Retinoic Acid Signaling Plays A Restrictive Role In Zebrafish Primitive Myelopoiesis

Being one of the best clinic treatment reagents of acute promyelocytic leukemia, retinoic acid (RA) is known to regulate normal myelopoiesis. In vitro study shows that RA promotes the differentiation of myeloid progenitor cells. Several in vivo models showed that RA is involved in the primitive erythropoiesis and definitive hematopoiesis. However, the role of RA in vertebrate primitive myelopoiesis remains unclear.To explore the role in the primitive myelopoiesis, we exposed zebrafish embryos to excessive RA. Our results showed that zebrafish primitive myelopoiesis is reduced by exogenous RA dose-dependently and also by knocking down endogenous cyp26s. Examining the formation of anterior hemangioblasts in the RA-treated embryos, we found that expressions of the anterior hemangioblast markers scl, lmo2, gata2 and etsrp are all significantly reduced. Consistently, investigation on the timing of RA action reveals that RA inhibits the primitive myelopoiesis before 1 lhpf when anterior hemangioblasts are initiated. Interestingly, the cardiac markers nkx2.5 and hand2 are ectopically expressed in the the rostral end of anterior lateral plate mesoderm (ALPM) of RA treated embryos though they exhibit greatly reduced expression in the caudal portion. The results suggest that the anterior hemangioblast can be transformed to cardiac progenitor cells under RA treatment.To determine the molecular mechanism of RA underlying zebrafish primitive myelopoiesis, we overexpressed hoxb5b, which is one of the RA target genes that can induce posteriorization of the ALPM, into zebrafish embryos. Our results showed that forced expression of hoxb5b can phenocopy the RA treatment in term of inhibiting zebrafish primitive myelopoiesis.However, our multiple evidences suggest that RA restrict the primitive myelopoiesis by playing a role in the regulatory network other than posteriorizing the ALPM. To determine the epistatic relationships between RA signaling and the genes controlling zebrafish primitive myelopoiesis, we first microinjected mRNAs of scl and lmo2 into the RA treated embryos, our results showed that the forced expression of scl and lmo2 can fully rescue the expression of anterior hemangioblast markers such as gata2 and etsrp and partially rescue primitive myelopoiesis in the RA treated embryos. The result suggest that RA signaling works upstream of scl and lmo2.When knocking down gata4/5/6, we found that the expression of raldh2, the gene that is responsible for RA synthesis, is significantly upregulated in the lateral plate mesoderm. However, treating the embryos with DEAB, ALDH1A inhibitor, successfully rescues the phenotype of gata4/5/6 morphants. The results suggest that RA works downstream of gata4/5/6.Examining the gene expression changes in cloche embryos that lack primitive myelopoiesis, we found that raldh2 is normally expressed whereas DEAB treatment fails to rescue the primitive myelopoiesis in either lycat morphants or cloche embryos. The results suggest RA acts upstream of, or parallel to, cloche to affect the primitive myelopoiesis.Taken together, our results demonstrate that RA signal restricts zebrafish primitive myelopoiesis mainly through acting downstream of gata4/5/6, upstream of scl/lmo2 and upstream of or parallel to cloche.