Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo

The transcriptional repressors G fi 1(a) and G fi 1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, G fi 1 and G fi 1b restrict or promote cell proliferation, prevent apoptosis, in fl uence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebra fi sh, prRBCs express 2 of 3 zebra fi sh G fi 1/ 1bparalogs,G fi 1aaandG fi 1b.Therecentlyidenti fi edzebra fi sh gfi1aa gene trap allele qmc551 drives erythroid green fl uorescent protein (GFP) instead of G fi 1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of G fi 1b in gfi1aa qmc551 homozygous embryos. To study the transcriptome of the G fi 1aa/1b double-depleted cells, we performed an RNA-Seq experi- ment on GFP-positive prRBCs sorted from 20-h our-old embryos that were heterozygous or homozygous for gfi1aa qmc551 ,aswellas wt or morphant for gfi1b .Wesubsequentlycon fi rmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combi ned, the data showed that in the absence of G fi 1aa, the synchronously developing prRBCs were delayed in activating late erythr oid differentiation, as they struggled to suppress early erythroid and endothelial transcripti on programs. The latter highlighted the bipotent natu re of the progenitors from which prRBCs arise. In the absence of G fi 1aa, G fi 1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed G fi 1aa-depleted prRBCs even further, showing that G fi 1aa and G fi 1b together set the pace for prRBC diffe rentiation from hemangioblasts.