br Results The RAS MEK ERK pathway is
Results The RAS/MEK/ERK pathway is a potential target of FGFR signaling. Indicative for its activation is the presence of double phosphorylated (activated) ERK (dpERK), which is generated in a series of phosphorylation events downstream of FGFR (op. cit. (Lemmon and Schlessinger, 2010)). Following ligand binding FGFR docks to proteins, which activate the GTPase RAS. RAS activates the serine-threonine kinase Raf1, which phosphorylates MEK. Finally, activated MEK phosphorylates ERK and generates its activated, double phosphorylated form, dpERK. The spatio-temporal activation of RAS/MEK/ERK signaling can thus be analyzed in whole tissue or single cells simply by immunohistochemistry against dpERK. To investigate local ERK activation in Hydra, we used an antibody directed against double phosphorylated ERK1/2 (Thr 202/Tyr 204 in the highly conserved mouse epitope HTGFLpTEpYVAT). This epitope is conserved in the predicted single Hydra ERK (syn. MAPK3, NCBI reference XP_002164372.2, partial EST) with a single F/M amino Z-YVAD-FMK exchange. In a Western blot the antibody detected a single band of 45kd in Hydra, which corresponds to about the expected size of ERK (Fig. S1).
Discussion Budding in Hydra is an astonishing process in which, within four days, a complete young polyp forms by mass tissue movement from the parent and finally detaches. Preceding tissue separation, a sharp boundary between bud and parent is defined by crosstalk between FGFR and Notch signaling (Münder et al., 2010, Prexl et al., 2011, Sudhop et al., 2004). The establishment of this boundary is a prerequisite for the formation of a tissue constriction between bud and parent. How this constriction narrows to a thin tube and finally completes separation of the continuous, single layered ecto- and endodermal epithelia with their acellular extracellular matrix (ECM, mesogloea) is unknown. Here, we provide evidence that initiation of the constriction and tissue separation directly depend on FGFR and, in the late phase, on FGFR/RAS/MEK/ERK signaling. We will discuss the role of FGFR (i) in formation of the constriction and (ii) in tissue separation and present a model (Fig. 8).
Conclusion Polyps transgenic for either the full length or a putatively dominant-negative Kringelchen FGFR corroborate the hypothesis that tissue separation during Hydra budding critically depends on this FGFR. Ectopic expression of Kringelchen induces tissue separation within the polyp׳s body column, probably by a transient local activation of MEK-ERK signaling in a small cluster of cells proximal to the separation site. The actin cytoskeleton is strongly affected in autotomizing polyps and thus, it seems to be another target for Kringelchen. Our study indicates a novel function of FGFR—not only in establishing a boundary between parent and young polyp (by Notch signaling) but in controlling the complete separation of continuous epithelia in a process inverse to the formation of anastomoses in circulatory systems.
Acknowledgements We thank Arno Müller and Anna Klingseisen, Dundee, for advice on using the anti-dpERK antibody. Rob Steele and Robert Campbell kindly provided Hydra vulgaris 1184A. We are grateful to Anja Rudolf for help with the kLSM and Katja Gessner for graphics. Supported by the DFG Grant HA 1732-11.
Main Text Puzzlingly, many of the key cellular signaling modules initiated by membrane-bound receptor tyrosine kinases, like FGFR, activate overlapping sets of downstream pathways, but with distinct outcomes. Consequently, a central question in growth-factor-mediated signal transduction is how a similar set of downstream signaling cascades can elicit diverse yet specific cellular outcomes. In this issue of Chemistry & Biology, Kim et al. (2014) introduce a new tool for addressing this question, showing that light-controlled activation of signal transduction enables superior spatial and temporal regulation, thus enabling dissection of the roles of specific receptor types.