br Materials and methods br Grant support Research

Materials and methods
Grant support Research was supported by intramural funding from the National Cancer Institute. > Acknowledgments
Introduction The mechanism which regulates the organization of the embryonic body plan was identified nearly a century ago by Spemann and Mangold, who showed that the dorsal lip of the amphibian gastrula induces the anterior–posterior (A–P) axis of the embryo (Spemann and Mangold, 2001). Accordingly, this group of rxr receptor was termed “the organizer”. Two of the major regulators of the amphibian organizer are Siamois and Twin (Laurent et al., 1997; Lemaire et al., 1995). These two highly similar proteins of the paired type homeodomain family have the ability to activate the entire organizer genetic network, as ventral injection of mRNA of either one of them induces a complete secondary axis (Engleka and Kessler, 2001). Conversely, simultaneous ablation of both genes causes severe A–P malformations (Bae et al., 2011; Ishibashi et al., 2008). Siamois and Twin directly regulate the organizer gene expression, as they were shown to bind the promoters of both Goosecoid (Bae et al., 2011) and Cerberus (Yamamoto et al., 2003) — two prominent organizer related genes. Surprisingly, no orthologue to any of the two was ever identified outside the amphibians. In mammals, studies performed primarily on mouse had shown that the gastrula organizer is divided between two centers: the anterior primitive streak (APS) on the posterior side of the embryo, and the anterior visceral endoderm (AVE) on its anterior (Kinder et al., 2001; Thomas and Beddington, 1996). Ectopic transplantation of the former into mouse or frog embryos induces secondary axial structures (Beddington, 1994; Blum et al., 1992). However, these fail to include the anterior most structures, suggesting the AVE has a crucial role in forebrain formation (Popperl et al., 1997). Molecularly, both structures are barely distinguishable, as their gene expression signature is nearly identical. Due to the scarcity in research material, very little is known about the establishment of the human body plan in general, and about the human gastrula organizer in particular. Human embryonic stem cells (hESCs) are pluripotent cells derived from the pre-implantation blastocyst (Thomson et al., 1998). When allowed to aggregate into embryoid bodies (EBs) they spontaneously differentiate into progenies of the three germ layers and thus mimic gastrulation in vitro (Dvash et al., 2004; Itskovitz-Eldor et al., 2000; Kopper et al., 2010). Recently, we isolated human gastrula organizer cells derived from hESCs. Through genetic labeling with eGFP fused to the GOOSECOID (GSC) promoter, we obtained a population of early differentiating hESCs that expressed GSC, the canonical organizer marker (Sharon et al., 2011). A whole transcriptome microarray analysis of the human organizer cells showed expression of numerous genes known to be organizer-related in model organisms, alongside with genes previously unknown to partake in this process. Upon transplantation into blastula stage Xenopus embryos, the GSC–GFP+ cells induced a partial A–P axis, thus establishing their organizer identity (Sharon et al., 2011). In this study, we focused on genes which were differentially expressed between the GSC–GFP+ and the GSC–GFP–cell populations. This brought to the identification of DUXO, a novel gene of the double homeobox family — a protein family whose members posses two homeodomains. DUXO is expressed in the human organizer cells, and both its homeodomains share high similarity with those of Siamois and Twin. Furthermore, like the two amphibian genes, DUXO regulates the expression of other organizer related genes, and binds the GSC promoter at a region conserved between frogs and man.
Results
Discussion Based on our previous isolation of cells equivalent to the human gastrula organizer from differentiated hESCs (Sharon et al., 2011), we now describe the identification of DUXO, a novel double homeobox protein. Our results indicate that DUXO fulfills several criteria that position it as a master regulator of the organizer: A. It is a nuclear protein specifically expressed in human organizer cells. B. Its expression may initiate organizer differentiation, as shown by GSC overexpression. C. DUXO knockdown results in the attenuation of the organizer cells and their mesendodermal progenies. D. It binds the promoter of GSC, a key transcription factor of the gastrula organizer. E. Its DNA binding domain is highly similar to that of Siamois and Twin, the two regulators of the frog\'s organizer.