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. 2020 Jan 3;9:e50065. doi: 10.7554/eLife.50065

Figure 4. A genetic screen identifies the mid-body Hox protein LIN-39 (Scr/Dfd/Hox4-5) as necessary for ectopic expression of VD and VC terminal features.

(A) Representative images of L4-stage WT, unc-3(n3435), unc-3(n3435); kas1, and unc-3(n3435); lin-39(n1760) animals carrying flp-11::gfp (VD/VC marker). Arrowheads point to MN cell bodies with gfp marker expression. (B) Quantification graph summarizing results from panel A. The two right-most bars show quantification of two independent transgenic lines driving lin-39 RNAi specifically in cholinergic MNs (Punc-3 >lin-39 RNAi) of unc-3 (n3435) mutants. N > 15. ***p<0.001. N.S: not significant. (C) Genetic locus of lin-39. Molecular lesions for kas1 and n1760 alleles are shown, as well as the AID::3xFLAG::mNG cassette inserted at the C-terminus (endogenous reporter). (D) Quantification of two VD (ser-2::gfp, oig-1::gfp) and one VC (ida-1::gfp) markers in WT, unc-3 (n3435), unc-3(n3435); lin-39(n1760) animals at L4. The three right-most bars show quantification of three independent transgenic lines driving lin-39 RNAi specifically in cholinergic MNs (Punc-3 >lin-39 RNAi) of unc-3 (n3435) mutants. N > 15. ***p<0.001. (E) Summary of unc-3 and lin-39 expression in cholinergic MNs. See Figure 4—figure supplement 2 for raw data. (F) Schematic that summarizes our findings. In the wild type (Faumont et al., 2011) panel on the left, lin-39 is normally expressed in cholinergic MNs but unable to induce expression of VD or VC genes. In the unc-3 mutant, lin-39 is now able to induce expression of alternative identity features (VD or VC) in distinct MN populations.

Figure 4.

Figure 4—figure supplement 1. Ectopic expression of VD terminal identity markers in unc-3 mutants requires LIN-39 but not UNC-30.

Figure 4—figure supplement 1.

(A) Quantification of unc-30::gfp and VD marker ser-2::gfp in WT and unc-3 (n3435) animals at L4, with ser-2::gfp further examined in unc-3 (n3435); unc-30 (e191) double mutants. Expression of ser-2::gfp is equally affected in unc-3 (n3435) single and unc-3 (n3435); unc-30 (e191) double mutants. N > 15. ***p<0.001. N. S: not significant. (B) Quantification of the VD/VC marker flp-11::gfp expression in L4 stage WT, unc-3 (n3435) mutant, unc-3 (n3435); kas1, and transgenic animals that rescue unc-3 (n3435); kas1 background with expression of lin-39 in cholinergic MNs (Punc-3 >lin-39) or expression of GFP-tagged lin-39 fosmid. Two independent transgenic lines were used for Punc-3 >lin-39 OE and lin-39 fosmid. Of note, the partial rescue observed with the Punc-3 >lin-39 OE lines likely arises because the fragment of unc-3 promoter used to drive lin-39 is not expressed in all 39 cholinergic MNs.
Figure 4—figure supplement 2. LIN-39 is continuously required to activate distinct terminal identity genes in sex-shared and sex-specific cholinergic MNs.

Figure 4—figure supplement 2.

(A) Co-localization analysis to determine the lin-39 expression pattern with single-cell resolution. Each column represents an individual MN in the VNC of a hermaphrodite worm. Top seven rows represent seven randomly-selected worms carrying a GABA marker (unc-47::RFP) and the lin-39::mNG::3xFLAG::AID endogenous reporter allele. Bottom seven rows represent seven randomly-selected worms carrying a cholinergic marker (cho-1::RFP and the lin-39 fosmid-based reporter wgIs18 [lin-39fosmid::GFP]. A color-filled lattice indicates co-expression of the known identity marker and lin-39 in the individual MN. (B) Representative images of L1- and L4-stage animals co-expressing unc-47::rfp (labels only DD at L1, labels both DD and VD at L4) and endogenous lin-39 marker (lin-39::mNG). Arrowheads point to cell bodies of DD and VD MNs co-expressing both markers. White dotted line indicates the boundary of intestinal tissue (gut), which tends to be autofluorescent in the green channel. (C) Auxin or ethanol (control) were administered at larval stage 4 (L4) on lin-39::mNG::3xFLAG::AID; eft-3::TIR1 animals carrying the sex-shared cholinergic MN markers acr-2::gfp and unc-77::gfp. Images were taken at the young adult stage (day 1 for acr-2::gfp and day 2.4 for unc-77::gfp) and the number of MNs expressing these markers was quantified. A statistically significant decrease is evident in the auxin-treated animals compared to EtOH-treated controls. For comparison, quantification of marker expression is also provided in WT and lin-39 (n1760) animals. N > 12. *p<0.05, **p<0.01, ***p<0.001. (D) Auxin or ethanol (control) were administered at larval stage 3 (L3) on lin-39::mNG::3xFLAG::AID; eft-3::TIR1 animals carrying the VC marker srb-16::gfp. Quantification was performed at the young adult stage (day 1.5). A significant decrease in the number of MNs expressing the VC marker was evident in the auxin-treated animals compared to EtOH-treated controls. For comparison, quantification of marker expression is also provided in WT animals. N > 15. **p<0.01, ***p<0.001. N. S: not significant. (E) Quantification of VD/VC marker flp-11::gfp in WT, lin-39 (n1760), unc-30 (e191) and lin-39 (n1760); unc-30 (e191) double mutant animals at L4 stage. Double mutants showed a more severe reduction in flp-11::gfp expression compared to each single mutant. N > 20. ***p<0.001. (F) Expression of GABA biosynthesis markers (unc-25, unc-47) is not affected in lin-39 (n1760); mab-5 (e1239) double mutants at the L4 stage. N > 15; N. S: not significant.