SignificanceWe explore the utility of expansion microscopy (ExM) in neuroscience and developmental biology using the zebrafish model. Regarding neuroscience studies, ExM enables the tracing of cellular processes in the zebrafish brain, as well as the imaging of synapses and their biomolecular content and organization. Regarding development, ExM enables the resolving of nuclear compartments, particularly nuclear invaginations and channels, and helps relate such cellular nanostructures to proteins of the cytoskeleton during embryogenesis.

Highlights d wnt-3 is upregulated early in posterior wounds and is required for tail regeneration d wnt-3 is expressed in stem cells and is needed for stem cell proliferation at wound sites d A general wound response factor, egr, activates wnt-3 during regeneration d Pre-existing patterning information is required for wnt-3 upregulation

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...