A reevaluation of the relationship between EGL-43 (EVI1/MECOM) and LIN-12 (Notch) duringC. elegansanchor cell invasion

Abstract

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.

An expandable FLP-ON::TIR1 system for precise spatiotemporal protein degradation inC. elegans

“…We elected to optimize the FLP/FRT 3 recombination system in this context as this FLP/FRT 3 combination has previously been demonstrated to work efficiently in C. elegans (Munoz-Jimenez et al 2017). We then generated a rpl-28 p::>STOP>:: At TIR1(F79G)::T2A::DHB::2xmK2 construct that harbors a >STOP> cassette (“>” denotes the FRT 3 site) immediately upstream of the At TIR1(F79G)::T2A::DHB::2xmK2 sequence (Martinez et al 2022). Next, using CRISPR/Cas9 genome engineering we inserted this construct into the C. elegans genome at a defined safe harbor site on chromosome II, corresponding to the ttTi5605 MosSCI insertion site (Frokjaer-Jensen et al 2008).…”

“…For example, our attempts to use the ckb-3 promoter to drive At TIR1 expression in these cell types failed to achieve significant LIN-12::mNG::AID depletion in the somatic gonad and also failed to produce the associated cell transformation phenotypes (2 AC phenotype) (Fig. S2C) (Martinez et al 2022; Taylor N Medwig et al 2022). This inability to recapitulate the lin-12(0) phenotype is likely caused by an ineffective expression of At TIR1 in Z1.ppp and Z4.aaa or their precursors during the experimental time course.…”

“…Plasmid pYX023_ rpl-28p ::>STOP>:: At TIR1(F79G)::T2A::DHB::2xmK2 contains the At TIR1(F79G) coding sequencing and a fragment of human DNA helicase B (DHB) fused with 2xmKate2 (Hills-Muckey et al 2022; Martinez et al 2022) (STOP cassette flanked by FRT3 sites includes two stop codons, TAATAG, followed by the let-858 -3’UTR).…”

“…Invasion of the AC into the vulval epithelium was scored by the visualization of the CDK activity sensor (Adikes et al, 2020; Martinez et al 2022) and a gap in the basement membrane underneath the AC. In strains with the endogenous labeled LAM-2::mNG, an intact yellow fluorescent barrier under the AC was used to assess invasion.…”

“…>STOP>:: At TIR1(F79G)::T2A::DHB::2xmK2 construct that harbors a >STOP> cassette (">" denotes the FRT 3 site) immediately upstream of the At TIR1(F79G)::T2A::DHB::2xmK2 sequence(Martinez et al 2022). Next, using CRISPR/Cas9 genome engineering we inserted this construct into the C. elegans genome at a defined safe harbor site on chromosome II, corresponding to the ttTi5605 MosSCI insertion site(Frokjaer-Jensen et al 2008).…”