Analysis of keratin dynamics in the early mouse embryo: regulatory mechanisms and functional consequences

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The epithelial keratin cytoskeleton is crucial for cell adhesion and cell type-specific functions through interactions with desmosomes, the actin cytoskeleton and associated proteins. Architecture and interactions of keratins are spatiotemporally regulated by Src, PKCalpha and MAPK family members in response to intra- and extracellular signals. Despite the well-documented importance of keratins in embryo development, wound healing and tumor progression, assembly and dynamics of the keratin network and its regulation have so far been studied only in vitro. Although morphological and genetic evidence supports interdependence between keratins, desmosomes and actin, the mechanism underlying de novo keratin network formation, its regulation and significance during differentiation, adhesion and invasion have not been investigated in vivo. Our goal is to unravel interactions between the keratin filament network, desmosomes and actin organization in the physiological context of mouse embryos. The proposal is based on extensive collaborative work from our laboratories on keratin network regulation in keratinocytes. Here, we want to analyze keratin network formation, dynamics, function and regulation in the trophectoderm of live mouse embryos before and during implantation using auto-fluorescent keratin 8 K8-YFP as a novel in vivo detection tool.In early embryos (morula to blastocyst stages) and embryo-derived trophoblast cell lines from K8-YFP knock-in mice, that we recently established, we will pursue three major objectives: 1. Analysis of de novo keratin network formation in live murine embryos2. Examination of the dynamic organization and function of the keratin-desmosome scaffold in the trophoblast during implantation and invasion 3. Analysis of molecular mechanisms regulating keratin network biogenesis, organization and interactions using pharmacological and genetic approaches in cultured trophoblast cells and developing blastocystsWe expect that our project provides novel mechanistic insights into the spatiotemporal organization and regulation of the keratin cytoskeleton in vivo, using an accessible in vivo model system. In the long run, our approach provides the ground to address dynamic keratin regulation during wound healing and during tumor metastasis, conditions in which understanding principles of keratin-desmosome reorganization likely provide novel therapeutic targets.