Abstract
Hydra polyps regenerate lost body parts, including the head. In addition, Hydra head tissue has organizer properties thus being able to recruit body column tissue from a host polyp to produce ectopic hydranths after transplantation. These pattern forming processes involve Notch- and Wnt/β-catenin-signaling. Hydra head regeneration consists of two parts, hypostome/organizer and tentacle development. Previous work had shown that the Notch inhibitor DAPT blocks hypostome regeneration and organizer formation, but not the appearance of tentacle genes and tentacle tissue. Here we show that the β-catenin inhibitor iCRT14 blocks tentacle regeneration, but not regeneration of hypostome and organizer tissue. Using RT-qPCR gene expression analyzes during head regeneration we found that DAPT inhibits HyWnt3- and HyBMP2/4 expression and expression of transcriptional repressor genes including CnGsc, Sp5 and HyHes, while increasing expression of HyBMP5/8b and the c-fos-related gene HyKayak. ICRT14 blocks expression of the tentacle specification factor HyAlx, but not expression of HyWnt3. Thus, in accordance with regeneration of two head structures we find two signaling and gene expression modules with HyWnt3 and HyBMP4 part of a hypostome/organizer module, and BMP5/8, HyAlx and β-catenin part of a tentacle module. We conclude that Notch functions as an inhibitor of tentacle production to allow regeneration of hypostome/head organizer. Furthermore, with HyKayak we present a candidate target gene for HvNotch induced repressor genes. Using siRNA and the Fos/Jun-inhibitor T5224 we show that HyKayak attenuates the expression of HyWnt3. Finally, Notch signaling was not required for head regeneration of fresh water polyps of Craspedacusta. Polyps of Craspedacusta do not have tentacles and thus, after head removal only regenerate a hypostome with a crescent of nematocytes around the mouth opening. This corroborates the idea that Notch-signaling mediates between two pattern forming processes during Hydra head regeneration.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Our major revision included the following main points: (1)Concerns regarding clarity of the manuscript: we have substantially edited the abstract, introduction and discussion part of the manuscript and added many more references to previous work by other authors, especially Cazet 2021, Tursch 2022 and Gahan 2017. We focused our introduction and discussion on organizer function and on the Gierer-Meinhardt-Model for pattern formation. We think that the conclusions are of great general interest because they suggest a function of the Hydra head organizer according to the original definition by Hans Spemann, that is -harmonious interlocking of separate processes which makes up development-. Notch signaling, in our interpretation, is an instrument for this function of the organizer. Comparison with Craspedacusta compellingly illustrates this idea. (2)Concerns regarding Craspedacusta experiments: we have isolated four Craspedacusta transcripts (CsSp5, CsWnt3, CsAlx and CsNOWA) and analyzed their response to DAPT during head regeneration in Craspedacusta. This revealed that DAPT did not inhibit CsWnt3 expression, in accordance with it not having an effect on head regeneration in Craspedacusta polyps. However, DAPT inhibited expression of the other potential CsNotch target genes, confirming that DAPT generally works in Craspedacusta polyps as Notch-inhibitor. (3)Concerns regarding HyKayak function: we have conducted a rescue experiment to show the function of Hykayak as a target for Notch-regulated repressor genes and a local inhibitor of Wnt-3 expression, which revealed that the expected up-regulation of HyWnt3 in DAPT-treated animals was very weak and did not rescue the DAPT-regeneration phenotype-this was discussed, but data were not included.
