Specification and maintenance of quiescent progenitor cells set aside for the biphasic life cycle of an invertebrate chordate

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PROJECT SUMMARY/ABSTRACT The objective of this proposal is to characterize the molecular mechanisms that specify, maintain, and protect a discrete compartment of pre-patterned but quiescent and undifferentiated progenitor cells (termed the “Neck”) in the tunicate Ciona. Tunicates like Ciona are the invertebrates most closely related to vertebrates, and possess a unique biphasic life cycle alternating between a short-lived, motile larva and a sessile adult. During metamorphosis, a mass wave of programmed cell death eliminates most differentiated larval cells while sparing set-aside undifferentiated adult progenitor cells that go on to form the majority of adult cell types and structures. Despite this heterochrony in differentiation and the complete remodeling of the post-metamorphic adult, the larval and adult body plans are contiguous and simultaneously patterned during embryonic development. This peculiar arrangement offers a unique opportunity to study how discrete stem cell compartments can be set aside and protected for later developmental potential in spite of the differentiation and cell death around them. The central hypothesis is that the stereotyped specification, maintenance, and survival of quiescent progenitor cells of the Neck are controlled by precise developmental regulation of genes encoding rate-limiting components of diverse biochemical pathways. The rationale underlying the proposed research is that, by exploiting the genomic and cellular simplicity of Ciona and their radical yet naturally stereotyped transition from a motile larval body plan to a sessile adult one, we can understand tissue remodeling and cellular turnover in much greater spatial and temporal detail. The central hypothesis will be tested by pursuing three specific aims: 1) We will test whether Pax2/5/8 is required for establishing the Neck as a discrete compartment of adult progenitor cells. 2) We will test the roles of FGF and Hedgehog signaling in maintaining Neck cells as a quiescent population of undifferentiated cells. 3) We will test whether the survival of Neck cells during the wave of apoptosis that occurs in metamorphosis requires the anti-apoptotic effects of Nitric oxide signaling and a unique class of metalloproteins that bind vanadium, a heavy metal that has been observed to accumulate in tunicate cells but without a known biological function. These aims will be pursued using an innovative approach that combines cell lineage-specific CRISPR/Cas9-based somatic gene knockouts with next- generation sequencing, in vivo fluorescence microscopy and in vitro biochemical assays. The expected outcomes of the proposed work would be to 1) reveal conserved biological mechanisms that might be shared with humans, and/or 2) identify highly divergent proteins and pathways that could nonetheless be harnessed for novel therapeutic tools to treat human disease and injury.