© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4533-4543 doi:10.1242/dev.092866 4533 ABSTRACT Ectoderm is one of the three classic germ layers in the early mouse embryo, with the capacity to develop into both the central nervous system and epidermis. Because it is a transient phase of development with few molecular markers, the early ectoderm is the least understood germ layer in mouse embryonic development. In this work, we studied the differentiation potential of isolated ectoderm tissue in response to BMP signaling at various developmental stages (E6.5, E7.0 and E7.5), and identified a transient region in the anterior- proximal side of the embryo at E7.0 that possesses the ability to become neural or epidermal ectoderm in response to the absence or presence of BMP4, respectively. Furthermore, we demonstrated that inhibition of Nodal signaling could direct the pluripotent E6.5 epiblast cells towards ectoderm lineages during differentiation in explants in vitro. Our work not only improves our understanding of ectodermal layer development in early embryos, but also provides a framework for regenerative differentiation towards ectodermal tissues. KEY WORDS: Ectoderm, BMP4, Epidermis, Nodal INTRODUCTION During early vertebrate development, initially pluripotent cells become progressively restricted in their developmental choices. Central to this transition is the process of gastrulation, during which the epiblast develops into the three primary germ layers (Tam and Loebel, 2007). In mouse embryos, the epiblast at embryonic day (E) 5.5 is pluripotent, and epiblast stem cells (EpiSCs) can be derived from this stage of development (Brons et al., 2007; Tesar et al., 2007). At E6.5, gastrulation is initiated with the formation of the primitive streak on the posterior side of the embryo. Epiblast cells that ingress through the primitive streak form the mesoderm and the endoderm. The cells that do not pass through the primitive streak and remain on the anterior side of the epiblast form the ectoderm (Lu et al., 2001; Tam and Loebel, 2007). RESEARCH ARTICLE STEM CELLS AND REGENERATION 1 Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, ON M5G 1X8, Canada. 2 State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China. 3 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. *Present address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. ‡ Present address: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Obstetrics and Gynecology Center for Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA. § Present address: Department for Clinical Science, Intervention and Technology Division of Obstetrics and Gynecology, Karolinska Institutet Fertility Unit, Karolinska University Hospital, Huddinge K57, 14186 Stockholm, Sweden. ¶ Author for correspondence (janet.rossant@sickkids.ca) Received 5 December 2012; Accepted 30 August 2013 In Xenopus, the development of ectoderm proceeds through an ectodermal progenitor stage, which then differentiates to form the two major ectodermal lineages: surface ectoderm and neurectoderm (Hemmati-Brivanlou and Melton, 1997a; Hemmati-Brivanlou and Melton, 1997b; Wilson and Hemmati-Brivanlou, 1995). BMP4, a member of the transforming growth factor β (TGFβ) ligand superfamily, induces epidermal differentiation from the ectoderm. By contrast, suppression of bone morphogenetic protein (BMP) signaling, accomplished by BMP antagonists, leads to the specification of the neural ectoderm (Chang and Hemmati- Brivanlou, 1998; Wilson and Hemmati-Brivanlou, 1995). In the mouse embryo, the pathways patterning the ectoderm have been less extensively studied. In early investigations, the fate map of the ectoderm was established using cell labeling and orthotopic and heterotopic grafts (Beddington, 1982; Beddington, 1981; Tam, 1989). At E6.5, the epiblast cells at the distal tip are fated to form neurectoderm, whereas the cells at the adjacent region anterior to the distal cap contribute to surface ectoderm. Other epiblast cells at E6.5 are multipotent and do not appear to be restricted to a single lineage outcome (Lawson et al., 1991; Quinlan et al., 1995). At E7.5, the proximal part of the ectodermal layer, which is close to the extra- embryonic ectoderm (ExE), is mostly restricted to becoming surface ectoderm. The remaining regions of the anterior ectodermal layer can be mapped into progenitor regions for forebrain, midbrain, hindbrain and spinal cord (Tam, 1989; Tam and Quinlan, 1996). Based on these fate-mapping studies, it remains unclear whether a transient ectodermal progenitor potential region exists in mouse embryo. Recently, Cajal and colleagues have discovered a small number of cells in mouse embryo that could contribute to both surface ectoderm and neural ectoderm during normal embryonic development. These cells were positioned between the proximal and distal regions of the anterior ectoderm layer at late gastrulation stage (Cajal et al., 2012). This would suggest that the majority of the ectoderm cells are biased to neural or epidermal fate except for this small subset of cells positioned in the narrow intermediate zone. However, it is important to note that in the intact embryo, cell fate regionalization does not necessarily indicate lineage commitment. Although local signals may lead to early separation of the surface ectoderm and neurectoderm cell fate in the intact embryo, these cells may retain a broader potential when explanted in vitro in response to new signals or the removal of repressive signals. For example, Osorno et al. revealed that presomitogenesis-stage embryo (E7.5- E8.0) tissue can also be cultured as pluripotent EpiSCs when explanted in activin/fibroblast growth factor (FGF) conditions (Osorno et al., 2012). In this study, we isolated anterior ectodermal tissue from E6.5, E7.0 and E7.5 mouse embryos, and studied their differentiation potential by culturing these tissue fragments in chemically defined medium with or without BMP4. We found that, at E6.5, the anterior part of the mouse embryo still retained pluripotency, giving rise to Location of transient ectodermal progenitor potential in mouse development Lingyu Li 1, *, Chang Liu 2 , Steffen Biechele 1,3,‡ , Qingqing Zhu 2 , Lu Song 2 , Fredrik Lanner 1,§ , Naihe Jing 2 and Janet Rossant 1,3,¶ Development