Characterizing cellular protrusion based stem cell communication in Schmidtea mediterranea

Abstract

Regeneration is widespread across the animal kingdom with diverse organisms such as zebrafish, snails, axolotls and mammals showing varying degrees of regenerative potential which ranges from whole body regeneration to that restricted to specific tissue types. Regeneration encompasses recognition of missing tissue, wound healing, repatterning of body axes followed by cell proliferation and differentiation. This process involves communication between various cells of the body to successfully replace lost tissue. Stem cells are a key component of this process. The planarian Schmidtea mediterranea is a master at whole body regeneration capable of regeneration the entire body from a fragment consisting of ~1000 cells within a span of two weeks making it an exceptional model to study regeneration. This ability is conferred by the presence of adult pluripotent stem cells called neoblasts. Here we show that neoblasts form distinct subpopulations based on cytoskeletal staining. We also find that neoblasts make long projections which connect them to each other both in vivo and cell culture. Through proteomics we identify 213 differentially expressed genes in neoblast subpopulations based on cytoskeletal stains. We then conducted an RNAi screen to identify potential genes regulating the formation of neoblast protrusions. We observed size defect/ degrowth phenotypes and regeneration defect phenotypes. These genes were highly expressed in protonephridia, pharynx and intestinal progenitors. Using various cytoskeletal inhibitors, we showed that neoblast protrusions can be ablated both in whole mounts and in cell culture. These protrusions may play a role in stem cell communication during regeneratioRegeneration is widespread across the animal kingdom with diverse organisms such as zebrafish, snails, axolotls and mammals showing varying degrees of regenerative potential which ranges from whole body regeneration to that restricted to specific tissue types. Regeneration encompasses recognition of missing tissue, wound healing, repatterning of body axes followed by cell proliferation and differentiation. This process involves communication between various cells of the body to successfully replace lost tissue. Stem cells are a key component of this process. The planarian Schmidtea mediterranea is a master at whole body regeneration capable of regeneration the entire body from a fragment consisting of ~1000 cells within a span of two weeks making it an exceptional model to study regeneration. This ability is conferred by the presence of adult pluripotent stem cells called neoblasts. Here we show that neoblasts form distinct subpopulations based on cytoskeletal staining. We also find that neoblasts make long projections which connect them to each other both in vivo and cell culture. Through proteomics we identify 213 differentially expressed genes in neoblast subpopulations based on cytoskeletal stains. We then conducted an RNAi screen to identify potential genes regulating the formation of neoblast protrusions. We observed size defect/ degrowth phenotypes and regeneration defect phenotypes. These genes were highly expressed in protonephridia, pharynx and intestinal progenitors. Using various cytoskeletal inhibitors, we showed that neoblast protrusions can be ablated both in whole mounts and in cell culture. These protrusions may play a role in stem cell communication during regeneration and emerge as novel facilitators of stem cell fate specification.n and emerge as novel facilitators of stem cell fate specification.