CONICET | Buscador de Institutos y Recursos Humanos

The Wnt and Bone Morphogenic Protein (BMP) pathways have been shown to direct the differentiation of ectodermal tissues towards epidermal fates. Wnt signaling requires multivesicular endosomes, also called multivesicular bodies (MVBs) in order to sequester Glycogen Synthase Kinase 3 beta (GSK3-beta) from the cytosol, inhibiting its activity. Without Wnt stimulation, GSK3-beta phosphorylates and primes both beta-catenin and Smad1/5/8, the effectors of the Wnt and BMP pathways, respectively for proteasomal degradation. We propose that these two pathways that drive epidermal induction are linked through a common mechanism: the sequestration of GSK3-beta into MVBs. Thus, knocking down MVB formation should inhibit epidermal induction. In order to test this hypothesis, knock-down of core components necessary for MVB formation, were conducted in Xenopus laevis embryos. Thus, hepatocyte growth factor regulated tyrosine related substrate (HRS), Ras-related protein 7 (Rab7) and Vacuolar sorting protein 4 (Vps4) were knocked down by injecting antisense morpholinos and DNA coding for the dominant negative Vps4 into 2-4 cell stage embryos. To determine the effects of the knock down, epidermal differentiation was induced either through Wnt8 DNA microinjections or BMP4 protein treatment. Analysis of epidermal gene expression levels by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) in dissociated animal cap ectodermal cells showed that knock-down of HRS, Rab7, and Vps4 function inhibits Wnt induced epidermal induction to a greater extent than BMP-induced induction. If preliminary experiments are confirmed, these experiments show for the first time that MVB formation is required for epidermal induction.

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