Invasive AC fate correlates to high levels of NHR-67.

(A) Schematic of C. elegans anchor cell (AC, magenta) and ventral uterine (VU, blue) cell fate specification from the Z1 and Z4 somatic gonad precursor cell lineages (p, posterior daughter; a, anterior daughter). (B) Micrographs depicting AC and VU cell differentiation over developmental time. AC/VU precursors express LAG-2 (H2B::mTurquoise), which eventually becomes restricted to the AC, whereas VU cells express LAG-1 (mNeonGreen) post-specification. The differentiated AC (cdh-3p::mCherry::moeABD) then invades through the underlying basement membrane (LAM-2::mNeonGreen). (C-D) Representative heat map micrographs (C) and quantification (D) of GFP-tagged HLH-2 and NHR-67 expression in the AC and VU cells at the time of AC invasion. (E) Expression of Notch (lin-12::mNeonGreen) and Delta (lag-2::P2A::H2B::mTurquoise2) following RNAi-induced knockdown of nhr-67 compared to empty vector control. (F) Micrographs depicting the ectopic invasive ACs (cdh-3p::mCherry::moeABD, arrowheads) and expanded basement membrane (laminin::GFP, arrows) gap observed following heat shock induced expression of NHR-67 (hsp::NHR-67::2x-BFP) compared to non-heat shocked controls. (G) Schematic summarizing AC and VU cell fates that result from perturbations of NHR-67 levels. For all figures: asterisk (*), AC/VU precursor; plus (+), VU precursor; solid arrowhead, AC; open arrowhead, VU cell; arrows, basement membrane breach. Statistical significance determined by Student’s t-test (*p > 0.05, **p > 0.01, ***p > 0.001). Scale bars, 5 µm.

NHR-67 expression is downregulated in VU cells through direct transcriptional regulation by HLH-2.

(A-B) Representative heat map micrographs (A) and quantification (B) of NHR-67::GFP expression in VU cells following heat shock induced expression of HLH-2 (2x-BFP) compared to non-heat shocked controls. (C) Schematic of a 276 bp putative regulatory element within the promoter of nhr-67 (Bodofsky et al., 2018), annotated with the location of three hypomorphic mutations (pf2, pf88, and pf159). (D) Yeast one-hybrid experiment pairing HLH-2 Gal4-AD prey with the 276 bp fragment of the nhr-67 promoter as bait on SC-HIS-TRP plates with and without competitive inhibitor 3-AT (175 mM).

NHR-67 dynamically compartmentalizes in nuclei of VU cells.

(A) Heat-map maximum intensity projection of NHR-67::GFP showing protein localization in the AC and VU cells. (B) Spatial color coded projection of NHR-67::GFP punctae in VU, with nuclear border indicated with a dotted line. (C) Schematic of DNA Helicase B (DHB) based CDK sensor and its dynamic localization over the cell cycle. (D) Graphs depicting CDK activity levels and corresponding cell cycle state (top), and percentage of cells exhibiting NHR-67::GFP punctae (bottom) over time, aligned to anaphase. (E) Representative time-lapse of NHR-67::GFP over the course of a cell cycle, with cell membranes indicated with dotted lines. (F) Time-lapse depicting NHR-67::GFP punctae fusion prior to cell division. Bottom panels are pseudo-colored. (G-H) Quantification (G) and representative images (H) depicting fluorescence recovery of NHR-67::GFP following photobleaching of individual punctae (arrow).

Groucho homologs LSY-22 an UNC-37 colocalize with NHR-67 punctae and contribute to maintenance of VU cell fate.

