Stem Cell Reports Ar ticle Targeted Disruption of TCF12 Reveals HEB as Essential in Human Mesodermal Specification and Hematopoiesis Yang Li, 1,2,3 Patrick M. Brauer, 1,3 Jastaranpreet Singh, 1 Sintia Xhiku, 1 Kogulan Yoganathan, 1 Juan Carlos Zu ´n ˜iga-Pflu ¨cker, 1,4, * and Michele K. Anderson 1,4, * 1 Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada 2 Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, Peking University, Beijing, 100191, China 3 Co-first author 4 Co-senior author *Correspondence: jczp@sri.utoronto.ca (J.C.Z.-P.), manderso@sri.utoronto.ca (M.K.A.) http://dx.doi.org/10.1016/j.stemcr.2017.07.011 SUMMARY Hematopoietic stem cells arise from mesoderm-derived hemogenic endothelium (HE) during embryogenesis in a process termed endo- thelial-hematopoietic transition (EHT). To better understand the gene networks that control this process, we investigated the role of the transcription factor HEB (TCF12) by disrupting the TCF12 gene locus in human embryonic stem cells (hESCs) and inducing them to differentiate toward hematopoietic outcomes. HEB-deficient hESCs retained key features of pluripotency, including expression of SOX2 and SSEA-4 and teratoma formation, while NANOG expression was reduced. Differentiation of HEB / hESCs toward hematopoi- etic fates revealed a severe defect in mesodermal development accompanied by decreased expression of regulators of mesoendodermal fate choices. We also identified independent defects in HE formation at the molecular and cellular levels, as well as a failure of T cell devel- opment. All defects were largely rescued by re-expression of HEB. Taken together, our results identify HEB as a critical regulator of human mesodermal and hematopoietic specification. INTRODUCTION During embryogenesis, multiple waves of mesodermal pro- genitors generate hematopoietic cells with different char- acteristics. Two early waves of hematopoietic progenitors give rise to primitive progenitors, followed by distinct myeloid and erythroid populations known as EMPs (Ditadi et al., 2017). At a later time point, hematopoietic stem cells (HSCs), which are distinguished by their self-renewing ca- pacity, arise from mesodermal precursors with endothelial characteristics. This process, termed the endothelial-to-he- matopoietic transition (EHT), involves a transient develop- mental intermediate known as hemogenic endothelium (HE) (Gritz and Hirschi, 2016). Early mesodermal specifica- tion from embryonic stem cells (ESCs) and generation of HE are both dependent on signaling through transforming growth factor b (TGFb) family members (Hadjimichael et al., 2016; Hong et al., 2011; Monteiro et al., 2016). As mesodermal precursors differentiate toward the hemato- poietic lineage, the transcription factors Runt-related tran- scription factor 1 (Runx1) and Notch1 are upregulated, and these factors are essential for HE generation in an evolu- tionarily conserved manner (Burns et al., 2005; Butko et al., 2016; Ditadi et al., 2015). Without Runx1, HE does not form (Yzaguirre et al., 2017). Furthermore, Runx1 and its target Spi1 (Huang et al., 2008) are among four factors that can reprogram adult endothelial cells into HSCs with long-term engrafting and lymphoid potential (Lis et al., 2017). The expression of a specific isoform of Runx1 also marks HE as distinct from arterial vascular endothelium in human ESC (hESC)-derived progenitors (Ditadi et al., 2015). Notch1 is also a key regulator of HE. Notch1 directly up- regulates Runx1 and controls the HSC-associated factor GATA3 (Burns et al., 2005; Butko et al., 2016; Ditadi et al., 2015; Frelin et al., 2013). Consequently, the generation of HE and the process of EHT are severely compromised in the absence of Notch signaling (Butko et al., 2016). The transcription factor HEB operates in the context of Notch1 and Runx1 during T cell development (Braunstein and An- derson, 2012), and has been shown to act cooperatively with the SMAD factors, downstream of TGFb family signaling, in mouse ESCs (mESCs) (Yoon et al., 2015). Furthermore, HEB and Notch1 operate in a positive feed- back loop during early T cell development (Braunstein and Anderson, 2012). In addition, HEB has been implicated in mesodermal development from mESCs (Yoon et al., 2015), potentially placing it upstream of HE formation. HEB belongs to the E protein transcription factor family, which also includes E2A (TCF3) and E2-2 (TCF4)(Murre et al., 1989). E proteins regulate transcription of their target genes as obligate dimers, dimerizing with each other or with class II basic helix-loop-helix factors. HEB factors are the products of the TCF12 gene locus, which encodes both the canonical HEB protein (HEBCan) and a shorter variant (HEBAlt) by way of alternative transcriptional initi- ation and alternative splicing (Hu et al., 1992; Wang et al., 2006). HEB is important in various developmental pro- cesses, including T-lymphopoiesis, neurogenesis, and myo- genesis (Barndt et al., 1999; Parker et al., 2006; Ravanpay Stem Cell Reports j Vol. 9 j 779–795 j September 12, 2017 j ª 2017 The Authors. 779 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).