Development of the C. elegans reproductive tract is orchestrated by the anchor cell (AC). Among other things, this occurs through a cell invasion event that connects the uterine and vulval tissue. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. EGL-43 is the C. elegans homolog of the human EVI1/MECOM proto- oncogene, and recently, a mechanistic connection has been made between its loss and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling is mitogenic in the absence of EGL-43. To reevaluate the relationship between EGL-43 and LIN-12, we designed and implemented a heterologous co expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating the AIDHB approach using AID-tagged GFP, we sought to test this approach using AID-tagged alleles of egl-43 and lin-12. Auxin-inducible degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively. This combined approach revealed that LIN-12 is not required for AC proliferation following loss of EGL-43, which contrasts with a double RNAi experiment directed against these same targets. The addition of AIDHB to the C. elegans transgenic toolkit should facilitate functional in vivo imaging of cell-cycle associated phenomena.

The auxin-inducible degradation system has been widely adopted in the Caenorhabditis elegans research community for its ability to empirically control the spatiotemporal expression of target proteins. This system can efficiently degrade auxin-inducible degron (AID)-tagged proteins via the expression of a ligand-activatable AtTIR1 protein derived from A. thaliana that adapts target proteins to the endogenous C. elegans proteasome. While broad expression of AtTIR1 using strong, ubiquitous promoters can lead to rapid degradation of AID-tagged proteins, cell type-specific expression of AtTIR1 using spatially restricted promoters often results in less efficient target protein degradation. To circumvent this limitation, we have developed a FLP/FRT3-based system that functions to reanimate a dormant, high-powered promoter that can drive sufficient AtTIR1 expression in a cell type-specific manner. We benchmark the utility of this system by generating a number of tissue-specific FLP-ON::TIR1 drivers to reveal genetically separable cell type-specific phenotypes for several target proteins. We also demonstrate that the FLP-ON::TIR1 system is compatible with enhanced degron epitopes. Finally, we provide an expandable toolkit utilizing the basic FLP-ON::TIR1 system that can be adapted to drive optimized AtTIR1 expression in any tissue or cell type of interest.

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that must exit the cell cycle and enter a post-mitotic to initiate contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the negative cell cycle regulators that maintain the AC in a post-mitotic, invasive state. Although our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in maintaining the post-mitotic state of the AC, loss of CKI-1 alone or in combination with other negative cell cycle regulators-including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (Beta-TrCP) resulted in proliferating ACs that retained their invasive abilities. Upon examination of the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs maintain pro-invasive gene expression. We therefore report that maintenance of the post-mitotic state is not necessary for AC invasion, breaking the previously established C. elegans invasion/proliferation dichotomy.

The auxin-inducible degradation system has been widely adopted in theC. elegansresearch community for its ability to empirically control the spatiotemporal expression of target proteins. This system can efficiently degradeauxin-inducibledegron (AID)-tagged proteins via the expression of a ligand-activatableAtTIR1 protein derived fromA. thalianathat adapts target proteins to the endogenousC. elegansproteosome. While broad expression ofAtTIR1 using strong, ubiquitous promoters can lead to rapid degradation of AID-tagged proteins, cell type-specific expression ofAtTIR1 using spatially restricted promoters often results in less efficient target protein degradation. To circumvent this limitation, we have developed a FLP/FRT3-based system that functions to reanimate a dormant, high-powered promoter that can drive sufficientAtTIR1expression in a cell type-specific manner. We benchmark the utility of this system by generating a number of tissue specific FLP-ON::TIR1 drivers to reveal genetically separable cell type-specific phenotypes for several target proteins. We also demonstrate that the FLP-ON::TIR1 system is compatible with enhanced degron epitopes. Finally, we provide an expandable toolkit utilizing the basic FLP-ON::TIR1 system that can be adapted to drive optimizedAtTIR1expression in any tissue or cell type of interest.

Development of the C. elegans reproductive tract is orchestrated by the anchor cell (AC). This occurs in part through a cell invasion event that connects the uterine and vulval tissue. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. Recently, a mechanistic connection has been made between loss of EGL-43 and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling has mitogenic effects in the invasive AC. To reexamine the relationship between EGL-43 and LIN-12, we first designed and implemented a heterologous co-expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating AIDHB using AID-tagged GFP, we sought to test it by using AID-tagged alleles of egl-43 and lin-12. Auxin-induced degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively, which revealed that LIN-12 is not required for AC proliferation following loss of EGL-43.