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    • angiogenesis In the phylum Cnidaria which

    2021-09-10

    In the phylum Cnidaria, which separated from the main line of Metazoa early during evolution, FGFR signaling is essential during development and morphogenesis. Two FGFRs and two FGFs antagonistically control formation of the apical organ in the anthozoan Nematostella and target the MAPK pathway (Rentzsch et al., 2008). In Hydra at least one of two FGFRs (Rudolf et al., 2012) is required for proper detachment of a young polyp in the final phase of the angiogenesis process (Münder et al., 2010, Sudhop et al., 2004). Budding, the vegetative mode of propagation in Hydra, is enabled by mass tissue and cell movement from the parent body column to an evaginating bud, which also involves rearrangement of the extracellular matrix (Aufschnaiter et al., 2011). The molecular control mechanisms are just becoming elucidated. Bud initiation and evagination depends on canonical and noncanonical WNT signaling (Hobmayer et al., 2000, Philipp et al., 2009) with the extracellular matrix playing an essential role (Aufschnaiter et al., 2011). While it became clear that the detachment process is initiated by FGFR signaling (Sudhop et al., 2004) and boundary formation is allowed by crosstalk of FGFR to Notch (Münder et al., 2010, Prexl et al., 2011), it is still obscure how tissue separation is controlled. To elucidate its mechanism, downstream targets of Hydra FGFR have to be identified. FGFRs are canonically activated by binding of an FGF ligand and subsequent receptor-dimerisation, which activates the intracellular tyrosine kinase domain. Transphosphorylation of highly conserved tyrosine residues in the intracellular domain generates docking sites for intracellular binding proteins, which mediate coupling to signaling partners. Depending on cell type or stage mostly three alternatively used signaling pathways are targeted: PI/PKC signaling is activated by direct binding of PLC-γ to tyrosine-phosphorylated docking sites, activation of the RAS/MAP- kinase or the PI3-kinase pathways requires, in contrast, docking proteins (Lemmon and Schlessinger, 2010). In Hydra, analysis of the distribution and function of FGF ligands (Lange et al., in revision) and of downstream regulators like the branch suppressor Sprouty just started. Of the three alternative downstream pathways for FGFR signaling, neither PI/PKC signaling nor PI3-kinase are likely to be involved in budding as concluded from inhibitor experiments (Fabila et al., 2002). Activation of PI/PKC signaling by externally applied diacylglycerol or phorbol esters is known to strongly induce ectopic head formation along the body column and suppress budding, but effects on morphogenesis of existing buds have not been observed (Hassel et al., 1998, Müller, 1989). Inhibition of PI-3 kinase in Hydra upregulates apoptosis (David et al., 2005), without affecting bud morphogenesis (own unpublished observations). In early and mid bud stages CREB and MAPK signaling have been shown to be required for head formation (Fabila et al., 2002, Kaloulis et al., 2004, Manuel et al., 2006), but a role of MAPK in late budding has not been reported yet. Since the FGFR-dependent RAS/MAPK pathway is essential for morphogenesis in e.g. Drosophila (Mandal et al., 2004, Muha and Muller, 2013) we tested whether FGFR targets the RAS/MAPK signaling pathway in late budding stages. Our data indicate that FGFR-dependent activation of MEK and ERK are necessary to control the final steps of bud detachment and to complete tissue separation.
    Material and methods Polyps were kept at 18°C with 16h light, 8h dark cycles in Hydra medium (0.29mM CaCl2, 0.59mM MgSO4, 0.5mM NaHCO3 and 0.08mM K2CO3, pH 7.6) and fed five times a week to synchronize bud development (Sudhop et al., 2004). , a kind gift of Campbell and Steele, Irvine, was used for detection of dpERK in whole polyps, this strain allows a background-free detection of dpERK-stained cells. For all other experiments including transgenesis, Hydra vulgaris AEP was used. Since most of the previously described inhibitor experiments were done with Hydra vulgaris Zürich and since H.v. AEP is only distantly related to the Zürich strain (Hemmrich et al., 2007, Martínez et al., 2010), we repeated several experiments (inhibitor and regeneration) to ensure comparable responses.