The Wnt/ß-catenin/TCF/Sp5/Zic4 Gene Network That Regulates Head Organizer Activity in Hydra Is Differentially Regulated in Epidermis and Gastrodermis.

Hydra head formation depends on an organizing center in which Wnt/ß-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/ß-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 ß-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/ß-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.

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/ß-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.

Combining RNAi-Mediated ß-Catenin Inhibition and Reaggregation to Study Hydra Whole-Body Regeneration.

In addition to its ability to regenerate any amputated body part, the Hydra freshwater polyp shows the amazing ability to regenerate as a full polyp after a complete dissociation of its tissues. The developmental processes at work in reaggregates undergoing whole-body regeneration can be investigated at the molecular level by RNA interference (RNAi). Here we provide a protocol that combines ß-catenin RNAi with reaggregation. This protocol serves as a basis to generate "RNAi-reaggregates," followed by the extraction of high-quality RNA for the precise quantification of gene expression by real-time PCR. This protocol is efficient, providing both a molecular signature, with the significant downregulation of ß-catenin and Wnt3, as well as a robust phenotype, the lack of axis formation, which is observed in all reaggregates.

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

Repair, regenerate and reconstruct: meeting the state-of-the-art.

The seventh EMBO meeting on the Molecular and Cellular Basis of Regeneration and Tissue Repair took place in Valletta, Malta, in September 2018. Researchers from all over the world gathered together with the aim of sharing the latest advances in wound healing, repair and regeneration. The meeting covered a wide range of regeneration models and tissues, identification of regulatory genes and signals, and striking advances toward regenerative therapies. Here, we report some of the exciting topics discussed during this conference, highlighting important discoveries in regeneration and the perspectives for regenerative medicine.