Identification and characterization of downstream elements of Hydra FGFR signaling

Hydra polyps predominantly reproduce through budding in the lower half of the parent’s body column. FGFRa (Kringelchen), a member of FGF receptor tyrosine kinases, plays an essential role and controls bud detachment from the parent. Whether signal transduction through Hydra FGFR is comparable to FGFR signal-­ ing in vertebrate and fly is unknown. In both Bilateria, activated FGFRs recruit docking proteins to connect to downstream pathways and negative regulators. While vertebrates use FRS2 to dock FGFR to the Ras/MAPK or PI3K pathways, a completely unrelated protein, Downstream-­of-­FGFR (Dof/Stumps/Heartbro-­ ken), fulfills this function in Drosophila. In Drosophila, Dof couples FGFR to MAPK signaling and transcriptionally activates the negative regulator Sprouty (Spry). Spry proteins are necessary to modulate receptor tyrosine kinase activity by in-­ terfering with MAPK signaling downstream of RTK. To elucidate potential downstream signaling elements of ancestral FGFRs, I an-­ alyzed genomic and EST sequence databases and identified Spry, FRS2, and/or Dof proteins in phyla derived early from the main lineage of animals – including Hydra. Dof was found only within the Eumetazoa, while FRS2 proteins were also predicted in Metazoa and their sister taxon, the Choanoflagellata. For the known FRS2 proteins of Deuterostomia and Ecdysozoa an N-­terminal myristoylation site, a PTB domain and multiple C-­terminal Grb2 and Shp2 binding sites are typ-­ ical. This structure also applies to FRS2 in Choanoflagellata and sponges (Porif-­ era). A deviating domain structure of FRS2 proteins is predicted in Placozoa, Cnidaria, and Spiralia (Annelida, Mollusca). Their FRS2 proteins all carry an N-­ terminal PH, a PTB domain and few Grb2 and Shp2 binding sites. Phylogenetic analysis identified a novel protein family (PH-­FRS2). Expression analysis of Dof and FRS2 in Hydra revealed high levels of Dof transcripts in the upper body re-­ gion and the tentacle zone. FRS2 mRNA, in contrast, was detected only weakly at the tentacle bases. The presence of both putative docking proteins in Metazoa hints to an early toolkit for the transduction of FGFR signals. Their functional sig-­ nificance remains to be shown. I also identified four spry genes in Hydra, all positioned in the basal most position of the phylogenetic tree. They encode the typical features of Spry proteins namely a c-­Cbl TKB (Tyrosine kinase binding) site, a Raf1-­binding and a Spry domain. Transcripts of spry2 were detected at the bud base adjacent to Hydra FGFRa from mid to late stages. Since no spry-­encoding genes were found in the ge-­ nomes of Parazoa (Trichoplax), sponges (Oscarella, Amphimedon), or choanoflagellates (Salpingoeca), Sprouties might have occurred in the Cnidaria first -­ or been lost from the early derived taxa. Tissue dynamics and the spatiotemporal expression pattern of FGFRa, dof and spry2, reveals (a) spry2 and FGFRa are present in the same cells at the bud base, (b) co-­expression of FGFRa (weakly) and dof (strongly) in the head region. In summary, data suggest the existence of two FGFR pathways. The first path-­ way functions in bud detachment, as shown previously, and potentially links FGFRa to a negative feedback loop activating Spry2. A second pathway function for FGFR signaling in Hydra might be at the bud and at the adult head – in a zone, where the mRNA encoding the docking protein Dof is expressed at a high level, and where the two FGFRs are transcribed at a low level. Here, FGFR might func-­ tion to control or modulate cell migration towards the head and/or cell differentia-­ tion necessary to form and maintain a fully functional head. Further elucidation of these potential functions and the molecular network, in which Hydra FGFRs act, is a very interesting task for the future

Identification and characterization of downstream elements of Hydra FGFR signaling

Hydra polyps predominantly reproduce through budding in the lower half of the parent’s body column. FGFRa (Kringelchen), a member of FGF receptor tyrosine kinases, plays an essential role and controls bud detachment from the parent. Whether signal transduction through Hydra FGFR is comparable to FGFR signal-­ ing in vertebrate and fly is unknown. In both Bilateria, activated FGFRs recruit docking proteins to connect to downstream pathways and negative regulators. While vertebrates use FRS2 to dock FGFR to the Ras/MAPK or PI3K pathways, a completely unrelated protein, Downstream-­of-­FGFR (Dof/Stumps/Heartbro-­ ken), fulfills this function in Drosophila. In Drosophila, Dof couples FGFR to MAPK signaling and transcriptionally activates the negative regulator Sprouty (Spry). Spry proteins are necessary to modulate receptor tyrosine kinase activity by in-­ terfering with MAPK signaling downstream of RTK. To elucidate potential downstream signaling elements of ancestral FGFRs, I an-­ alyzed genomic and EST sequence databases and identified Spry, FRS2, and/or Dof proteins in phyla derived early from the main lineage of animals – including Hydra. Dof was found only within the Eumetazoa, while FRS2 proteins were also predicted in Metazoa and their sister taxon, the Choanoflagellata. For the known FRS2 proteins of Deuterostomia and Ecdysozoa an N-­terminal myristoylation site, a PTB domain and multiple C-­terminal Grb2 and Shp2 binding sites are typ-­ ical. This structure also applies to FRS2 in Choanoflagellata and sponges (Porif-­ era). A deviating domain structure of FRS2 proteins is predicted in Placozoa, Cnidaria, and Spiralia (Annelida, Mollusca). Their FRS2 proteins all carry an N-­ terminal PH, a PTB domain and few Grb2 and Shp2 binding sites. Phylogenetic analysis identified a novel protein family (PH-­FRS2). Expression analysis of Dof and FRS2 in Hydra revealed high levels of Dof transcripts in the upper body re-­ gion and the tentacle zone. FRS2 mRNA, in contrast, was detected only weakly at the tentacle bases. The presence of both putative docking proteins in Metazoa hints to an early toolkit for the transduction of FGFR signals. Their functional sig-­ nificance remains to be shown. I also identified four spry genes in Hydra, all positioned in the basal most position of the phylogenetic tree. They encode the typical features of Spry proteins namely a c-­Cbl TKB (Tyrosine kinase binding) site, a Raf1-­binding and a Spry domain. Transcripts of spry2 were detected at the bud base adjacent to Hydra FGFRa from mid to late stages. Since no spry-­encoding genes were found in the ge-­ nomes of Parazoa (Trichoplax), sponges (Oscarella, Amphimedon), or choanoflagellates (Salpingoeca), Sprouties might have occurred in the Cnidaria first -­ or been lost from the early derived taxa. Tissue dynamics and the spatiotemporal expression pattern of FGFRa, dof and spry2, reveals (a) spry2 and FGFRa are present in the same cells at the bud base, (b) co-­expression of FGFRa (weakly) and dof (strongly) in the head region. In summary, data suggest the existence of two FGFR pathways. The first path-­ way functions in bud detachment, as shown previously, and potentially links FGFRa to a negative feedback loop activating Spry2. A second pathway function for FGFR signaling in Hydra might be at the bud and at the adult head – in a zone, where the mRNA encoding the docking protein Dof is expressed at a high level, and where the two FGFRs are transcribed at a low level. Here, FGFR might func-­ tion to control or modulate cell migration towards the head and/or cell differentia-­ tion necessary to form and maintain a fully functional head. Further elucidation of these potential functions and the molecular network, in which Hydra FGFRs act, is a very interesting task for the future