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Comparative analysis of planarian regeneration - why some worms regenerate while others don’t

Periodic Reporting for period 3 - RegEvolve (Comparative analysis of planarian regeneration - why some worms regenerate while others don’t)

Reporting period: 2018-10-01 to 2020-03-31

Many animals have the ability to regenerate body parts lost as a result of accidents and injuries. However, many others (including us humans) do not have this ability. This raises a big and largely unresolved question: Why is it that in a world dominated by survival of the fittest, regeneration is the exception rather than the rule?

RegEvolve systematically explores this question by example of planarian flatworms. Planarians are a common group of worms that occur worldwide in fresh water, land and marine ecosystems. Some planarian species have the astonishing ability to regenerate complete and perfectly proportioned animals from tiny tissue pieces. However, other planarian species cannot regenerate. Comparisons between regenerating and non-regenerating species therefore provide unique opportunities to ask how regeneration works in the first place, what goes wrong in regeneration deficient species and finally, why some worms regenerate while others do not. RegEvolve will explore these three questions with a specific focus on the Wnt signalling pathway, which we have previously shown to be a critical mediator of planarian regeneration and regeneration defects. Overall, RegEvolve will provide fundamental insights into the molecular orchestration of regeneration and the principles that govern the evolution of the trait in the case of planarians.
The specific aims of RegEvolve are:
1) Comparisons of Wnt pathway activity between the regenerating model species S. mediterranea and the regeneration deficient species D. lacteum.
2) Comparative analysis of the genomic mechanisms regulating pathway deployment in the two species species;
3) Using our live collection of >50 species, to systematically explore the correlation of Wnt signalling levels and gene expression signatures with planarian regeneration defects.

With respect to the first aim, RegEvolve has so far established that Wnt signalling in the model species S. mediterranea establishes a long-range signalling gradient along the planarian 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 a patterning system in the head. Further, we have obtained new insights into the mechanism that trigger the b-Catenin switch appropriately at head or tail regenerating wounds. Together with our genome sequencing efforts (see below) and our ongoing efforts to identify Wnt targets and Wnt-responsive regulatory sequences, we are now in a stretgic position to investigate the gene regulatory dimension of planarian Wnt signalling.

We have also made significant progress with respect to the second aim. Our original plan was 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. During the course of RegEvolve, we have so far published the S. mediterranea genome in Nature, and completed the sequencing of a sister species of S. mediterranea (S. polychroa) and three additional Polycelis species. The latter were chosen by virtue of representing a closely related branch of planarian phylogeny that harbours a regeneration-deficient species. However, results obtained during this reporting period indicate that the Polycelis species may be refractory to RNAi, which complicates the functional aspects of the intended comparisons. However, the utility of the additional genomes is that their alignment against the Smed genome can reveal areas in the vicinity of genes that change more slowly than the surrounding seqwuences. Such areas, termed “Conserved Non-Coding Elements (CNEs) are often involved in regulating the expression of genes. Together with Chromatin immune-precipitation protocols against enhancer-associated histone marks that we have developed during this reporting period, we will enlist the CNEs to help us identify the Wnt-dependent regulatory regions in the Smed genome. As such, we will be able to complete at least part of the original objectives of Aim 2.

With respect to the third aim, we made significant progress towards the objective of understanding why regeneration-impairing levels of Wnt signalling have been repeatedly selected in planarian phylogeny. Specifically, we discovered that Wnt signalling exerts a pleiotropic effect on yolk production. Yolk provides the nutrients for embryonic development and more yolk likely improves the survival chances of hatchlings as they emerge at a larger body size. Our finding that yolk production is under positive regulation by Wnt signalling thus provides a putative proximate cause for the evolutionary selection of regeneration-impairing Wnt signalling levels. Unfortunately, our results indicate that neither the critical role of Wnt signalling is conserved outside the Dugesids, nor do yolk amounts correlate with the regenerative abilities of planarians in general. The Wnt/yolk hypothesis is therefore unlikely to answer the question of why some worms regenerate while so many do not and we will use the remaining time of RegEvolve to evaluate other possibilities.
RegEvolve is a basic research project that aims to contribute insights to the basic question why in a world shaped by survival of the fittest, regeneration is the exception rather than the rule. To our knowledge, our systematic analysis of the regenerative abilities of >50 planarian species in terms of phylogeny, genomic information and molecular mechanisms is so far unparalleled in breadth and depth.