(A) Co-visualization of NHR-67 with RNA Polymerase II (GFP::AMA-1), HP1 heterochromatin proteins (HPL-1::mKate2 and HPL-2::mKate2), and Groucho homologs (TagRFP-T::LSY-22 and mNeonGreen::UNC-37) in VU cells using endogenously tagged alleles. (B) Quantification of colocalization, with plot reporting Manders’ overlap coefficients compared to negative controls (90 degree rotation of one channel) and positive controls. (C) Schematic of the auxin inducible degron (AID) system, where AtTIR1 mediates proteasomal degradation of AID-tagged proteins in the presence of auxin. (D) Representative images of phenotypes observed following individual AID-depletion of UNC-37 and LSY-22 compared to control animals without AID-tagged alleles. All animals compared here are expressing TIR1 ubiquitously (rpl-28p::AtTIR1::T2A::mCherry::HIS-11) and an AC marker (cdh-3p::mCherry::moeABD). Insets depict different z planes of the same image. (E) Quantification of AC marker (cdh-3p::mCherry::moeABD) expression in ectopic ACs resulting from AID-depletion of UNC-37 and LSY-22 compared to control AC and VU cells.

POP-1 is enriched in VU cells and colocalizes with NHR-67 punctae.

(A-B) Expression of mNeonGreen::UNC-37 and mNeonGreen::LSY-22 (A) and eGFP::POP-1 (B) in the AC/VU precursors pre-specification (left), as well as in the AC and VU cells post-specification (right). (C) Quantification of UNC-37, LSY-22, and POP-1 expression at the time of AC invasion. (D) Co-visualization of NHR-67::mScarlet-I and eGFP::POP-1 in the VU. (E) Quantification of POP-1 and NHR-67 colocalization, with plot reporting Manders’ overlap coefficient compared to negative and positive controls. (F) Representative micrographs showing expression of POPTOP, a synthetic pop-1-activated reporter construct, in wild-type ACs, VU cells, and their precursors. Insets depict different z planes of the same image. (G-H) Micrographs (G) and quantification (H) of eGFP-tagged POP-1 expression in proliferative ACs following RNAi depletion of nhr-67 compared to empty vector control.

Ectopic ACs arise through VU-to-AC cell fate transformation.

(A) Representative images of ectopic AC (cdh-3p::mCherry::moeABD; LAG-2::P2A::H2B::mTurquoise2) phenotypes observed following RNAi depletion of POP-1. Schematics (right) depict potential explanations for observed phenotypes. (B) Expression of AC markers and a VU cell marker (LAG-1::mNeonGreen, inverted to aid visualization) in pop-1(RNAi) treated animals over time. (C) Quantification of LAG-2 (magenta) and LAG-1 (blue) expression in transdifferentiating cells produced by pop-1(RNAi) over time.

NHR-67 binds to UNC-37 through IDR-mediated protein-protein interaction.

(A) Predicted structure of NHR-67 generated by AlphaFold. (B) Measure of intrinsic disorder of NHR-67 using the PONDR VSL2 prediction algorithm. (C) Schematic of NHR-67 protein coding sequences used for Yeast two-hybrid experiments with reference to its intrinsically disordered region (IDR, magenta), DNA binding domain (DBD, green), and ligand binding domain (LBD, cyan). Scale bar, 10 amino acids. (D) Yeast two-hybrid experiment shows pairing of UNC-37 with either full-length NHR-67 or the IDR alone allows for yeast growth in the presence of competitive inhibitor 3-AT (20 mM). (E) Possible models of the roles of NHR-67, UNC-37, LSY-22, and POP-1 in maintenance of AC and VU cell fate. In the ventral uterine cells, the association of NHR-67 with the Groucho/TCF complex may result in repression of NHR-67 targets (top) or sequestration of NHR-67 away from its targets (bottom).

Expression of pro-invasive transcription factors EGL-43 and FOS-1 in the somatic gonad.

(A) Schematic of the AC pro-invasive gene regulatory network comprised of four transcription factors: EGL-43, FOS-1, HLH-2, and NHR-67. (B-C) Representative heat-map micrographs (B) and quantification (C) of GFP-tagged EGL-43 and FOS-1 expression in the AC and VU cells.

NHR-67-deficient ACs express both Notch and Delta.

Quantification of Notch (LIN-12::mNeonGreen) (A) and Delta (LAG-2::P2A::H2B::mTurquoise2) (B) expression in nhr-67(RNAi) treated ACs compared to empty vector control AC and VU cells.

