedra et al., 1998; Ryan et al., 1998; St Amand et al., 1998; Asymmetric expression of sonic hedgehog (Shh) in the left side of Hensen’s node, a crucial step for specifying the leftright (LR) axis in the chick embryo, is established by the repression of Shhexpression 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 ActivinβB 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 chickPcl2 expression in the early embryo results in randomized heart looping direction, which is accompanied by the ectopic expression of Shhin the right side of the node and Shh downstream genes in the right lateral plate mesoderm (LPM), while overexpression of chickPcl2 represses Shh expression in the node. The repression of Shh by chick Pcl2 was also supported by studies in which chickPcl2 was overexpressed in the developing chick limb bud and feather bud. Similarly, transgenic overexpression of chickPcl2 in the developing mouse limb inhibits Shhexpression 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 chickPcl2 was found to directly repress Shh promoter activity in vitro, our results demonstrate a crucial role for chickPcl2 in regulating LR axis patterning in the chick by silencing Shh in the right side of the node.