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. 2021 Sep 3;219(3):iyab127. doi: 10.1093/genetics/iyab127

Figure 2.

Figure 2

Ectopic expression of HLH-12 in the AC does not change its fate but compromises its function. WT represents animals lacking the +HLH-12 transgene but containing other markers as noted. (A) The number of apical invadopodia is reduced in ACs that ectopically express HLH-12. Invadopodia were examined in WT and +HLH-12 animals using lag-2p::2xNLS-yfp to mark the nucleus of the AC and zmp-1p::mcherry::MoesinABD to visualize invadopodia. ***P < 0.001, Kolmogorov–Smirnov; n = 31 (WT) or 35 (+HLH-12). (B) Endogenous GFP::FOS-1 expression is downregulated in ACs that ectopically express HLH-12. WT and +HLH-12 animals carry gfp::fos-1(bmd138) (Medwig-Kinney et al. 2020). In the bar graph, ***P < 0.001, Fisher’s exact test; n = 17 (WT) or 29 (+HLH-12). In the photomicrographs, the AC is surrounded by white boxes. Scale bar is 5 µm. (C) ACs are displaced horizontally by ectopic expression of HLH-12. The cartoon depicts the method used to quantify displacement of the AC with respect to P6.pxx nuclei as a ratio of two measurements in the anterior–posterior axis, B/A, as depicted in the cartoon (see Materials and Methods and Supplementary Figure S2A for further details); no dorsal-ventral displacement was seen at this stage of development (Supplementary Figure S1B). Photomicrographs show representative ACs for WT and +HLH-12; lag-2p::2xnls-tagRFP marks AC and P6.pxx nuclei; cdh-3p::gfp marks AC cytoplasm. (D) Expression of endogenous INA-1::GFP is activated in the AC by ectopic expression of HLH-12. The photomicrographs show early L2 hermaphrodites, with arIs222[lag-2p::2xnls-rfp] marking the nucleus of the AC; in the +HLH-12 hermaphrodite, the yellow arrow points to cell membrane of the AC showing ectopic INA-1::GFP (bottom). 9/16 +HLH-12 and 0/15 WT hermaphrodites showed ectopic INA-1::GFP expression. Scale bar is 5 µm. (E) RNAi against factors known to be required for fDTC migration suppresses displacement of ACs caused by ectopic expression of HLH-12. In +HLH-12 animals treated with negative control RNAi (lacZ), the AC is displaced compared to animals lacking the +HLH-12 transgene [genotype arIs222[lag-2p::2xnls-tagrfp] him-5(e1490), labeled “WT”]; treatment of animals carrying the +HLH-12 transgene with the positive control RNAi (hlh-12), or RNAi against ina-1, pdf-1, or cdc-42 abrogates this displacement. **P < 0.01, ***P < 0.001, Fisher’s exact test. The statistics shown for the positive control and experimental RNAis are compared to +HLH-12 treated with lacZ(RNAi). n = 20–52 for each genotype. This panel shows one trial; an additional trial for each RNAi is shown in Supplementary Figure S2. (F) Loss of FOS-1 does not affect AC displacement in +HLH-12 animals at the L3 stage (Pn.pxx). n = 18–22 for each genotype. Note that fos-1(0) hermaphrodites were segregated from balanced heterozygotes (see Materials and Methods). Bars show mean.