Abstract
The TGF-beta-related molecule Nodal plays an essential and conserved role in left-right patterning of the vertebrate embryo. Previous reports have shown that the zebrafish and mouse Cerberus-related proteins Charon and Cerberus-like-2 (Cerl-2), respectively, act in the node region to prevent the Nodal signal from crossing to the right side, whereas chick Cerberus (cCer) has an unclear function in the left-side mesoderm. In this study, we investigate the transcriptional regulation and function of cCer in left-right development. By analyzing the enhancer activity of cCer 5' genomic sequences in electroporated chick embryos, we identified a cCer left-side enhancer that contains two FoxH1 and one SMAD binding site. We show that these Nodal-responsive elements are necessary and sufficient for the activation of transcription in the left-side mesoderm. In transgenic mouse embryos, cCer regulatory sequences behave as in chick embryos, suggesting that the cis-regulatory sequences of Cerberus-related genes have diverged during vertebrate evolution. Moreover, our findings from cCer overexpression and knockdown experiments indicate that cCer is a negative-feedback regulator of Nodal asymmetric signaling. We propose that cCer and mouse Cerl-2 have evolved distinct regulatory mechanisms but retained a conserved function in left-right development, which is to restrict Nodal activity to the left side of the embryo.
Highlights
Chick Cerberus is a member of the Cerberus-Dan family of cysteine-knot-secreted proteins (Rodriguez Esteban et al, 1999; Yokouchi et al, 1999; Zhu et al, 1999)
Our present findings demonstrate that Chick Cerberus (cCer) asymmetric expression is directly activated by Nodal signaling and suggest that the cisregulatory sequences of Cerberus-related genes have diverged among vertebrates
Beads soaked in recombinant mouse Nodal protein were implanted on the right side of HH6 chick embryos and cCer expression was examined by whole-mount in situ hybridization
Summary
Chick Cerberus (cCer; known as Caronte) is a member of the Cerberus-Dan family of cysteine-knot-secreted proteins (Rodriguez Esteban et al, 1999; Yokouchi et al, 1999; Zhu et al, 1999). At later stages, Cerberus-related genes have very distinct patterns: XCer expression is no longer detected, mouse Cerl-1 transcripts are found in nascent somites and presomitic mesoderm, zebrafish charon and mouse Cerl-2 are expressed around the node region (Cerl-2 expression levels are higher on the right side), and chick Cer is expressed in the left paraxial and lateral plate mesoderm (Bouwmeester et al, 1996; Belo et al, 1997; Rodriguez Esteban et al, 1999; Marques et al, 2004; Hashimoto et al, 2004). The understanding of how these different patterns of expression are generated may bring some insights into the evolution of Cerberus-related genes and their functions in the different vertebrate species
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