Mechanisms underlying the establishment of whole-body regeneration competence

Division Of Basic Sciences - Nci

Investigators

Abstract

We initiated studies to determine when and how whole-body regenerative abilities are established during development and published our findings in Current Biology. We report that planarians, unlike mammals, gradually acquire whole-body regenerative abilities during development. We conducted amputation assays to address when embryos were capable of regenerating missing adult tissues, and discovered differences in onset of anterior (head) and posterior (tail) regenerative abilities during development. Posterior regeneration was observed by Stage 6.5 and was axial position-independent by Stage 7.5. In contrast, we observed marked axial position- and stage-dependent effects on anterior regeneration prior to hatching (Stage J0). Fragments cut within the anterior or along the midline that kept more of the main body axis intact were capable of head regeneration sooner than fragments that retained only posterior patterning information. Recut assays demonstrated that head regeneration-incompetent fragments acquired anterior regenerative abilities as the tissue matured. Anterior regeneration abilities were axial position-independent by juvenile stages. We determined that irradiation-sensitive cells are required for regeneration of new tissues during development, but that their presence is not sufficient for head regeneration. Instead, the ability to perform anteroposterior axis reset dictated onset of head regeneration competence during development. Prior to hatching, posterior fragments have difficulty reforming the anterior pole, a signaling center that effects local inhibition of canonical Wnt signaling to promote head regeneration. Strikingly, RNAi knock-down of the Wnt effector gene ß-catenin-1 elicited precocious head formation in posterior fragments that are normally head regeneration-incompetent. Next, we determined developmental stage-dependent differences in the transcriptional injury response and showed that regeneration is blocked upstream of the axis reset decision in head regeneration-incompetent fragments. Wound-responsive expression of immediate early genes (jun-1, fos-1, and egr-2), occurred similarly in regeneration-incompetent and -competent fragments. Patterning genes pivotal for polarity determination, including notum, wnt-1, and follistatin, were not induced in embryonic Stage 7 fragments but they were expressed in Juvenile J2 fragments, in irradiation-sensitive cells proximal to the wound site. Interestingly, in adult S. mediterranea these patterning genes are induced in longitudinal (notum, follistatin) and circular muscle fibers (wnt-1). These data led us to hypothesize that onset of head regeneration competence is linked to body wall musculature development. As a companion piece to our Current Biology publication, we wrote "Protocol combining RNA Interference and Regeneration Assays in Planarian Embryos" (https://doi.org/10.1101/2025.07.21.666018). Here we provide a how-to guide for RNAi construct design, double stranded (ds)RNA synthesis, embryo staging, amputations, double stranded (ds)RNA soaking of amputated fragments, and downstream assays for qualitative characterization and quantitative determination of knock-down efficacy. We demonstrate utility and efficacy of dsRNA soaking to elicit penetrant knock-down phenotypes in regenerating Stage 7 fragments for Spol-ß-catenin-1, Spol-ovo, and Spol-foxA1. Consistent with reports that Smed-ovo is required for eye maintenance and regeneration, decapitated Stage 7 posterior fragments treated with Spol-ovo(RNAi) regenerated eyeless animals. Similarly, Stage 7 anterior fragments treated with Spol-foxA1(RNAi) failed to regenerate the pharynx , phenocopying Smed-foxA1(RNAi) regenerates. Moving forward, we will continue adapting RNAi and ex vivo embryo culturing protocols for use with intact embryos at earlier developmental stages (S5-S6.5).