The major objectives of the proposed research are to illuminate the cellular and molecular mechanisms that control plasticity of developing and differentiated cells in vivo and to investigate how post-mitotic differentiated cells can be transdifferentiated and remodeled into new cell types. The well-elucidated pathway for endoderm development in C. elegans will be applied to the molecular dissection of transdifferentiation and transorganogenesis (conversion of one organ into another). One component in this pathway, the ELT-7 GATA-type transcription factor, is capable of overriding the embryonic multipotency commitment transition (MCT), which normally locks cells into their differentiated states and prevents them from being reprogrammed. ELT-7 can cause differentiated pharynx cells in adults to be remodeled into cells with ultrastructural characteristics, and gene expression patterns, of intestinal cells, without intercession of a dedifferentiated intermediate or cell division. ELT-7 can also convert the developing uterus and vas deferens into mini-guts, which show typical fine-structure gut morphology and that can be physically isolated. With these preliminary findings in hand, we will probe the mechanisms of transdifferentiation through three Specific Aims.
In Aim 1, we will evaluate the specificity of targeting ELT-7 to the uterus and will test the hypothesis that uterus gut transorganogenesis involves superimposition of gonadal gene repression and recapitulation of normal gut development through analysis of transdifferentiated organs and transcriptional profiling of isolated mini-guts. We will investigate the mechanisms of cellular remodeling during transdifferentiation, including the requirement for protein degradation, and will test the hypothesis that a discrete domain of ELT-7 allows for its ability to promote transdifferentiation.
In Aim 2, we will investigate the role of cell-autonomous regulators, including PHA-4/FoxA, genes in the endoderm cascade, and iPS-promoting genes, as well cell-cell signaling processes, including Notch, EGF/ras and cell fusion, in modulating susceptibility to transdifferentiation.
In Aim 3, we will undertake a comprehensive analysis of molecular components that regulate reprogramming by classical and functional genomics (RNAi-based) screens and direct genetic selections and, with our collaborators, will test the generality of the C. elegans transdifferentiation mechanisms in the zebrafish vertebrate model and in reprogramming of human fibroblasts to iPS cells. These studies may advance our understanding of the mechanisms involved in pre-cancerous metaplasias of the digestive tract. They could also lead to methods for generating patient-specific replacement organs and other strategies that advance regenerative medicine, and will provide insights into the mechanisms of organ malformation in birth defects.
During their development, cells that are initially non-specialized undergo differentiation, causing them to adopt identities that endow them with the properties that are essential to perform their specialized functions. Once cells have selected a particular identity, they typically remain committed to that role and do not subsequently switch to a completely different type of cell. We propose to reveal the genetic and molecular mechanisms that cause cells to lock onto particular differentiated, specialized identities and will investigat how they can be provoked to switch to adopt entirely different identities. The findings from these studies could lead to new methods for creating replacement tissues and organs in regenerative medicine, will contribute to our understanding of malformed tissues and organs in birth defects, and may provide important information about particular cancers of the digestive tract.
|Spickard, Erik A; Joshi, Pradeep M; Rothman, Joel H (2018) The multipotency-to-commitment transition in Caenorhabditis elegans-implications for reprogramming from cells to organs. FEBS Lett 592:838-851|
|Dineen, Aidan; Osborne Nishimura, Erin; Goszczynski, Barbara et al. (2018) Quantitating transcription factor redundancy: The relative roles of the ELT-2 and ELT-7 GATA factors in the C. elegans endoderm. Dev Biol 435:150-161|
|Riddle, Misty R; Spickard, Erik A; Jevince, Angela et al. (2016) Transorganogenesis and transdifferentiation in C. elegans are dependent on differentiated cell identity. Dev Biol 420:136-147|