Figure 2. A double-negative gene-regulatory circuit: BX-C miRNAs prevent Hth from excluding Dsx in the VNC.

(A) Transcriptome analysis reveals substantial similarity in the larval iab-8 domain of the BX-C miRNA and hth[BSmut] mutants, compared with Canton-S (three biological replicates per genotype). Only genes > 1 RPKM and p < 0.05 are plotted. Dotted gray lines indicate fold change = 1.5.

(B) A notable downregulated transcript in both mutants is the sex-specific differentiation factor doublesex (dsx).

(C) Hth and Dsx proteins are spatially complementary in the adult VNC.

(D) Derepression of Hth in BX-C miRNA mutant (mir[Δ/C11]) and hth[BSmut] compromises accumulation of Dsx in abdominal segments of adult VNCs, especially in dorsal planes as shown here.

(E) Quantification of Dsx⁺ nuclei in wild-type, BX-C miRNA mutant, and hth[BSmut] VNC; total VNC and dorsal half are shown. The number of Dsx+ neurons is compromised in the mutant conditions.

(F) Conversely, the number of other identified abdominal subpopulations that mediate PMRs (VT-switch lines) are not affected by miRNA deletion.

(G) Quantification of Hth and Dsx protein levels in individual abdominal neurons relevant to the female post-mating switch (abdominal SAG-1 neurons). Depression of Hth correlates with reduction of Dsx within identified neurons.

(H) Representative GFP-labeled SAG-1 neurons from higher (Q3) and lower (Q1) quartiles of Dsx, as quantified in (G), with corresponding Dsx and Hth levels. The nuclear levels of each factor (Int) were calculated by subtracting the signals in cytoplasm (marked in green) from the corresponding nucleus (dotted line) for each antigen; samples were co-stained and imaged in parallel within the linear range.

t test with Welch’s correction (B, E, and F); Mann-Whitney non-parametric test (G). *p < 0.05, ***p < 0.001, ****p < 0.0001; ns, not significant. Error bars, SEM. Scale bars, 60 μm (C); 30 μm (D); 2 μm (H).