Onset of expression and regulatory interaction between NHR-67 and HLH-2 in the somatic gonad.

(A) Micrographs depicting onset of GFP-tagged HLH-2 and a wCherry-labeled NHR-67 transgene (inverted to aid visualization) in Z1.pp and Z4.aa cells at early (top) and late (bottom) stages. (B-D) Representative micrographs (B) and quantification (C-D) of GFP-tagged HLH-2 and TagRFP-T-tagged NHR-67 in AC (C) and VU cells (D) following uba-1(RNAi) compared to control. Insets depict different z planes of the same image.

Knock-in alleles of nhr-67.

(A) Representative images of VU cells exhibiting punctae formed by NHR-67 tagged with GFP, mNeonGreen, mScarlet-I, and TagRFP-T. (B) Schematics of the new endogenously tagged loci generated in this paper for nhr-67. Scale bar, 100 base pairs (bp).

Knock-in alleles of lsy-22.

Schematics of the new endogenously tagged loci generated in this paper for lsy-22. Scale bar, 100 base pairs (bp).

UNC-37 mutants show ectopic expression of AC markers.

Ectopic expression of AC marker (cdh-3p::mCherry::moeABD) in hypomorphic (unc-37(e262wd26)) and null (unc-37(wd17wd22)) alleles of unc-37 compared to wild-type unc-37. Insets depict different z planes of the same image.

Expression of LSY-22, UNC-37, and POP-1 over developmental time.

(A-B) Developmental series (A) and quantified expression (B) of mNeonGreen::UNC-37, mNeonGreen::LSY-22, and eGFP::POP-1 expression in the AC/VU precursors, AC, and VU cells over time. Following AC/VU cell specification, animals are staged by the division of the underlying primary vulval precursor cells (1° VPCs).

POP-1 function in VU cells is distinct from activating role in distal somatic gonad.

(A) Schematics representing the dual functions of POP-1. In the presence of Wnt signaling, POP-1 binds to its co-activator β-catenin (e.g., SYS-1) and activates transcription of its target genes. In the absence of Wnt signaling, POP-1 binds to its co-repressor Groucho (UNC-37) and represses transcription of its target genes. (B) Representative micrographs of eGFP::POP-1 and POPTOP (pes-10::7x-TCF::mCherry) expression in the AC, dorsal uterine cells (DU), spermatheca/sheath cells (SS), and VU cells. (C) Schematic of SYS-1 (β-catenin) expression in the Z1/Z4 lineage (based on Philips et al., 2007).

POP-1 expression is regulated by the cell cycle-dependent pro-invasion pathway.

(A-B) Representative micrographs of eGFP::POP-1 and POPTOP (pes-10::7x-TCF::mCherry) following RNAi-induced knockdown of pro-invasive transcription factors and chromatin modifiers compared to control AC and VU cells. (B) Quantification of eGFP::POP-1 expression in ACs following RNAi treatments. Here, the presence of multiple ACs are the result of failure of the AC to exit the cell cycle.

POP-1 functions to regulate AC/VU cell fates post-specification.

(A) Schematic of the anti-GFP nanobody protein degradation system (based on Wang et al., 2017). (B) Micrographs demonstrating that the anti-GFP nanobody (driven under the egl-43L promoter) is not expressed pre-specification or even shortly after when the presumptive AC begins to express its differentiated cell reporter (cdh-3). (C) With decreased levels of pop-1, a low penetrance (∼7%) of multi-AC phenotypes were observed.

Ectopic ACs resulting from pop-1 perturbation express VU cell markers.

Expression of AC markers (cdh-3p::mCherry::moeABD; LAG-2::P2A::H2B::mTurquoise2) and VU marker (LAG-1::mNeonGreen) in pop-1(RNAi) treated animals compared to empty vector control.

NHR-67 exhibits protein-protein interaction with UNC-37.

Yeast two-hybrid experiment pairing UNC-37 and NHR-67 Gal4-AD prey with LSY-22, NHR-67, and POP-1 Gal4-DBD bait on SC-HIS-TRP-LEU plates with and without competitive inhibitor 3-AT (20 mM).