Ectoderm specification
In Xenopus laevis, the specification of the three germ layers (endoderm, mesoderm and ectoderm) occurs at the blastula stage. Great efforts have been made to determine the factors that specify the endoderm and mesoderm. On the other hand, only a few examples of genes that are required for ectoderm specification have been described in the last decade. The first molecule identified to be required for the specification of ectoderm was the ubiquitin ligase Ectodermin (Ecto, TIF1-γ, TRIM33); later, it was found that the deubiquitinating enzyme, FAM/USP9x, is able to overcome the effects of ubiquitination made by Ectodermin in Smad4 (Dupont et al., 2009). Two transcription factors have been proposed to control gene expression of ectodermal specific genes: POU91/Oct3/4 and FoxIe1/Xema. A new factor specific for the ectoderm, XFDL156, has shown to be essential for suppression of mesoderm differentiation from pluripotent cells. Ectodermin and FAM Biological role of Ectodermin and FAM The protein Ectodermin, firstly identified in Xenopus embryos, promotes ectodermal fate and suppresses the mesoderm formation mediated by the signaling of Transforming Growth Factor β (TGFβ) and Bone Morphogenic Proteins (BMP), members of the TGFβ-superfamily. When the TGFβ ligands bind to TGFβ receptors, they cause the activation of the signal transducers R-Smads (Smad2, Smad3). Smad4 forms a complex with activated R-Smads and activates transcription of specific genes in response to TGFβ signal. The BMP pathway transmits its signals in a similar way but through other types of R-Smads (Smad1, Smad5 and Smad8). The transcription factor Smad4 is the only common mediator shared between both TGFβ and the BMP pathways. During ectoderm specification, the function of Smad4 is regulated by ubiquitination and deubiquitination made by ectodermin and FAM, respectively. The ubiquitination state of Smad4 will determine if it is able to respond to signals derived from TGFβ and BMP. The equilibrium of the activity, localization and timing of TGFβ and BMP transducers, Smad4, FAM and of Ectodermin should be achieved in order to be able to modulate the gene expression of genes required for germ layer formation. Identification of Ectodermin and FAM A cDNA library from the blastula stage of a frog embryo was cloned into RNA expression plasmids to generate synthetic mRNA. The mRNA was then injected into several Xenopus embryos at a four-cell stage and looked in early blastula embryos for an expansion of the region of the ectodermal marker Sox2 and diminution of the expression of the mesodermal marker Xbra. Ectodermin was one out of 50 clones to present this phenotype when injected into embryos. The identification of FAM was done through a siRNA screen to find deubiquitinases that regulate the response to TGFβ. Ectodermin and FAM localization Ectodermin mRNA is maternally deposited in the animal pole of the egg. In the early blastula stage of the embryo, Ectodermin mRNA and protein forms a gradient that goes from the animal pole (highest concentration) down to the marginal zone (lowest concentration) to prevent TGFβ and nodal signals that induce mesoderm originating from the vegetal pole. Ectodermin mRNA is enriched