Whole mount in situ hybridization analysis of tbx5a expression in wildtype, cdx4 and cdx1a/4-deficient embryos shows expansion of cardiogenic anterior-lateral plate mesoderm. The intermediate mesoderm field, marked by pax2a expression, shows a posterior shift and reduction in cell number, as reported previously. The effect was more pronounced in cdx1/4 double deficient embryos than in cdx4-/- embryos. Shown are zebrafish embryos at 15-somite stage. Purple staining was used for tbx5 and pax2a and red staining for krox20 and myoD as landmarks of other tissues.

Excessive tbx5 positive anterior-lateral plate mesoderm formed in cdx-deficient embryos requires additional inhibition of the retinoic acid pathway in order to adopt cardiac fate and form nkx2.5 expressing cardiac cells. Shown are wildtype, cdx4-/- and cdx1/4 deficient embryos analyzed at 15-somite stage after treatment with the retinoic acid inhibitors DEAB, Ro-41-5253, or DMSO (vehicle) as a control. cdx-dose-dependent effects are observed with both inhibitors.

Suppression of RA levels expands cardiac myosin expressing-cells (cmlc2) in cdx-deficient embryos, and can be rescued by exogenous RA treatment. Expression of cmlc2 expression in wildtype, cdx4 and cdx1a/4-deficient embryos incubated with DMSO (control), DEAB, or DEAB and RA, was assayed by whole mount in situ hybridization at the 16 somite stage. cdx mutants evinced dramatic expansion of cardiac mesoderm when RA production was abrogated, and this phenotype was rescued by concomitant DEAB and RA treatment.