PROJECT SUMMARY / ABSTRACT The ability of cells to traverse basement membranes (BMs) is a key part of fertility, development, immunity, and disease. BM invasion is facilitated through expression of extracellular matrix proteins, upregulation of matrix metalloproteinases, polarization of the F-actin cytoskeleton, and cell cycle arrest. Precise coordination of these pro-invasive programs is largely achieved through transcriptional regulation; however, our understanding of the gene regulatory networks (GRNs) involved is limited due to the lack of model systems in which cell invasion can be visualized live. Here, I propose to fill this gap in knowledge by utilizing morphogenesis of the Caenorhabditis elegans uterine-vulval connection as a tractable and visually amenable model to examine cell invasion in vivo. During development of the hermaphroditic somatic gonad, a specialized uterine cell called the anchor cell (AC) invades through the underlying BM to connect the uterus to the vulval epithelium. The AC itself is specified in a cell fate decision event earlier in development, in which two initially equipotent cells diverge via stochastic Notch asymmetry, giving rise to the presumptive AC and a proliferative ventral uterine (VU) cell. Prior research by our lab and others has identified six transcription factors (TFs) that regulate AC invasion. These include the basic leucine zipper TF fos-1 (Fos), the basic helix-loop-helix TF hlh-2 (E/Daughterless), two nuclear hormone receptors, nhr-67 (NR2E1/Tailless/TLX) and sex-1 (RARB/NR1B2), as well as two zinc-finger TFs, egl-43 (EVI1/MEL1) and mep-1. These TFs appear to be functioning in at least three distinct GRN sub-circuits to regulate AC invasion, one of which involves NHR-67, which functions upstream of CKI-1 (p21/p27) to induce G1 cell cycle arrest. Remarkably, five of the six pro-invasive TFs function reiteratively during the AC/VU cell fate decision. These include the three TFs comprising the NHR-67/cell cycle-dependent pro-invasive pathway (EGL- 43S, MEP-1, and NHR-67), as well as HLH-2 and SEX-1, which have predicted binding sites within the nhr-67 promoter. Thus, based on the literature and my preliminary studies, my central hypothesis is that the AC invasive program is dependent on the function of multiple GRN sub-circuits, one of which modulates cell cycle arrest and is reiteratively used in AC fate specification. In Aim 1 of this project, I will dissect the cis- and trans-regulation of AC invasion, focusing on the cell cycle-dependent GRN sub-circuit involving the pro-invasive TF nhr-67/TLX. In Aim 2, I will examine the roles of pro-invasive TFs that reiteratively function in AC specification and investigate if cell cycle control is the common denominator underlying these two processes. Cutting-edge functional tools, including an endogenous protein depletion system and a novel cell cycle state sensor, paired with the ability to perform high-resolution subcellular visual analyses, will allow f...