Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development

James C. Cross 0 4 Margaret L. Flannery 4 Michael A. Blanar 2 3 Eirikur Steingrimsson 1 Nancy A. Jenkins 1 Neal G. Copeland 1 William J. Rutter 3 Zena Werb 4 0 Present address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue , Toronto, Ontario M5G 1X5 , Canada 1 Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center , Frederick, Maryland 21702 , USA 2 Present address: Department of Cardiovascular Molecular and Cellular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute , Route 206 and Provinceline Road, Princeton NJ 08543-4000 , USA 3 Hormone Research Institute, University of California , San Francisco, California 94143 , USA 4 Laboratory of Radiobiology and Environmental Health, Department of Anatomy, and Program in Developmental Biology - Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development SUMMARY Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed The first cell lineages to be established in the mammalian embryo contribute only to extraembryonic structures that form the placenta (see Cross et al., 1994). It is only after implantation that significant differentiation occurs in the embryonic ectoderm, cells that give rise to all the structures of the embryo proper. The placenta is formed from three extraembryonic lineages: trophoblasts that lie adjacent to maternal cells in the implantation site, endoderm that migrates across the inner surface of the trophoblast layer and mesoderm that gives rise to placental blood vessels. The development of these extraembryonic lineages has largely been ignored in the molecular analysis of development. Nonetheless, abnormalities in trophoblast development or in formation of the placenta account for the most common gestational abnormalities in humans and animals, including failures of implantation, intrauterine growth retardation and stillbirth. Human fecundity is only around 25%, owing partially to fertilization failure but more significantly to failures in early development. For example, even after careful selection of embryos, the success of embryo transfer in humans is only 20-25% (Wilcox et al., 1993) and approximately one quarter of human embryos that begin to implant die before the pregnancy is recognized clinically (Wilcox et al., 1988). In by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells. farm animals, failures in development at implantation account for almost 80% of the embryonic loss that occurs throughout the entire length of gestation (Roberts et al., 1990). Factors that regulate the differentiation of placental cell lineages are largely unknown. The POU-domain transcription factor Oct-4 is expressed in undifferentiated cells during cleavage stage development and is downregulated as cells differentiate into trophoblast and endoderm (Palmieri et al., 1994). Although Oct-4 may play a direct role in preventing differentiation into the trophoblast lineage, other factors must play a positive role. Pem is a homeodomain transcription factor that is expressed in trophoblast and yolk sac in the mouse placenta (Wilkinson et al., 1990), but its expression peaks after implantation. GATA-3 is a zinc finger transcription factor that is expressed in the placenta and in trophoblast cell lines which may regulate placental lactogen-1 transcription (Ng et al., 1994). However, GATA-3 is also expressed in other cell types (Ko et al., 1991). Genetic evidence indicates that basic-helix-loop-helix (bHLH) transcription factors function as cell-lineage determinants in skeletal muscle development in mammals (MyoD, myogenin, mrf-4, myf-5) and in mesoderm and neuronal cell differentiation in Drosophila (achaete-scute) (Olson, 1990, 1992; Jan and Jan, 1993). Cell-specific bHLH factors have also been identified in other mammalian cell types, where it is likely they function as regulators of lineage commitment and differentiation. This suggested to us that such factors might also regulate the trophoblast cell lineage. Members of the bHLH transcription factor family function as heterodimers, typically between cell-specific factors and the widely expressed E factors, such as E12 and E47, which are products of the E2A gene (Murre et al., 1991), HEB (Hu et al., 1992), and ITF2 (Henthorn et al., 1990). To take advantage of the ability of cellspecific factors to heterodimerize with E factors, we used the HLH domain of E47 as protein probe to identify two novel bHLH factors, by means of the so-called interaction cloning procedure (Blanar and Rutter, 1992). One of these factors, Hxt, is specifically expressed in trophoblast cells. We present evidence that Hxt regulates trophoblast differentiation and suggest a model for the role of bHLH factors in trophoblast development. MATERIALS AND METHODS Interaction cloning and sequencing of Hxt and Hed Phage expression libraries prepared from day 13 ovine conceptuses (Kramer et al., 1994) or differentiated mouse embryoid bodies (Robbins et al., 1990) were screened by interaction cloning (Blanar and Rutter, 1992). A detailed description of the mutant shPan-1 (E47; German et al., 1991) protein probe is provided elsewhere (Blanar et al., 1995). Briefly, the bHLH domain of E47 was cloned into an E. coli expression vector downstream of a sequence encoding the recognition sequence for cAMP-dependent protein kinase (heart muscle kinase). Basic residues in the bHLH domain were mutated to block its ability to bind DNA. This protein was labeled with heart muscle kinase (Sigma) and g -[32P]ATP. The original Hxt isolate from the ovine conceptus library (l 8.1) did not contain a full-length Hxt cDNA. A 5 end fragment was used to probe the original library by using standard procedures (Sambrook et al., 1989). cDNAs cloned into pBluescript (Stratagene) were sequenced by using the dideoxy chain termination method. A cytomegalovirus promoter-Hxt expression vector (pCMVHxt) was prepared by ligating an XhoI-EcoRI fragment of the ovine Hxt cDNA into pcD (...truncated)