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 for thorough and rigorous testing of this hypothesis.
Invasion through basement membranes is a key cellular behavior required for the establishment of pregnancy, tissue patterning during development, and immune response to infection and injury, and is also aberrantly deployed during cancer metastasis. The research proposed here is poised to elucidate the transcriptional programs that coordinate cell invasive differentiation in vivo and how they interface with cell cycle control. The knowledge expected to be gained from this work will inform efforts to develop therapies for various invasion- associated pathologies, including preeclampsia, congenital abnormalities, immune disorders, and malignant cancers.
