What lies beneath: Hydra provides cnidarian perspectives into the evolution of FGFR docking proteins.

Across the Bilateria, FGF/FGFR signaling is critical for normal development, and in both Drosophila and vertebrates, docking proteins are required to connect activated FGFRs with downstream pathways. While vertebrates use Frs2 to dock FGFR to the RAS/MAPK or PI3K pathways, the unrelated protein, downstream of FGFR (Dof/stumps/heartbroken), fulfills the corresponding function in Drosophila. To better understand the evolution of the signaling pathway downstream of FGFR, the available sequence databases were screened to identify Frs2, Dof, and other key pathway components in phyla that diverged early in animal evolution. While Frs2 homologues were detected only in members of the Bilateria, canonical Dof sequences (containing Dof, ankyrin, and SH2/SH3 domains) were present in cnidarians as well as bilaterians (but not in other animals or holozoans), correlating with the appearance of FGFR. Although these data suggested that Dof coupling might be ancestral, gene expression analysis in the cnidarian Hydra revealed that Dof is not upregulated in the zone of strong FGFRa and FGFRb expression at the bud base, where FGFR signaling controls detachment. In contrast, transcripts encoding other, known elements of FGFR signaling in Bilateria, namely the FGFR adaptors Grb2 and Crkl, which are acting downstream of Dof (and Frs2), as well as the guanyl nucleotide exchange factor Sos, and the tyrosine phosphatase Csw/Shp2, were strongly upregulated at the bud base. Our expression analysis, thus, identified transcriptional upregulation of known elements of FGFR signaling at the Hydra bud base indicating a highly conserved toolkit. Lack of transcriptional Dof upregulation raises the interesting question, whether Hydra FGFR signaling requires either of the docking proteins known from Bilateria.

Bud detachment in hydra requires activation of fibroblast growth factor receptor and a Rho-ROCK-myosin II signaling pathway to ensure formation of a basal constriction.

BACKGROUND: Hydra propagates asexually by exporting tissue into a bud, which detaches 4 days later as a fully differentiated young polyp. Prerequisite for detachment is activation of fibroblast growth factor receptor (FGFR) signaling. The mechanism which enables constriction and tissue separation within the monolayered ecto- and endodermal epithelia is unknown. RESULTS: Histological sections and staining of F-actin by phalloidin revealed conspicuous cell shape changes at the bud detachment site indicating a localized generation of mechanical forces and the potential enhancement of secretory functions in ectodermal cells. By gene expression analysis and pharmacological inhibition, we identified a candidate signaling pathway through Rho, ROCK, and myosin II, which controls bud base constriction and rearrangement of the actin cytoskeleton. Specific regional myosin phosphorylation suggests a crucial role of ectodermal cells at the detachment site. Inhibition of FGFR, Rho, ROCK, or myosin II kinase activity is permissive for budding, but represses myosin phosphorylation, rearrangement of F-actin and constriction. The young polyp remains permanently connected to the parent by a broad tissue bridge. CONCLUSIONS: Our data suggest an essential role of FGFR and a Rho-ROCK-myosin II pathway in the control of cell shape changes required for bud detachment. Developmental Dynamics 246:502-516, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Dynamic expression of a Hydra FGF at boundaries and termini.

Guidance of cells and tissue sheets is an essential function in developing and differentiating animal tissues. In Hydra, where cells and tissue move dynamically due to constant cell proliferation towards the termini or into lateral, vegetative buds, factors essential for guidance are still unknown. Good candidates to take over this function are fibroblast growth factors (FGFs). We present the phylogeny of several Hydra FGFs and analysis of their expression patterns. One of the FGFs is expressed in all terminal regions targeted by tissue movement and at boundaries crossed by moving tissue and cells with an expression pattern slightly differing in two Hydra strains. A model addressing an involvement of this FGF in cell movement and morphogenesis is proposed: Hydra FGFf-expressing cells might serve as sources to attract tissue and cells towards the termini of the body column and across morphological boundaries. Moreover, a function in morphogenesis and/or differentiation of cells and tissue is suggested.

FGFR-ERK signaling is an essential component of tissue separation.

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.