Analysis of Lineage Specification of Planarian Stem Cells

Abstract

Regeneration is a fascinating mystery. Planarians are freshwater flatworms that show extensive regeneration capacity. They comprise a population of adult pluripotent stem cells called neoblasts that are necessary for regeneration. Neoblasts are a heterogeneous population of stem cells, with individually pluripotent as well as multipotent stem cells. Gene expression studies have been the primary tool for investigating the cellular complexity and neoblast lineages and a vast amount of literature exists that describes different types of neoblasts based on their transcription profile. Neoblast differentiation is found to be closely related to the expression of fate-specific transcription factors (FSTFs) that are upregulated as a neoblast specifies. Despite extensive work that describes the significance of FSTFs for regeneration and differentiation of respective lineages, there is little to no understanding of the regulators of lineage choice during neoblast specification. We hypothesized that transcription factors upstream of FSTFs could be determining lineage choice. Two approaches were used to test this hypothesis: Chromatin Fiber Sequencing and Motif Enrichment Analysis. Since Chromatin Fiber Sequencing had not been attempted in planarians before, this study aimed to optimize the protocol of Fiber-seq for S.med. While the resultant protocol is much simplified and more optimized, further experiments are required to generate a good quality dataset. The second approach resulted in the identification of three different transcription factor families, namely, C2H2 Zinc Finger Factors, Rfx-6 related factors and Nuclear Factor Y Complex C. Forty-five candidate S.med. genes were found corresponding to these TF families after appropriate thresholding. An RNAi screen was conducted for these 45 predicted factors to screen for regeneration and differentiation defects. Six out of forty-five candidate factors presented with diverse regeneration abnormalities and potential differentiation defects. Downstream analysis to narrow down the expression of the validated transcription factors resulted in enrichment in both neoblast and non-neoblast clusters, indicating a broader role of the TFs in neoblast function.