Mezzo, a paired-like homeobox protein is an immediate target of Nodal signalling and regulates endoderm specification in zebrafish.

Endoderm specification in zebrafish is mediated by the zygotic transcription factors Bon/Mixer, Faust/Gata5, Casanova and Sox17, whose expression is induced by Nodal signalling. Bon/Mixer and Gata5 require Casanova in order to promote endoderm formation and all three factors act upstream of sox17, but it is not clear whether Casanova acts downstream of or in parallel to Bon/Mixer and Gata5. An additional factor induced at the margin of the blastoderm by Nodal signalling is thought to be required to induce casanova expression. We show that Mezzo, a novel paired-like homeobox protein, may be this missing transcription factor. The homeobox of Mezzo is mostly related to the homeodomain of the Mix-like and Mixer homeoproteins, but Mezzo is distinct from Bon/Mixer, the product of the bonnie and clyde gene. Like bon/mixer, mezzo is expressed transiently in mesendoderm precursors. By analysing the expression of mezzo in various mutants of Nodal signalling, we show that its expression strictly depends on a functional Nodal signalling pathway. By expressing a constitutively active Nodal receptor in the presence of translation inhibitors, we further demonstrate that mezzo, bonnie and clyde, and casanova are all immediate early targets of Nodal signalling, while sox17 requires post-MBT protein synthesis in order to be induced. Overexpression of mezzo mRNA can induce ectopic expression of casanova and sox17 and can also turn on the pan mesodermal marker gene ntl. We show that the function of mezzo is redundant with that of bonnie and clyde and that mezzo RNA can partially rescue bonnie and clyde mutants. Injection of antisense Morpholino oligonucleotides targeted against mezzo into bonnie and clyde mutant embryos abolishes all sox17 expression and aggravates their mutant phenotype. These results highlight the complexity of the transcriptional network operating during endoderm formation. They place mezzo as a new transcription factor with unique properties acting in parallel with bonnie and clyde, faust and casanova in the Nodal signalling pathway that controls specification of mesoderm and endoderm in zebrafish.