GDF 3 , a BMP inhibitor , regulates cell fate in stem cells and early embryos

INTRODUCTION To begin to understand the molecular character of totipotent cells and the mechanisms of the primary cell fate decisions of the early mammalian embryo, we previously performed a global analysis of all genes expressed in human embryonic stem (ES) cells when cultured under undifferentiated versus differentiated conditions (Sato et al., 2003). The examination of signaling components specific to the state of pluripotency revealed a very small number of ligands belonging to the main signaling pathways involved in early embryogenesis. Notably, only three secreted factors of the TGF superfamily are expressed at high levels during pluripotency and decline dramatically when the cells are allowed to differentiate, suggesting a role during this process: LEFTY1, LEFTY2 and GDF3 (Sato et al., 2003). Both LEFTY proteins have been previously shown to be secreted inhibitors of TGF ligands (reviewed by Branford and Yost, 2004). The role of GDF3, however, is unknown, as this TGF family member has not been as extensively studied as the others. In this study, we dissect the cellular and biochemical functions of GDF3 in the context of embryonic development and ES cells. GDF3 is a TGF superfamily member subclassified into the bone morphogenetic protein/growth and differentiation factor (BMP/GDF) branch of this family (Jones et al., 1992). GDF3 is most similar to VG1 (57% amino acid identity, 79% similarity) and has 53 and 50% identity to BMP2 and BMP4, respectively. GDF3 has only six of the classical seven cysteines present in other TGF superfamily members (Jones et al., 1992). It is missing the fourth cysteine – the one involved in inter-molecular interactions among TGF family members and with their secreted inhibitors (Groppe et al., 2002). GDF3 is a mammalian-specific TGF ligand as it has not been found in the genomes of fugu, frog, zebrafish or chick. It was first isolated from a mouse embryonic day (E) 6.5 cDNA embryonic library by homology to Xenopus VG1, although neither GDF3 mRNA expression nor GDF3 protein has ever been detected before mid-gestation mouse development (Jones et al., 1992). Later, GDF3 is expressed in embryonic bone and adult bone marrow, thymus, spleen and fat. It is also expressed in the pluripotent mouse teratocarcinoma cell line, F9, where its expression is reduced upon retinoic acid-induced differentiation (Jones et al., 1992; McPherron and Lee, 1993). Human GDF3 was identified through subtractive hybridization in activin-treated embryonic carcinoma (EC) cell lines (Caricasole et al., 1998). Although very little is known about GDF3, the roles of its other subfamily members, classic BMPs, have been well characterized. In the early Xenopus embryo, one of the earliest known functions of BMP signaling occurs in the late blastula and leads to the inhibition of neural fate in the dorsal ectoderm and induction of epidermal fate in ventral ectoderm (Munoz-Sanjuan and Brivanlou, 2002). In mammalian embryogenesis, the first in-vivo cell fate decision of the blastocyst is between inner cell mass and trophoblast fates. Recent evidence suggests that BMP signaling also plays a role in this crucial balance, as the addition of classic BMPs to human ES cells results in differentiation to trophoblast (Xu et al., 2002). Conversely, in mouse ES cells, BMPs have been shown to promote the maintenance of pluripotency, or ‘stemness’, in cooperation with LIF (Ying et al., 2003). To study the potential function of GDF3 in the undifferentiated, pluripotent state of ES cells, we characterized GDF3 expression throughout very early murine embryogenesis. We also show that GDF3 is a direct BMP inhibitor in early embryos and pluripotent cells. In addition to its embryological and biochemical characterization, we found that GDF3 acts in human ES cells to maintain markers of pluripotency, while, paradoxically, in mouse ES cells it acts to maintain the ability to differentiate in vitro into cell types representing all germ layers.