Control and Implementation of a Morphogenetic Program

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PROJECT SUMMARY / ABSTRACT Cellular morphogenesis is essential to animal development as well as wound healing and cancer. During this process, cells coordinately change shape, migrate, and may fuse. Cellular morphogenesis is executed by molecules called "effectors" involved in junctions, cytoskeleton, cell polarity, vesicular trafficking, and other modules/processes of the cytological machinery. The sex-, tissue-, position-, and time-specificity of morphogenesis is determined by "regulators", such as transcription factors. There are two main knowledge gaps: (1) how morphogenetic events are temporally scheduled, and (2) how the transcriptional regulatory input is linked to the effectors. Here, the overall objective is to elucidate an intriguing part of the timing mechanism that involves a cytoplasmic long-noncoding RNA in post-translational regulation, and to delineate the "missing links" between regulators and effectors for an experimentally accessible structure, the tail tip of C. elegans. The tail tip is made of 4 cells which, in males only, change shape and migrate at the juvenile-to-adult transition (J/A), a process called Tail Tip Morphogenesis (TTM). Prior studies showed that (1) a long non-coding RNA (lep-5) is an instructive switch for TTM onset at the J/A, and (2) transcription factor DMD-3 is required and sufficient for TTM and coordinates several processes of the cytological machinery. The overall approach is to (Aim 1) test the hypothesis that the lep-5 lncRNA acts to schedule TTM cell-autonomously by scaffolding the ubiquitination of a central, conserved node in the heterochronic pathway, LIN-28, by the C. elegans Makorin protein, LEP-2 (another highly conserved but under-characterized protein), and (Aim 2) to determine what are the direct targets of DMD-3 and how they function in TTM. Aim 1 will use primarily co-immunoprecipitation (coIP) and ubiquitination assays to test binding partners of lep-5 lncRNA. Aim 2 will use chromatin immunoprecipitation (ChIP-seq) to identify candidate targets of DMD-3; the roles of these candidates in TTM will then be tested by labeling them or genetically perturbing them in a strain with subcellular landmarks tagged with different fluorescent proteins. The expected outcomes include (1) a mechanistic understanding for a novel paradigmatic role for a lncRNA acting instructively and post-translationally in the regulation of a key conserved temporal regulator, and (2) the foundation of a systems network model for morphogenesis connecting transcriptional control by DMD-3 (a conserved transcription factor homologous to human DMRTs) to the cytological machinery involved in morphogenetic cell behaviors. These results are expected to have additional positive impacts on biology and medicine by (a) determining how sexual dimorphism occurs by linking sex determination to morphogenetic effectors; (b) identifying key conserved genes controlling morphogenesis and its timing, thus potentially identifying new targets for treating defects in pubertal timing, sex-reversal or cancer, or to aid wound healing; and (c) providing candidate genes that may underlie evolutionary diversity.