Proposed model for the regulation of blood development by cdx4 as mediated by RA and hoxa9a. (A). cdx4 directly modulates expression of numerous hox genes, including hoxa9a. cdx4 also inhibits expression of raldh2, thereby determining the extent of the RA gradient in the developing posterior mesoderm. As a result, cdx4 regulates 3 aspects of primitive blood development that are mediated by RA and/or hoxa9a: (1) specification of competent mesoderm, mediated by hoxa9a; (2) regulating the onset of the blood program, mediated by both hoxa9a and RA; and (3) setting the anterior edge of the hematopoietic stripe in the posterior mesoderm, mediated by RA. It is probable that other hox genes regulated by cdx4 have similar function, although here we report results with hoxa9a only. (B). Schematic representation of gata1⁺ cells in the posterior mesoderm of flat-mounted zebrafish embryos at the 10 ss. Red boxes indicate expression of gata1 in the lateral plate mesoderm flanking the paraxial mesoderm of each embryo. Solid filled boxes represent strong expression, and checked boxes represent relatively sparse expression. Numbers indicate the somite pair adjacent to the most anterior gata1⁺ cells. scl overexpression rescues gata1⁺ cells after exogenous RA treatment but not in cdx4 mutants. This can be explained by taking into account the notion that posterior hox genes may act as “competence factors” for blood cell differentiation. In cdx4 mutants, the posterior hox gene expression domains are decreased and shifted posteriorly so that even in the presence of exogenous scl, the mesoderm is unable to activate the blood program. In RA-treated embryos, the cdx-hox pathway is intact, and the posterior mesoderm remains competent to respond to scl and form blood, albeit with the anterior margin of the gata1⁺ cells still shifted posteriorly. Likewise, hoxa9a overexpression rescues blood development in cdx4^−/− embryos, but not RA-treated embryos. Normally expressed throughout the posterior mesoderm, hoxa9a expression in cdx4^−/− embryos is isolated in the very tip of the tailbud.¹⁶  Overexpression of hoxa9a in cdx4^−/− embryos adequately corrects the competency of the posterior mesoderm so that gata1⁺ cells are expanded and appear very similar to wild-type embryos. In contrast, expression of hoxa9a is not decreased after RA treatment. Therefore the cdx4-hox pathway remains relatively intact in RA-treated embryos, so increased hoxa9a expression would not be expected to have a significant effect on erythropoiesis.