Hydra head formation depends on an organizing center in which Wnt/β-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in either the epidermis or gastrodermis, we show that Sp5 promoter activity is differentially regulated in each epithelial layer. In intact animals, epidermal HySp5:GFP activity is strong apically and weak along the body column, while in the gastrodermis, it is maximal in the tentacle ring region and maintained at a high level along the upper body column. During apical regeneration, HySp5:GFP is activated early in the gastrodermis and later in the epidermis. Alsterpaullone treatment induces a shift in apical HySp5:GFP expression towards the body column where it forms transient circular figures in the epidermis. Upon β-catenin(RNAi), HySp5:GFP activity is down-regulated in the epidermis while bud-like structures expressing HySp5:GFP in the gastrodermis develop. Sp5(RNAi) reveals a negative Sp5 autoregulation in the epidermis, but not in the gastrodermis. These differential regulations in the epidermis and gastrodermis highlight the distinct architectures of the Wnt/β-catenin/TCF/Sp5/Zic4 network in the hypostome, tentacle base and body column of intact animals, as well as in the buds and apical and basal regenerating tips.

Hydra are freshwater polyps widely studied for their amazing regenerative capacity, adult stem cell populations, low senescence and value as ecotoxicological marker. Many wild-type strains of H. vulgaris have been collected worldwide and maintained effectively under laboratory conditions by asexual reproduction, while stable transgenic lines have been continuously produced since 2006. Efforts are now needed to ensure the genetic characterization of all these strains, which despite similar morphologies, show significant variability in their response to gene expression silencing procedures, pharmacological treatments or environmental conditions. Here, we established a rapid and reliable procedure at the single polyp level to produce via PCR amplification of three distinct microsatellite sequences molecular signatures that clearly distinguish between Hydra strains and species. The TG-rich region of an uncharacterized gene ( ms-c25145 ) helps to distinguish between Eurasian H. vulgaris strains ( Hm-105 , Basel1 , Basel2 and reg-16 ), between Eurasian and North American H. vulgaris strains ( H. carnea, AEP ), and between the H. vulgaris and H. oligactis species. The AT-rich microsatellite sequences located in the AIP gene ( Aryl Hydrocarbon Receptor Interaction Protein, ms-AIP ) also differ between Eurasian and North American H. vulgaris strains. Finally, the AT-rich microsatellite located in the Myb-Like cyclin D-binding transcription factor1 gene ( ms-DMTF1 ) gene helps to distinguish certain transgenic AEP lines. This study shows that the analysis of microsatellite sequences provides a barcoding tool that is sensitive and robust for the identification of Hydra strains. It is also capable of identifying cryptic species by tracing microevolutionary events within the genus Hydra .

In Hydra , head formation depends on Wnt/β-catenin signaling, which positively regulates Sp5 and Zic4 , with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in the epidermis or gastrodermis, we show that in intact animals, epidermal HySp5: GFP is expressed strongly apically and weakly along the body column, while gastrodermal HySp5: GFP is also maximally expressed apically but absent from the oral region, and remains high along the upper body column. During apical regeneration, gastrodermal HySp5 :GFP appears early and diffusely, epidermal HySp5 :GFP later. Upon alsterpaullone treatment, apical HySp5:GFP expression is shifted to the body column where epidermal HySp5: GFP transiently forms ectopic circular figures. After β-catenin (RNAi), only epidermal HySp5: GFP is down-regulated, while pseudo-bud structures expressing gastrodermal HySp5: GFP develop. Sp5 (RNAi) highlights the negative autoregulation of Sp5 in epidermis, involving direct binding of Sp5 to its own promoter as observed in human HEK293T cells. In these cells, HyZic4, which can interact with huTCF1, regulates Wnt3 negatively and Sp5 positively. This differential regulation of the Wnt/β-catenin/TCF/Sp5/Zic4 network in epidermis and gastrodermis highlights distinct architectures and patterning roles in the hypostome, tentacle and body column, as well as distinct regulations in homeostatic and developmental organizers.

The molecular mechanisms that maintain cell identities and prevent transdifferentiation remain mysterious. Interestingly, both dedifferentiation and transdifferentiation are transiently reshuffled during regeneration. Therefore, organisms that regenerate readily offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4 silencing is sufficient to induce transdifferentiation of tentacle into foot cells. We identified a Wnt-controlled Gene Regulatory Network that controls a transcriptional switch of cell identity. Furthermore, we show that this switch also controls the re-entry into the cell cycle. Our data indicate that maintenance of cell fate by a Wnt-controlled GRN is a key mechanism during both homeostasis and regeneration. One-Sentence Summary: A Wnt-controlled GRN controls fate maintenance in Hydra .

The Hydra polyp regenerates its head by transforming the gastric tissue below the wound into a head organizer made of two antagonistic cross-reacting components. The activator, previously characterized as Wnt3 , drives apical differentiation by acting locally and auto-catalytically. The uncharacterized inhibitor, produced under the control of the activator, prevents ectopic head formation. By crossing RNA-seq data obtained in a β-catenin (RNAi) screen performed in planarians and a quantitative analysis of positional and temporal gene expression in Hydra , we identified Sp5 as a transcription factor that fulfills the head inhibitor properties: a Wnt/β-catenin inducible expression, a graded apical-to-basal expression, a sustained up-regulation during head regeneration, a multi-headed phenotype when knocked-down, a repressing activity on Wnt3 expression. In mammalian cells, Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also auto-activates its expression, possibly via β-catenin and/or Tcf/Lef1 interaction. This work identifies Sp5 as a novel potent feedback loop inhibitor of Wnt/β-catenin signaling across eumetazoans.