Our ChIP seq data indicate that Cdx and Sall engage

Our ChIP-seq data indicate that Cdx4 and Sall4 engage in auto- and cross-regulatory loops. Such regulatory circuits are frequently seen during embryonic development. For example, during segmentation of rhombomere 4 in the mouse hindbrain, retinoic g-quadruplex signaling first turns on HoxB1, followed by HoxB1, HoxB2, and HoxA2, maintaining their expression through tight auto- and cross-regulatory loops (Agarwal et al., 2011; Gavalas et al., 2003; Tümpel et al., 2007). The Cdx4-Sall4 circuit during zebrafish development may function in an analogous way with upstream regulators such as Wnt, BMP, and FGF, activating expression of Cdx4 and Sall4, followed by the stabilization of their expression by auto- and cross-regulatory loops. We hypothesize that a stable Cdx4-Sall4 circuit is required to make the mesoderm competent for the specification of posterior tissue lineages such as blood by turning on the hematopoietic master TFs Scl and Lmo2 (Figure 6). Our current model also gives insights into leukemogenesis. Previously, Hox gene misregulation was the main focus of attempts to understand the mechanism behind Cdx genes causing leukemia (Bansal et al., 2006; Scholl et al., 2007). Our current model suggests another possibility, i.e., that Cdx genes directly regulate Scl and Lmo2. As mutation of both of these genes has been linked to various leukemias, such as T cell acute lymphoblastic leukemia (Bash et al., 1995; Boehm et al., 1991; Royer-Pokora et al., 1991), the model raises the possibility that these two genes may be aberrantly regulated in leukemias that are caused by Cdx or Sall4 mutation. In conclusion, our data establish a Cdx4-Sall4 circuit that acts in the posterior mesoderm to facilitate blood cell formation and axial elongation. We propose that during the early gastrula stage, Cdx4 and Sall4 are activated by upstream regulators such as Wnt, and their auto- and cross-regulation act to stabilize the mesoderm state. At the end of gastrulation, Cdx4 and Sall4 bind to scl and lmo2 to induce the hematopoietic program in the mesoderm. As upstream signaling dissipates, the Cdx4-Sall4 regulatory loops are disrupted, with a concomitant reduction in cdx4 and sall4 expression, and further blood differentiation is driven by factors such as Scl and Lmo2 (Figure 6). This model is likely applicable to the formation of other tissues and provides a molecular framework to understand how Cdx and Sall factors induce leukemia.
Experimental Procedures
Acknowledgments We thank members of the Zon laboratory for helpful discussions, J. Ganis for providing Tg(lcr:GFP), and M. Logan for providing the sall4 cDNA construct. We also thank the Whitehead Genome Technology Core for data production and analysis support. Microarray studies were performed by the Molecular Genetics Core Facility at Children’s Hospital Boston, supported by NIH grants P50-NS40828 and P30-HD18655. This work was supported by grants from the NHLBI (5R01HL048801-21 to L.I.Z. and 5P01HL32262-31), NIDDK (5P30 DK49126-19, DK53298-15, and R24 DK092760-02), and HHMI (to L.I.Z.). L.I.Z. is a founder and stockholder of Fate, Inc., and Scholar Rock, and a scientific advisor for Stemgent.
Introduction The ability to generate human oligodendrocyte precursor cells (OPCs) and oligodendrocytes in vitro, and thereby study the signals that promote OPC differentiation, maturation, and myelination, could provide new insights into human demyelinating diseases such as multiple sclerosis (MS), as well as other neurological disorders in which oligodendrocyte lineage cells play a key role, including periventricular multifocal leukoencephalopathy, multiple system atrophy, and malignant gliomas (Liu et al., 2011; Papp and Lantos, 1994; Mázló and Tariska, 1980). Human embryonic stem cells (hESCs), by virtue of their dual characteristics of self-renewal and pluripotency, have the greatest potential to provide the large numbers of these cells that are required for such studies. However, techniques that were developed in mouse ESC-based systems (Billon et al., 2002; Brüstle et al., 1999; Glaser et al., 2005) have not readily translated to human cells in culture. Few studies have reported successful specification of human OPCs from hESCs (Nistor et al., 2005; Kang et al., 2007; Izrael et al., 2007; Hu et al., 2009; Sundberg et al., 2010; Wang et al., 2013), and still fewer have convincingly shown in vitro generation of mature human oligodendrocytes (and then only in small numbers; Izrael et al., 2007; Hu et al., 2009; Wang et al., 2013).