Identification of a crystal cell-specific enhancer of the black cells prophenoloxidase gene in Drosophila

In Drosophila, Black cells (Bc) encodes a Prophenoloxidase and is expressed late in the maturation of crystal cells, which are blood cells involved in wound healing and immune encapsulation. Enhancer analysis of Bc revealed a 1,025-bp upstream sequence that regulates gene expression in a crystal cell exclusive pattern. Expression of this fragment is altered by mutations in the GATA family serpent (srp) and RUNX family lozenge (lz) genes; Srp and Lz are required for crystal cell specification. Deletional analysis uncovered a 330-bp crystal cell-specific sequence, which contains two GATA and three Lz binding sites. Mutational analysis revealed that both GATA sites are necessary, but not sufficient for crystal cell expression. However, one of the Lz sites is essential for crystal cell expression. Thus, Srp and Lz do not just specify the crystal cell lineage, but also regulate the later differentiation of these cells. Additionally, we now have a sensitive tool for marking crystal cells in live animals.     

Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.     

Functional conservation of hematopoietic factors in Drosophila and vertebrates

The GATA, Friend of GATA, and Runt homology domain protein families function during hematopoiesis to promote progenitor cell development and regulate lineage commitment and differentiation. The hematopoietic functions of these factors have been remarkably conserved across taxonomic groups, ranging from flies to humans. Furthermore, aspects of hematopoiesis and hemocyte function appear to be conserved. Thus, comparative studies using Drosophila and vertebrate models should enhance our understanding of blood cell development.     

JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila

The lymph gland is the major site of hematopoiesis in Drosophila. During late larval stages three types of hemocytes are produced, plasmatocytes, crystal cells, and lamellocytes, and their differentiation is tightly controlled by conserved factors and signaling pathways. JAK/STAT is one of these pathways which have essential roles in vertebrate and fly hematopoiesis. We show that Stat has opposing cell-autonomous and non-autonomous functions in hemocyte differentiation. Using a clonal approach we established that loss of Stat in a set of prohemocytes in the cortical zone induces plasmatocyte maturation in adjacent hemocytes. Hemocytes lacking Stat fail to differentiate into plasmatocytes, indicating that Stat positively and cell-autonomously controls plasmatocyte differentiation. We also identified the GATA factor pannier (pnr) as a downstream target of Stat. By analyzing the phenotypes resulting from clonal loss and over-expression of pnr in lymph glands, we find that Pnr is positively regulated by Stat and specifically required for the differentiation of plasmatocytes. Stat and Pnr represent two essential factors controlling blood cell maturation in the developing lymph gland and exert their functions both in a cell-autonomous and non-cell-autonomous manner.     

Friend of GATA (FOG) interacts with the nucleosome remodeling and deacetylase complex (NuRD) to support primitive erythropoiesis in Xenopus laevis

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.     

Potential roles for RUNX1 and its orthologs in determining hematopoietic cell fate

Runx1 (also known as AML1, Cbfa2 and Pebpa2b) and Cbfb encode a DNA-binding alpha subunit and the non-DNA-binding beta subunit of a mammalian core-binding factor (CBF). The discovery of RUNX1 and CBFB as genes rearranged in human leukemias prompted predictions that both genes would play important roles in normal hematopoiesis. These predictions were borne out, as indeed Runx1 and its Xenopus and Drosophila homologs, Xaml and lozenge (lz), appear to determine hematopoietic cell fate during development. We will review what is known about Runx1 function in hematopoiesis in three model organisms, mouse, frog and fly, focusing on the earliest events of hematopoietic cell emergence in the embryo.