Developmental reprogramming and transorganogenesis

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SUMMARY The major objectives of the proposed research are to illuminate the cellular and molecular mechanisms that control developmental plasticity and to investigate how post-mitotic differentiated cells in an intact animal can be reprogrammed and remodeled into new cell types in the process of transdifferentiation (Td). The well-described pathway for endoderm development in C. elegans will be applied to molecularly dissecting Td and “transorganogenesis” (conversion of one organ into another). One component in this pathway, the C. elegans ELT-7 GATA transcription factor, is capable of converting differentiated, post-mitotic cells of two organs, the pharynx and uterus, into cells with gene expression patterns and ultrastructural characteristics of normal gut cells. Highly dynamic changes in the transcriptome occur during this remarkable process, and six stages (promiscuity, attenuation, extinction, rebound, persistence, and remodeling) can be resolved during Td based on gene expression and altered cellular and organ morphology. These events define Td-competent (Td+) cells that convert to gut-like cells and Td-resistant (Td-) cells that only transiently permit ELT-7 to activate its target. Among the genes whose expression undergoes upregulation during Td are sets of genes associated with protein turnover, autophagy, and the intracellular pathogen response (IPR), suggesting that these pathways play a role in Td. On the basis of ELT-7-induced developmental arrest, two genetic selections were developed that identify large numbers of mutants defective in Td. With these preliminary findings in hand, we will probe the mechanisms of Td through three Specific Aims. In SA1, we will investigate the dynamics of cellular remodeling, test the hypothesis that turnover processes of protein degradation and autophagy participate in key stages of Td and remodeling, assess the role of the IPR in Td, and investigate the action of cell-cycle exit in the Td process. In SA2, we will evaluate whether differences between single Td+ and Td- cells of varied differentiated cell types can be ascribed to their unique transcriptome dynamics and will test the hypothesis that changes in chromatin architecture of Td- cells, assessed by ATAC-seq, initially and transiently proceed through similar patterns to those of Td+ cells, with elastic reversion to the original state. In SA3, we will characterize mutants from two genetic selections that are defective in ELT-7-mediated developmental arrest and Td and will use them to investigate interdependence of the six stages of Td. We will identify the causal gene(s) underlying large numbers of mutants defective in Td by a high-throughput method based on statistical analysis of variants in pooled genome-wide sequences, including those coding for proteins and non-coding RNAs. These studies may advance our understanding of mechanisms involved in pre-cancerous metaplasias of the digestive tract. They will also provide insights into the mechanisms of organ malformation in birth defects and could lead to methods for reprogramming differentiation for organ generation in regenerative medicine.