The specific aims of RegEvolve are:
1) Understanding the mechanistic contribution of Wnt pathway activity to the regenerative prowess of the model species S. mediterranea and the differences in pathway regulation that cause the regeneration defect in D. lacteum.
2) Comparative analysis of the genomic mechanisms regulating Wnt pathway deployment in the two species;
3) Using our live collection of >50 species, to systematically explore the correlation of Wnt signalling levels and planarian regeneration defects to ultimately understand why some worms regenerate while others cannot.
With respect to the first aim, RegEvolve has significantly advanced our understanding of why Wnt signaling is so important in planarian regeneration. In the regenerating model species S. mediterranea, we have shown that establishes a long-range signalling gradient along the A/P axis, that Wnt-mediated Wnt expression is criticial in the establishment and maintenance of the gradient and that the switch-like function of the pathway during regenerative head/tail specification involves mutual antagonism with an unknown patterning system deployed from the head tip. Further, we have established critical tools for the requisite dissection of the gene regulatory consequences of Wnt signaling in planarians (see below). With respect to identifying differences in pathway regulation that cause regeneration defects, we had to deviate from the originally envisaged Smed/Dlac comparisons for the reason that we discovered that the genomic distances between the two species were too great for the envisaged comparative genomics approaches. We instead focused on understanding the regeneration defect in the species G. tigrina, which in a body size, position and piece-length dependent manner regenerates heads instead of tails. We could show that this defect involves the expression of the Wnt inhibitor notum at both head and tail wounds, consistent with the guiding hypothesis that gene regulatory changes of Wnt pathway components cause species-specific regeneration defects (J. Cleland, in preparation).
The second aim of RegEvolve has significantly advanced the state of planarian genomics. A major achievement has been the assembly and publication of the reference genome of the model species S. mediterranea in 2017. In addition, RegEvolve has supported a significant expansion of PlanMine, the sequence database that we maintain for the planarian research community. As platform for sharing and mining genome and transcriptome data, PlanMine constitutes a lasting contribution of RegEvolve to the field. In terms of specific aims, we originally intended to sequence and compare a single, regeneration-deficient species, D. lacteum. However, our discovery of deep splits and consequently large genomic distances between planarian lineages necessitated a broadening of our genome sequencing efforts to include additional species. We have sequenced and assembled 6 additional planarian genomes during the course of RegEvolve as a basis for comparative genomics approaches in planarian systems. In addition, we have developed CHIPseq and ATACseq protocols to annotate and study gene regulatory sequences that we are currently using to sprobe the gene regulatory dimension of Wnt-signaling in the S. mediterranea genome (M. Ivancovik, in preparation). Although the various technical challenges that we encountered during these pioneering efforts have delayed the completion of the objectives, the publications and release of the genomes on PlanMine are now in preparation.
With respect to the third aim, RegEvolve came a long way to answering the question of why some worms regenerate, while others cannot. Based on our large live collection of planarian species, the results of this multi-year effort include a highly accurate multi-gene phylogeny of the taxon, the assaying and mapping of regenerative abilities on planarian phylogeny, the conclusion that head regeneration emerged at the base of the lineage and was lost secondarily at multiple independent points of the lineage, the demonstration that demonstration that Wnt signalling can rescue head regeneration all across planarian phylogeny and an exploration of the roles of Wnt signalling pleiotropies in the reproductive system as a potential explanation as to why regeneration-impairing levels of pathway activity might have been selected at multiple independent points. Overall, our multi-scale effort suggests that the question of why some worms regenerate while others cannot may be linked i) to the choice amongst two reproductive strategies, i.e. asexual reproduction by fission and subsequent regeneration or sexual reproduction via eggs and embryos, for which regeneration is dispensible; ii) conflicting selection pressures on the activity of signalling pathways with pleiotropic roles in regeneration and sexual reproduction, such as Wnt signalling.
Besides the published and soon-to-be published manuscripts, RegEvolve findings have been disseminated via multiple international conference presentation, including the Kestone Conference on Regeneration and tissue repair in 2016, the Euro EvoDevo conference in Gallway in 2018, the international Planarian meeting in Madison in 2018 or most recently in multiple talks of Rink department members during the world-wide flatworm Friday seminar series. Further important outreach activities included the participation of my group in the annual “long night of science” outreach event in Dresden or the reglar “Max Planck goes to school” event in Goettingen.
