Dokument

Summary:
Tissue separation is an essential process during embryonic development. The underlying mechanism is highly conserved and mostly controlled by apical constriction of cells. It requires a rearrangement of the actin cytoskeleton and the asymmetric establishment of cortical actomyosin complexes. These actomyosin complexes are essential for cell shape changes, morphogenesis, boundary formation and tissue separation. In adult Hydra, morphogenesis is well observable during the asexual budding process. Various receptor systems are involved and FGFR signaling controls bud detachment in which intact epithelia separate from each other without a wound. This morphogenetic process is regulated by bifunctional epitheliomuscle cells which have to ensure movement and morphogenetic processes. The knowledge of intracellular signaling pathways targeting these processes in Hydra was limited and its elucidation is the main subject of my thesis. A candidate signaling pathway through Rho, ROCK and myosin II was identified which controls bud base constriction and therewith also bud detachment. Gene expression analysis confirmed that candidate genes of an FGFR-coupled pathway are expressed in overlapping regions undergoing morphogenesis. In addition, pharmacological inhibition of any component of the pathway prevented bud detachment and led to stable non-detaching, Y-shaped phenotypes. Accompanying bud detachment, a strong accumulation of F-actin in the constricting cells at the bud base was detected which undergo dynamic cell shape changes. In parallel the myosin regulatory light chain (MLC) was phosphorylated (pMLC20) at the late bud base. The MLC signal was detected by gene expression data and antibody staining in all morphogenetic active regions. The pMLC20 antibody revealed the phosphorylated protein in the basal contractile processes of ectodermal epitheliomuscle cells which control body movement as well as the essential constriction of the bud base. Here, pMLC20 was detected in the apical and basolateral compartments of a small cell population. Pharmacological inhibition revealed that MLC phosphorylation occurs in distinct subcellular compartments during movement and morphogenesis and is controlled by at least two independent pathways. During contraction of the basal cell protrusions (movement) MLC is mainly phosphorylated by myosin light chain kinase (MLCK). In contrast, apical and cortical MLC phosphorylation is stimulated via a Rho-ROCK pathway during morphogenesis in constricting cells of the bud base. The present data provide a new, complex picture of the function of epitheliomuscle cells with Rho-dependent phosphorylation of MLC in bud morphogenesis and a housekeeping function fulfilled by MLCK in the normal basal contractility of ectodermal epitheliomuscle cells during body movement.

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