Autonomous and non-autonomous regulation on planarian growth and regeneration: Smed-bls, canonical Wnt signalling and Fox family

Abstract

Development requires an increment of cell growth and cell number, concomitant to a tightly control of cell differentiation. Thanks to cell communication, cells can be spatiotemporal patterned to acquire the required fate. Planarians are a unique model to study developmental processes due to their ability to regenerate and modulate their body size according to the nutrient availability. This body plasticity is based on the presence of pluripotent adult stem cells (neoblasts) and the continuous activation of the intercellular communication mechanisms. This active regulation of stem cells fate make them perfect models to study processes as growth, patterning, differentiation cell proliferation or cell death. In this thesis we have studied different molecular mechanisms that control planarian growth and pattern.

We have described a novel gene family, blitzschnell (bls), formed by de novo and taxonomically restricted genes, which control cell number trough the regulation of cell proliferation and cell death. Nutrient intake controls its expression suggesting that bls family have evolved in planarians as a mechanism by which to restrict cell number in nutrient-fluctuating environments. During growth and regeneration, planarians are not only able regulate their body and organ size accordingly but they also maintain a proper pattern. This regulation is mediated by different signalling centres that specify different regions along the 3 body axes (AP, DV and ML). Particularly, after an amputation, the anterior and the posterior planarian tips behave as organizers (signalling centre), specifying the fate of each planarian pole. The anterior organizer is defined by notum (a Wnt inhibitor) and the posterior by wnt1 expression. The inhibition of any of those elements leads to a shift in polarity. During the first hours of regeneration both notum and wnt1 are expressed in both poles, and it’s around 36 hours that their expression becomes restricted to their respective tip. To decipher the molecular interactions that restrict the expression of wnt1 to the posterior tip and confer the organizing activity we used genome wide approaches. ATAC-seq and RNA-seq analysis of regenerating wild-type and wnt1 (RNAi) planarians allowed the identification of specific Cis-Regulatory Elements (CREs) of posterior regeneration. We found that already at 12 hours of regeneration the accessible CREs in posterior and anterior blastemas have essentially changed, indicating that specific posterior chromatin changes induced by amputation occur much earlier than the formation of the organizers. Furthermore, we have identified specific transcription factors (TF) of the Otx and Fox families, which are enriched in posterior CREs. Particularly, pitx and foxG regulates wnt1+ cells and are essential for the specification of the posterior cells.

TFs regulate patterning events and developmental specification, particularly the Fox Family exerts crucial roles defining cell types of all germ cell layers or regulating cell cycle. Before this Thesis, poorly was known about the Fox family in Schmidtea mediterranea (Smed) neither in the Lophocotrozoan clade. In this study we have identified 27 Fox genes in Smed, classified in 13 families: A, At, C, D, E, G, L1t, QD, J1, N2/3, Nt, O and P. We have performed an extensive phylogenetic study of the family to understand the evolution of the Fox family in this clade. Furthermore, we have studied the sequence, expression and function of several planarian Fox genes.

Overall, we studied different molecular mechanisms that regulate planarian growth and regeneration, and that provide novel data concerning development and evolution.