Analysis of candidate genes potentially supporting the putative plasticity of epithelial cells in Hydra

L'Hydre est un polype d'eau douce formé de deux couches cellulaires contenant trois populations de cellules souches et possédant d'incroyables capacités de régénération. Il a été démontré qu'un traitement à l'hydroxyurée (HU) était capable d'éliminer les cellules interstitielles, sans affecter les cellules épithéliales. De même, après une amputation transversale au milieu de la cavité gastrique, une vague d'apoptose est visualisée dans les cellules interstitielles de l'extrémité régénerant la tête. De telles données indiquent que les cellules de la lignée interstitielle sont d'avantage affectées par la mort cellulaire que les cellules épithéliales chez l'Hydre. Une analyse quantitative basée sur le séquençage d'ARN a été effectuée sur des Hydres régénérantes et sur des Hydres dépourvues de cellules interstitielles. Les données ont montré que de nombreux gènes, qui n'étaient pas, ou très peu exprimés dans l'Hydre intacte, se trouvaient fortement surexprimés en l'absence de cellules interstitielles ou en conditions de régénération, impliquant une possible plasticité des cellules épithéliales après une apoptose massive ou localisée des cellules interstitielles. Pour vérifier cela, nous avons sélectionné, caractérisé avec des outils bioinformatiques, et analysé par hybridation in situ une série de gènes candidats à la plasticité dans le but de mettre en évidence leur patron d'expression. L'hybridation in situ confirme les résultats obtenus après séquençage d'ARN et nous avons pu identifier 11 gènes différentiellement exprimés chez des Hydres non traitées ou traitées au HU ainsi qu'après quelques heures de régénération suite à une amputation, pour 4 autres gènes. Nous avons ensuite tenté d'établir un nouveau protocole d'interférence à ARN où les Hydres sont directement plongées dans une solution d'ARN double brin. Cette approche devait nous fournir un outil de travail pratique pour tester la fonction des gènes candidats, cependant cette méthode pour éteindre un gène n'est pas robuste chez l'Hydre. En résumé, les résultats démontrent que la méthode d'hybridation in situ confirme les résultats obtenus par séquençage d'ARN et permettent de mettre en évidence une liste de gènes surexprimés, comme par exemple TCTP ou antistasin, impliqués dans différents contextes mais pointant tous vers une forme de plasticité des cellules épithéliales lorsqu'elles pallient l'absence des cellules interstitielles

The polymorphism of Hydra microsatellite sequences provides strain-specific signatures

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

The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra

Hydra vulgaris (Hv) has a high regenerative potential and negligible senescence, as its stem cell populations divide continuously. In contrast, the cold-sensitive H. oligactis (Ho_CS) rapidly develop an aging phenotype under stress, with epithelial stem cells deficient for autophagy, unable to maintain their self-renewal. Here we tested in aging, non-aging and regenerating Hydra the activity and regulation of the ULK1 kinase involved in autophagosome formation. In vitro kinase assays show that human ULK1 activity is activated by Hv extracts but repressed by Ho_CS extracts, reflecting the ability or inability of their respective epithelial cells to initiate autophagosome formation. The factors that keep ULK1 inactive in Ho_CS remain uncharacterized. Hv_Basel1 animals exposed to the ULK1 inhibitor SBI-0206965 no longer regenerate their head, indicating that the sustained autophagy flux recorded in regenerating Hv_AEP2 transgenic animals expressing the DsRed-GFP-LC3A autophagy tandem sensor is necessary. The SBI-0206965 treatment also alters the contractility of intact Hv_Basel1 animals, and leads to a progressive reduction of animal size in Hv_AEP2, similarly to what is observed in ULK1(RNAi) animals. We conclude that the evolutionarily-conserved role of ULK1 in autophagy initiation is crucial to maintain a dynamic homeostasis in Hydra, which supports regeneration efficiency and prevents aging

Generic and context-dependent gene modulations during <i>Hydra</i> whole body regeneration

The cnidarian Hydra is a classical model of whole-body regeneration. Historically, Hydra apical regeneration has received more attention than its basal counterpart, most studies considering these two regenerative processes independently. We present here a transcriptome-wide comparative analysis of apical and basal regeneration after decapitation and mid-gastric bisection, augmented with a characterization of positional and cell-type expression patterns in non-regenerating animals. The profiles of 25'637 Hydra transcripts are available on HydrATLAS (https://hydratlas.unige.ch), a web interface allowing a convenient access to each transcript profile. These data indicate that generic impulse-type modulations occur during the first four hours post-amputation, consistent with a similar integration of injury-related cues on both sides of the amputation plane. Initial divergences in gene regulations are observed in regenerating tips between four and eight hours post-amputation, followed by a dramatic transcriptomic reprogramming between eight and 16 hours when regulations become sustained. As expected, central components of apical patterning, Wnt3 and HyBra1, are among the earliest genes up-regulated during apical regeneration. During early basal regeneration, a BMP signaling ligand (BMP5-8c) and a potential BMP inhibitor (NBL1) are up-regulated, suggesting that BMP signaling is involved in the basal organizer, as supported by higher levels of phosphorylated Smad in the basal region and by the LiCl-induced extension of NBL1 expression. By contrast, upon ectopic activation of Wnt/b-catenin signaling, NBL1 is no longer expressed, basal differentiation is not maintained and basal regeneration is abolished. A tight cross-talk between Wnt/b-catenin apically and BMP signaling basally appears necessary for maintaining and regenerating Hydra anatomy

Loss of neurogenesis in aging <i>Hydra</i>

In Hydra the nervous system is composed of neurons and mechano‐sensory cells that differentiate from interstitial stem cells, which also provide gland cells and germ cells. The adult nervous system is actively maintained through continuous de novo neurogenesis that occurs at two distinct paces, slow in intact animals and fast in regenerating ones. Surprisingly Hydra vulgaris survive the elimination of cycling interstitial cells and the subsequent loss of neurogenesis if force‐fed. By contrast, H. oligactis animals exposed to cold temperature undergo gametogenesis and a concomitant progressive loss of neurogenesis. In the cold‐sensitive strain Ho_CS, this loss irreversibly leads to aging and animal death. Within four weeks, Ho_CS animals lose their contractility, feeding response and reaction to light. Meanwhile, two positive regulators of neurogenesis, the homeoprotein prdl‐a and the neuropeptide Hym‐355, are no longer expressed, while the “old” RFamide‐expressing neurons persist. A comparative transcriptomic analysis performed in cold‐sensitive and cold‐resistant strains confirms the down‐regulation of classical neuronal markers during aging but also shows the up‐regulation of putative regulators of neurotransmission and neurogenesis such as AHR, FGFR, FoxJ3, Fral2, Jagged, Meis1, Notch, Otx1, TCF15. The switch of Fral2 expression from neurons to germ cells suggests that in aging animals, the neurogenic program active in interstitial stem cells is re‐routed to germ cells, preventing de novo neurogenesis and impacting animal survival

An evolutionarily-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity in Hydra

Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when β-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra, acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/β-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via β-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/β-catenin signaling, a function conserved across eumetazoan evolution

The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra

The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration. </p