A regulatory program for initiation of Wnt signaling during posterior regeneration

Abstract

11Whole-body regeneration requires the re-establishment of body axes for appropriate patterning 12 of new and old tissue. Wnt signaling has been utilized to correctly regenerate tissues along the 13 primary axis in many animals. However, the causal molecular mechanisms that first launch Wnt 14 signaling during regeneration are poorly characterized. We used the acoel worm Hofstenia 15 miamia to identify processes that initiate Wnt signaling. Transcriptome profiling, in situ 16 hybridization, and functional studies revealed a Wnt ligand, wnt-3, as an early wound-induced 17 gene specifically activated in posterior-facing wound sites and was required for establishing 18 posterior identity during regeneration. wnt-3 was upregulated upon amputation in stem cells, 19 and its inhibition affected stem cell proliferation. Ectopic expression of anterior markers in wnt-3 20 RNAi head fragments was stem cell dependent. Chromatin accessibility data revealed that wnt-21 3 activation during regeneration required input from the general wound response. Additionally, 22 the expression of a different Wnt ligand, wnt-1, prior to amputation was required for activation of 23 wound-induced wnt-3 expression. Our study establishes a gene regulatory network for initiating 24 Wnt signaling in posterior tissues in a bilaterian. 25 26 Keywords 27 Regeneration, axial polarity, acoel, Wnt signaling, wound response, posterior identity 28 29 Although it has been hypothesized that transient suppression of the repressor must occur to 63 enable Wnt3 activation at oral-facing wound sites(Nakamura et al., 2011), and expression 64 analysis shows a corresponding absence of Sp5 prior to Wnt3 activation(Vogg et al., 2019), the 65 mechanisms leading to transcription of the Wnt3 locus upon amputation are not known in 66 cnidarians. 67 It is also unknown which transcriptional programs induce Wnt ligand expression upon 68 wounding in bilaterians. In planarian regeneration, mechanisms for inhibition of Wnt signaling pathways for initiation of Wnt 72 ligand expression, which occurs at both anterior-and posterior-facing wound sites, are yet to be 73 identified. The control of Wnt signaling during regeneration has not yet been investigated in 74 acoels. Here, we sought to assess the dynamics of Wnt pathway expression during 75 regeneration and to identify mechanisms that drive its activation upon amputation in Hofstenia. 76 Our analysis of the regeneration transcriptome of Hofstenia revealed that Wnt ligands 77 and other posterior markers are expressed at posterior-facing wound sites within six hours 78 following amputation. To find candidate genes for the initiation of this expression, we focused on 79 the earliest asymmetries between anterior-and posterior-facing wound sites during 80 regeneration. We found that a combination of a generic wound response factor and a pre-81 existing patterning gradient activates a Wnt ligand specifically at posterior-facing wound sites 82 within three hours upon wounding. Specific establishment of Wnt signaling at posterior-faci...

Transgenesis in the acoel worm Hofstenia miamia

“…The function of muscle cells in regeneration has recently been emphasized in planarians (Witchley et al, 2013;Scimone, Cote and Reddien, 2017;Tewari et al, 2019;Scimone et al, 2020), where they appear to guide progenitor cells to their correct location, notably by establishing a chemical coordinate system, through release of signaling molecules (Position Control Genes, PCGs; e.g., several wnt, fz-4, bmp4, admp). Quite remarkably, despite the ancient divergence of acoels and planarians (550 mya), Hofstenia body wall muscles seem to share, at least partially, this instructive role as they express homologs of PCGs that are required for patterning of tissue during regeneration, and, as in planarians, PCG expression is triggered in muscle by regeneration (Raz et al, 2017;Ramirez, Loubet-Senear and Srivastava, 2020). Our results show a high degree of interconnection between the different muscle compartments (Fig.…”

“…Hofstenia is capable of this "wholebody" regeneration and harbors adult stem cells referred to as "neoblasts". A sequenced genome, transcriptomic resources, and robust and systemic RNA interference (RNAi) have enabled functional studies of regeneration and stem cells in Hofstenia (Srivastava et al, 2014;Raz et al, 2017;Gehrke et al, 2019;Ramirez, Loubet-Senear and Srivastava, 2020), and we sought to leverage the biology of this system further by developing tools for transgenesis.…”

Genomic analyses reveal FoxG as an upstream regulator ofwnt1required for posterior identity specification in planarians

“…During Hydra head regeneration, an enhancer collection become accessible allowing the expression of cWNT genes in head organizing cells (69,70). In acoels, egr is expressed after amputation triggering the expression of wnt3, which participates in the posterior specification (67,71). As recently proposed, it could be that enhancers may be maintained as part of conserved gene regulatory network modules over evolution (72).…”

“…Genomic studies in different regenerative species have identified different sets of TFs as regulators of cWNT genes during regeneration. In Drosophila, injured imaginal disc required 'regenerative enhancers' to trigger wingless expression and the regeneration process (66)(67)(68). During Hydra head regeneration, an enhancer collection become accessible allowing the expression of cWNT genes in head organizing cells (69,70).…”

Oral Regeneration Is the Default Pathway Triggered by Injury inHydra

“…A conserved role for bZIPs in activating Wnt signaling during regeneration is supported by the findings that Erk is required for the injury-induced activation of Wnt signaling in planarians (Owlarn et al, 2017) and bZIP TFBMs are required for Wnt expression during imaginal wing disc regeneration in Drosophila (Harris et al, 2016). In contrast, it was recently reported that the zinc finger TF Egr is required for Wnt expression during acoel regeneration (Ramirez et al, 2020). It will therefore be important for future research to characterize injury-responsive GRNs across diverse animal taxa to better understand the evolutionary history of Wnt signaling and its regulation during wound repair.…”