Abstract
Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.
Highlights
Vertebrate blood development takes place in two waves, referred to as primitive and definitive hematopoiesis
We find that Xenopus FOG (xFOG) and its interaction with the Nucleosome Remodeling and Deacetylase complex (NuRD) complex are required for primitive erythropoiesis, but that direct interaction with C-terminal binding protein (CtBP) is dispensable
Levels of xFOG-MYC protein translated from wild type RNA were reduced in the presence of the morpholino oligonucleotides (MOs), whereas there was no effect on translation of a rescue RNA containing silent mutations that prevent MO annealing (Figure 1B), demonstrating that the MO targets wild type xFOG but not the xFOGr rescue RNA
Summary
Vertebrate blood development takes place in two waves, referred to as primitive and definitive hematopoiesis. To begin to address the discrepancy between the current results and previous studies in which overexpressed mFOG-2 or a truncated xFOG were shown to repress RBC development, we first asked whether overexpression of full-length Xenopus FOG resulted in loss of RBCs. We injected RNA encoding wild type xFOG into the two ventral vegetal blastomeres of eight-cell embryos and analyzed expression of globin by Northern blotting (Figure 2A).
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