Stem cell ontogeny and dynamics in Macrostomum lignano

Division Of Basic Sciences - Nci

Investigators

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Abstract

Macrostomum lignano (Mlig) is an ideal evolutionary and developmental counterpoint to the Schmidtea species for studying adult stem cell biology in different developmental stages and physiological contexts. My lab will make targeted investments in resource generation, establishment of transgenic lines, and technology adaptation to study Mlig adult stem cell regulation and dynamics in vivo. Mlig adults contain 'neoblasts,' a cycling somatic cell population that is ill-defined, but which we hypothesize to be homologous to the PSC and cycling progenitor populations found in Smed. Our first priority is to molecularly and functionally define Mlig neoblast population(s). We will create scRNA-Seq and ATAC-Seq atlases for wildtype and lethally irradiated Mlig adult hermaphrodites, resources necessary for identification of stem cell and tissue-specific biomarkers, and putative promoter and cis-regulatory sequences for neoblast population(s) that will inform construct design for transgenic reporters. These transcriptomic and epigenomic datasets will enable in silico prediction of the neoblast lineage repertoire and will identify candidate regulators of stem cell potency, lineage commitment, and tissue-specific differentiation programs for functional screening by RNAi in the future. Using established methods, we will generate fluorescent reporter lines for neoblast population(s) and will use these tools to analyze neoblast behavior during homeostasis, clonal repopulation, and regeneration. Using cell transplantation and clonal expansion assays following sublethal irradiation, we will determine whether neoblasts are collectively or individually pluripotent, and whether different neoblast subpopulations are functionally equivalent or distinct from one another. Is there progressive winnowing of potency within the neoblast population as lineage-priming occurs, or are presumptive progenitor populations capable of reverting to a more primitive fate? When does lineage commitment occur relative to cell cycle exit? We will perform lineage tracing experiments using inducible fluorescent markers to generate in vivo recordings of stem cell lineages. To monitor stem cell dynamics, we will partner with the Optical Light Microscopy and Image Analysis Lab to develop time-lapse imaging protocols to track stem cell movement and cell division. Live observation of stem cell behavior will provide answers to long-standing questions in the field and will set the stage for detailed characterization of RNAi knock-down phenotypes in the future. For example, how does the blastema form? Do stem cells migrate to wounds? Do progenitor cells undergo transit-amplifying divisions, and if yes, for which tissues, and in what contexts? Ultimately, we seek to address how stereotyped or tailored stem cell behaviors are in response to different physiological challenges, what role local and systemic environmental cues play in determining stem cell behavior, and finally to discover the genetic and molecular mechanisms underlying invariant and adapted behaviors.