Epidermal Induction Mediated Through Multivesicular Endosomes

The Wnt and Bone Morphogenic Protein (BMP) pathways have been shown to play a role in the differentiation of epidermal tissues. Wnt signaling requires multivesicular endosomes, also called multivesicular bodies (MVBs), in order to sequester Glycogen Synthase Kinase 3 (GSK3), its key negative regulator. GSK3 is part of a larger destruction complex that targets both Smad1/5/8 and β-Catenin, the effectors of the Wnt and BMP pathways respectively. We hypothesize that these two pathways that drive epidermal induction are linked through a common mechanism: the sequestration of GSK3 into MVBs. We propose that knocking down MVB components will inhibit epidermal induction. In order to test this, knock-down of key MVB components, like hepatocyte growth factor regulated tyrosine related substrate (HRS) is required. To induce epidermis, either BMP4 mRNA or Wnt DNA will be co-microinjected with antisense morpholinos (MO) directed against core components of the MVB machinery into 4 cell stage Xenopus laevis embryos. Analysis of gene expression levels from dissociated animal cap cells will be performed using Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Preliminary results indicate that an antisense morpholino directed against HRS, a core component necessary for MVB formation, inhibits epidermal induction. If preliminary experiments are confirmed, they would show for the first time how MVBs are required for Wnt signaling in a developmental context.