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    • br Experimental Procedures Drosophila stocks are described i

    2018-11-12


    Experimental Procedures Drosophila stocks are described in the Supplemental Experimental Procedures.
    Introduction Primitive hematopoietic progenitors arise from the yolk sac in mammals and generate red blood paf receptor (RBCs), thereby providing oxygen to the rapidly developing embryos (Orkin and Zon, 2008). In zebrafish, equivalent cells arise from the lateral plate mesoderm (LPM), where anteriorly located cells give rise to myeloid cells and posteriorly located cells produce mostly RBCs. The first hematopoietic progenitors appear bilaterally from the 2- to 3-somite stage and express the transcription factor (TF) genes fli1a, scl, and lmo2 (Liao et al., 1998; Thompson et al., 1998). By the 5-somite stage, posterior LPM cells are specified to the RBC lineage expressing gata1 (Davidson et al., 2003; Detrich et al., 1995). As embryos develop, these bilateral stripes merge, creating the intermediate cell mass (ICM) region (Detrich et al., 1995). By 24 hr postfertilization (hpf), the embryonic RBCs start circulating. Cdx4, a member of the caudal family, has been linked to embryonic hematopoiesis and leukemogenesis (Bansal et al., 2006; Davidson et al., 2003; Wang et al., 2005). The Cdx genes encode homeodomain-containing TFs that are known as master regulators of the Hox genes, and help establish the anterior-posterior (A-P) axis (Pownall et al., 1996; Subramanian et al., 1995). Mammals have three paralogs of the Cdx family (Cdx1, Cdx2, and Cdx4) that are expressed in the posterior tissues of the embryo (Young and Deschamps, 2009). Targeted knockout of Cdx genes demonstrated their roles in paraxial mesoderm, neurectoderm, and endoderm formation in mice (Chawengsaksophak et al., 2004; Gao et al., 2009; van den Akker et al., 2002; van Nes et al., 2006; Young et al., 2009). For example, Cdx2/4 compound knockout mice show a truncated axial skeleton, decreased presomitic mesoderm, and defective caudal hindgut endoderm and placenta, indicating that Cdx genes function redundantly in mesendodermal tissue formation (van Nes et al., 2006; Young et al., 2009). Zebrafish also have three cdx paralogs: cdx1a, cdx1b, and cdx4. cdx4 embryos display shortened tail and neurectoderm defects, which are enhanced when cdx1a is also knocked down (Davidson et al., 2003; Davidson and Zon, 2006; Shimizu et al., 2006). Zebrafish cdx4 embryos display a severe anemia, with decreased levels of scl, lmo2, and gata1 expression in the posterior LPM (Davidson et al., 2003). This function of Cdx4 in hematopoiesis is conserved in murine embryonic stem cells (ESCs), where Cdx4 overexpression increases erythroid, megakaryocyte, granulocyte, and macrophage lineage formation (Wang et al., 2005). Compound Cdx knockout in murine ESCs also leads to failures in hematopoietic differentiation (Wang et al., 2008). In addition to embryonic hematopoiesis, Cdx members are implicated in leukemogenesis, as shown by CDX2 translocations in acute myeloid leukemia (AML) patients, and leukemia seen in mice with Cdx4 overexpression in bone marrow (Bansal et al., 2006; Scholl et al., 2007). Many of the functions of Cdx have been linked to its ability to regulate Hox gene transcription (Young and Deschamps, 2009). However, an impact on Hox gene regulation does not explain all the defects in Cdx mutants. For example, conditional Cdx2 knockout mice show defects in endoderm and presomitic mesoderm formation independently of Hox genes (Gao et al., 2009; Savory et al., 2009a). In addition, overexpression of anterior hox genes does not rescue the hindbrain defects seen in cdx4/cdx1a zebrafish (Skromne et al., 2007). These results suggest that Cdx function cannot be explained solely by Hox genes, and there must be other critical downstream genes. Advances in chromatin immunoprecipitation sequencing (ChIP-seq) technology have helped investigators decipher complex transcriptional networks in many biological systems in a global manner. In ESCs, ChIP-seq experiments revealed that Oct4, Nanog, Sox2, and Sall4 share cis-regulatory modules and regulate each other to maintain pluripotency (Lim et al., 2008; Loh et al., 2006). Similar global studies were conducted in developing Drosophila and zebrafish embryos (Morley et al., 2009; Sandmann et al., 2006, 2007). Here, we used a genome-wide approach to identify direct Cdx4 target genes in zebrafish embryos by implementing ChIP-seq and microarray expression profiling. We show that the zinc-finger TF gene sall4 is a downstream target of Cdx4. ChIP-seq analysis of Sall4 similarly demonstrates that it binds to its own promoter and cdx4 regulatory elements. Gene-expression studies demonstrate that Cdx4 and Sall4 directly activate hox genes and hematopoietic genes. Cdx4 and Sall4 genetically interact during ventral mesoderm development, consistent with a role for these TFs in the early expression of hematopoietic TFs. Together, these results suggest that auto- and cross-regulatory interactions between cdx4 and sall4, as well as coactivation of common gene targets, establish a key regulatory module that directs the transition of the mesoderm into blood.