Xenbase Image ID: 147663

Figure 1. Functional Cloning of Ectodermin, a Maternal Determinant Regulating Cell Fates in Early Xenopus Embryos (A) Current model for the induction of primitive germ layers in Xenopus embryos. (B) Functional cloning strategy for candidate ectoderm determinants. (C) Schematic representation of the Ectodermin protein. The linker sequence is unique to Xenopus and mammalian Ectodermin (Ecto). Domains at the N terminus (RING finger, B boxes, coiled coil) and the C terminus (PHD, bromodomain) are shared with other members of the Tif1/TRIM protein family. (D–F) Expression analysis of Ectodermin by in situ hybridization. Arrowhead in (F) points to the dorsal lip. Embryos shown in (E) and (F) were bisected before in situ hybridization to allow penetration of the probe in the more vegetal regions. Controls for the in situs are provided in Figure S1. (G and H) Ectodermin protein is a localized determinant in the prospective ectoderm germ layer. (G) shows whole-mount immunolocalization of the endogenous Ectodermin protein on bisected blastula-stage embryos. Magnifications showing specific nuclear staining and additional controls are provided in Figure S1. (H) shows quantitative analysis of Ectodermin protein distribution along the animal-vegetal axis. Anti-Ectodermin immunoblot shows high enrichment of Ectodermin protein in explanted animal cells (An), low expression in marginal explants (MZ), and no expression in vegetal base (Vg), all explanted from blastula-stage embryos. Sibling explants were pretested by RT-PCR to verify their identity according to molecular markers. (I–N) Molecular characterization of the biological activity of Ectodermin by in situ hybridization. Sox2 is an ectoderm and neural plate marker; Xbra and Mix.1 are mesoderm markers. (I and J) Embryos at the four-cell stage were radially injected with Ecto mRNA (350 pg/blastomere) and harvested at the early gastrula stage (stage 10+). (K–N) Eight-cell-stage embryos were injected with Ecto mRNA (1 ng) in a single blastomere (marginal zone), together with lacZ mRNA (200 pg), to identify injected cells (in red). Embryos were harvested at the gastrula stage (stage 11). (O) RT-PCR analysis of marginal-zone explants (VMZ) expressing control (GFP mRNA, 1 ng) and Ecto mRNA (1 ng). Cells normally fated to express mesendoderm markers (Mix.2, Vent-1, Xwnt8, Eomes, Xbra) now change their fate, upregulating the ectoderm-specific marker Sox2. (P and Q) Long-term phenotypic effects of Ecto mRNA overexpression. Arrowheads indicate ruffled ventral epidermis. Image published in: Dupont S et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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