Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. We identified a zinc finger transcription factor, Smed-ZicA, as a downstream target of Smed-FoxD, a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. We suggest a model in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

Background: Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown. Results: To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning. Conclusions: Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians.

Wnt/β-catenin signaling regulates tissue homeostasis and regeneration in metazoans. In planarians-flatworms with high regenerative potential-Wnt ligands are thought to control tissue polarity by shaping a β-catenin activity gradient along the anterior-posterior axis, yet the downstream mechanisms are poorly understood. We performed an RNA sequencing (RNA-seq)-based screen and identified hundreds of β-catenin-dependent transcripts, of which several were expressed in muscle tissue and stem cells in a graded fashion. In particular, a teashirt (tsh) ortholog was induced in a β-catenin-dependent manner during regeneration in planarians and zebrafish, and RNAi resulted in two-headed planarians. Strikingly, intact planarians depleted of tsh induced anterior markers and slowly transformed their tail into a head, reminiscent of β-catenin RNAi phenotypes. Given that β-catenin RNAi enhanced the formation of muscle cells expressing anterior determinants in tail regions, our study suggests that this pathway controls tissue polarity through regulating the identity of differentiating cells during homeostasis and regeneration.

The freshwater polyp Hydra provides a potent model system for investigating the conditions that promote wound healing, reactivation of a developmental process and, ultimately, regeneration of an amputated body part. Hydra polyps can also be dissociated to the single cell level and can regenerate a complete body axis from aggregates, behaving as natural organoids. In recent years, the ability to exploit Hydra has been expanded with the advent of new live-imaging approaches, genetic manipulations that include stable transgenesis, gene silencing and genome editing, and the accumulation of high-throughput omics data. In this Primer, we provide an overview of Hydra as a model system for studying regeneration, highlighting recent results that question the classical self-enhancement and long-range inhibition model supposed to drive Hydra regeneration. We underscore the need for integrative explanations incorporating biochemical as well as mechanical signalling.

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.

Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here we show that members of the Integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that Integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.