Booth, C. L. T. | Search bioRxiv

bioRxiv2025

Protocol combining RNA interference and regeneration assays in planarian embryos

Planarian flatworms gradually acquire whole-body regeneration abilities during late embryonic and juvenile development. Here we show how to perturb gene function with RNA interference (RNAi) in S. polychroa embryos at developmental stages capable of wound healing following amputation. We describe embryo staging, embryo amputation and double-stranded RNA soaking, phenotype analysis, and techniques for qualitative and quantitative assessment of RNAi knock-down efficacy. This protocol is adaptable for use with embryonic stages amenable to ex vivo culturing and with other embryo-producing flatworm species. For complete details on the use and execution of this protocol, please refer to 1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/666018v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d4bf29org.highwire.dtl.DTLVardef@1524f6forg.highwire.dtl.DTLVardef@dd40b0org.highwire.dtl.DTLVardef@1c1f6bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Axis reset is rate limiting for onset of whole-body regenerative abilities during planarian development

Few studies have investigated whether or how regenerative abilities vary across developmental stages of animal life cycles. Determining mechanisms that promote or limit regeneration in certain life cycle stages may pinpoint the most critical factors for successful regeneration and suggest strategies for reverse-engineering regenerative responses in therapeutic settings. In contrast to many mammalian systems, which typically show a loss of regenerative abilities with age, planarian flatworms remain highly regenerative throughout adulthood. The robust reproductive and regenerative capabilities of the planarian Schmidtea polychroa (Spol) make them an ideal model to determine when and how regeneration competence is established during development. We report that Spol gradually acquires whole body regenerative abilities during late embryonic and early juvenile stages. Posterior (tail) regenerative abilities are constitutive, whereas anterior (head) regenerative abilities are dependent on developmental stage, tissue composition of the amputated fragment, and axial position of the cut plane. Stem-like cells are required, but not sufficient, for onset of head regeneration ability. We propose that regulation of main body axis reset, specifically the ability to remake an anterior organizing center, is a rate-limiting factor for establishment of whole-body regeneration competence. Supporting this hypothesis, knock-down of the canonical Wnt pathway effector Spol {beta}-catenin-1, a posterior determinant, induces precocious head regeneration under conditions that are normally head regeneration incompetent. Our results suggest that regeneration competence emerges through interactions between cycling stem-like cells, the cellular source of new tissue, and developing adult tissue(s) harboring axial patterning information.

developmental biology↗