Retinoic Acid Blockade Increases Primitive Blood Cell Formation in cdx4 Mutant Zebrafish Embryos, Murine Yolk Sac Explants and Differentiated Embryonic Stem Cells.

Abstract Retinoic acid signaling is critical for proper development, as well as the regulation of hematopoiesis in murine and human cells. It has been reported that retinoic acid expands definitive hematopoietic stem cells and confers an advantage in transplantation of murine HSCs. We demonstrate here that all trans retinoic acid (ATRA) blocks early hematopoiesis in zebrafish embryos as measured by decreased expression of scl and gata1. This loss of gata1 expression after exposure to ATRA is reminiscent of the lack of gata1 expression in cdx4 mutant zebrafish embryos, prompting the hypothesis that retinoic acid and cdx4 share a common signaling pathway regulating the emergence of the hematopoietic system, and that inhibition of ATRA might rescue blood development in cdx4 mutants. We tested 4-diethylamino benzaldehyde (DEAB), an inhibitor of retinoic acid biosynthesis, and AGN193109, a pan-RAR antagonist, for their ability to rescue gata1 expression in cdx4 mutant embryos. We found that treatment with either DEAB or AGN193109 results in increased gata1 expression in the posterior mesoderm of both cdx4 mutants and wild type zebrafish embryos. This rescue of gata1 expression in the cdx4 mutants is observed only when the DEAB treatment occurs during late gastrulation. Mutant embryos exposed to DEAB only during early gastrulation or somitogenesis did not have increased gata1 expression. While this observation may reflect the complex activity of ATRA in both hematopoiesis and in mesodermal patterning, we conclude that it is also consistent with a switch to the stimulatory effect of retinoic acid on hematopoiesis observed by others in adult mice. Similarly, DEAB treatment increased primitive erythroid cells in murine hematopoietic cultures. We show that E7.5 murine yolk sac explant cultures exposed to DEAB have increased primitive erythroid colony forming cells (EryP-CFC) and definitive erythroid burst forming units (BFU-E). In contrast, ATRA markedly inhibits all erythroid colony formation in murine yolk sac explants. Likewise, murine embryonic stem cells treated with DEAB during embryoid body development have an 8-fold expansion of EryP-CFC, and a 2-fold expansion of multipotent GEMM colonies. Previously we published that overexpression of hoxA9 mRNA rescues gata1 expression in cdx4 mutant embryos. However, the hematopoietic defect in cdx4 mutant embryos is not rescued by overexpression of scl. To test the hypothesis that hoxA9 and scl are both signaling downstream of retinoic acid in the stimulation of primitive erythroid development, we overexpressed hoxA9 or scl by microinjecting mRNA into single-cell wild type zebrafish embryos followed by treatment with ATRA. We find that overexpression of either hoxA9 or scl partially rescues the block of hematopoiesis induced by ATRA. Taken together, these data indicate that the commitment of mesodermal cells to hematopoietic fates is inhibited by retinoic acid, and that the retinoic acid signal is acting downstream of cdx4 in the zebrafish embryo, while scl and hoxA9 are acting downstream of retinoic acid signaling.