The role of lin-12 notch in C. elegans anchor cell proliferation

The gonadal anchor cell (AC) in C. elegans hermaphrodites is specified at the beginning of the second larval stage (L2) from one of two equivalent precursor cells (Z1.ppp and Z4.aaa) (Greenwald, 2005). Reciprocal lin-12 notch signaling between the two AC precursors determines the AC fate. The precursor cell exhibiting higher LIN-12 NOTCH activity adopts the ventral uterine (VU) cell fate, while the cell with lower LIN-12 NOTCH activity adopts the default AC fate. Hence, if LIN-12 NOTCH signaling is inactive at this stage, two ACs are specified at the expense of a VU cell (Greenwald et al., 1983). After its specification, the AC permanently arrests in the G1 phase of the cell cycle. Maintaining the AC arrested in G1 is necessary for its later AC functions during vulval development, especially for breaching the basement membranes (BMs) during AC invasion at the late L3 stage (Matus et al., 2015). The two transcription factors NHR-67, a nuclear receptor of the tailless family, and EGL-43, a homolog of the mammalian EVI1 proto-oncogene, are required to keep the AC arrested in the G1 phase (Medwig-Kinney et al., 2020; Deng et al., 2020). NHR-67 acts primarily by up-regulating the expression of the CDK inhibitor CKI-1. A search for EGL-43 targets identified the notch receptor lin-12 as a gene repressed by EGL-43. Loss of eg-43 resulted in an up-regulation of lin-12 expression in the AC and expression of a constitutively active LIN-12 intracellular fragment (NICDΔCT) induced AC proliferation (Deng et al., 2020; Martinez et al., 2022). However, there are conflicting reports on the consequences of inhibiting LIN-12 NOTCH signaling in the AC. While Deng et al. (2020) found that simultaneous inhibition of egl-43 and lin-12 by RNAi prevented AC proliferation, Martinez et al. (2022) reported that down-regulation of LIN-12 through the auxin-inducible degradation system (AID) did not suppress AC proliferation.

To resolve these conflicting observations, we used the lin-12(lf) (null) allele (n137n720) (Greenwald et al., 1983) in combination with egl-43 or vector control RNAi and scored AC proliferation at the late L3 (Pn.pxx) stage. Since lin-12(lf) results in the specification of two ACs during the early L2 stage, the numbers of proliferating ACs in lin-12(lf) mutants were normalized to the two ACs initially formed per animal. Our results indicated that a complete loss of lin-12 function reduces AC proliferation during the L3 stage to a similar degree as lin-12 RNAi (Fig. 1). However, neither lin-12(lf) nor lin-12 RNAi completely blocked AC proliferation, indicating that EGL-43 prevents AC proliferation not exclusively by repressing lin-12 expression.

Thus, a complete loss or strong down-regulation of lin-12 is required to reduce AC proliferation caused by inhibiting egl-43. AID-mediated degradation of LIN-12 may leave sufficient residual LIN-12 activity for the AC to proliferate. The observation that LIN-12 AID causes an early AC duplication phenotype (Martinez et al., 2022) could indicate that relatively strong LIN-12 NOTCH signaling in late L1 and early L2 larvae is necessary to prevent specification of the default AC cell fate. Hence, a partial reduction in LIN12 activity may be sufficient to permit both precursor cells to differentiate into ACs but not to prevent AC proliferation. Finally, it should be noted that not in every case reported AC proliferation depended on lin-12 notch activity. For example, lin-12 RNAi did not suppress the AC proliferation phenotype caused by nhr-67 RNAi (Deng et al., 2020), and overexpression of the hox gene lin-39 induced AC proliferation independently of lin-12 (Heinze et al., 2023), pointing to a variety of mechanisms that maintain the quiescent state of the AC.

Strain used: AH6848, genotype: +/hT2[bli-4(e937) let(q782) qIs48] (I;III); rrf-3(pk1426) II; unc-32(e189) lin-12(n137n720) III/ hT2[bli-4(e937) let(q782) qIs48] (I;III); qyIs10 IV; qyIs24 X.

The RNAi clone was obtained and sequence-verified from a C. elegans genome-wide RNAi library (Source BioScience). Bacteria were grown overnight in 2 ml of LB medium containing 200 µg/ml ampicillin and 25 µg/ml tetracycline at 37°C, diluted 1:100 in LB medium containing the antibiotics and 1 mM IPTG, and grown for another 4 to 6 h at 37°C before seeding them on NGM plates containing 1 mM IPTG. Embryos from strain AH6848 were isolated by hypochlorite treatment of gravid adults and allowed to hatch overnight in M9 buffer to obtain synchronized L1 larvae that were plated on NGM plates seeded with E. coli producing egl-43 dsRNA or containing the empty RNAi vector. Animals were grown for 38 to 42 h at 20°C until late L3 when they were analyzed. Heterozygous lin-12(lf)/+ siblings grown on the same RNAi plates as the lin-12(lf) homozygotes were used as controls.

Nomarski and fluorescent images of late L3 larvae at the Pn.pxx stage (before vulval invagination) were acquired with a LEICA DM6000B microscope equipped with a Hammamatsu ORCA FLASH 4.0LT sCMOS camera, a piezo objective drive (Piezosystems Jena, Germany) and a 63x (N.A. 1.32) oil-immersion lens. z-stacks were acquired with a spacing of 0.5 μm, and images were deconvolved using the YacuDecu implementation of CUDA-based Richardson Lucy deconvolution in MATLAB (www.github.com/bobpepin/YacuDecu). ACs were counted across the z-stacks using the qyIs24[Pcdh-3>mcherry::PLCdeltaPH] membrane marker (Ziel et al., 2008) overlaid on the Nomarski channel to visualize the nuclei.

We thank the members of the Hajnal lab for discussion and critical comments.