doublesex functions early and late in gustatory sense organ development

Abstract

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSX(M) and DSX(F), respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSX(M) and DSX(F) promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSX(F) then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.

Figure 1. dsx regulates the number of foreleg GSOs.

(A) The sex determination hierarchy directs the generation of sex-specific DSX and FRU isoforms. The 2∶2 ratio of X chromosomes to autosomes in females sets off a female-specific alternative RNA splicing cascade in which TRA directs splicing of dsx and fru transcripts into the female forms. The lack of TRA activity in males results in the production of male forms of these transcripts. (B–D) poxn-GAL4 driving expression of UAS-mCD8::GFP in a (B) male and (C) female foreleg at 48 h APF. Tarsal segments T1–T5 are indicated. Note that there are more clusters of neurons labeled in the male than in the female in T1–T4. (D) Magnified view of two distinct GSOs. The GRNs (arrows) of each GSO project their dendrites into the base of their cognate bristle (arrowheads). (E) Quantitation of foreleg GSOs in T1–T4. 3XP3DsRed was used to distinguish XY flies from XX flies in a dsx-deficient background where chromosomal sex could not otherwise be distinguished. All XY males had a sex chromosome genotype of w/Y. The genotype of the sex chromosomes of dsx-deficient chromosomal females was w/w, 3XP3DsRed, while all other females were w/y w, 3XP3DsRed. Genotype abbreviations: dsx ⁺ (UAS-mCD8::GFP; FRT82B dsx¹, poxn-GAL4/TM6B). dsx ⁻ (UAS-mCD8::GFP; FRT82B dsx¹, poxn-GAL4/dsx^M+R13). dsx^D (UAS-mCD8::GFP; FRT82B dsx¹, poxn-GAL4/dsx^D). dsx ⁺ and dsx^D are siblings from the same cross. Error bars indicate SEM. P-values are for comparisons between the indicated dsx mutant and dsx ⁺ of the same chromosomal sex. (*p = .07, **p<.0001, † p = .04, ‡ p = .14, Tukey multiple comparisons of means.).

Figure 2. The sexually dimorphic number of foreleg GSOs is specified by 8 h APF.

Male (A) and female (B) forelegs with poxn-GAL4 driving UAS-mCD8::GFP (green) were stained for 22C10 (magenta) at 8 h APF. Merged images on right show overlap in yellow and DAPI-stained DNA (blue). Tarsal segment boundaries indicated with blue lines. Cells marked with 22C10 were classified based both on colocalization of poxn-GAL4 and morphology of the cells or cell clusters: GSO lineage cells (magenta arrows); non-GSO cells that lack poxn-GAL4 in T3 (dark blue arrows); non-GSO cells marked by poxn-GAL4 but lacking GSO morphology in T4 (light blue arrows). Scale bars, 50 µm. (C) Averages and SEMs of quantitated GSO numbers in T2, T3, and T4 for both male (n = 8) and female (n = 9) forelegs at 8 h APF.

Figure 3. DSX^M is present in the foreleg disc epithelium when AC accumulates in proneural clusters.

(A–E) Male foreleg discs from the indicated time points of third instar larval development were stained for AC (green) and DSX^M (magenta). Merged images on right show overlap in white. (A and B) From 36–40 h 3I, DSX^M is present in a crescent within T1 and there is no overlap with AC. (C) At 44 h 3I, DSX^M signal increases across the epithelium of tarsal segments distal to T1 (i.e. toward disc center) and is present in some clusters of AC-positive cells (arrows). (D) At 48 h 3I, DSX^M is present in swaths of epithelial cells in T1–T4 and overlaps in these segments with subsets of the AC-positive cells that are proneural clusters (arrows). A candidate SOP with high levels of AC and DSX^M (barbed arrow) is seen in T2. (E) Magnified view of boxed region in (D). Candidate SOP in T2 (barbed arrow). (F) Same image as (E) with AC (green), DSX^M (red), and stained with DAPI (blue) to visualize all nuclei in the focal planes shown. All images are projections of only those focal planes that encompass the majority of DSX^M signal within the disc. Scale bars (A–D) 50 µm and (E and F) 10 µm.

Figure 4. DSX is present in SOP daughters of the foreleg disc.

(A and B) DSX (magenta) is present across the tarsal segment epithelium in male discs at 0 h APF as well as in subsets of cells expressing ase-lacZ (green) in T5 (arrows) and T4 (boxed area). (B) Magnified view of boxed region in (A) shown as a partial projection. Daughters of a recently divided SOP (arrows). (C) DSX (magenta) is present in the tarsal segment epithelium in male discs at 6 h APF. DSX overlaps with neur-lacZ expression (green) in several cells across T1–T5 (arrowheads) and in a transverse row of cells in T1 that likely correspond to the sex comb bristle lineages (small arrows). (D) T2–T3 from a separate male leg disc at 6 h APF marked as per (C) with DSX (red) in right panel and DAPI-stained DNA (blue). For A–D, images on right are a merge of the left and middle images. Projection of multiple focal planes shown. Projection of multiple focal planes shown. Scale bars (A, C, and D) 25 µm and (B) 10 µm.

Figure 5. dsx regulates axonal morphology independent of GSO number.

(A–F”) fru^GAL4 driving UAS-mCD8::GFP (green) labels GRN cell bodies and axons. Forelegs of (A) control male (UAS-mCD8::GFP/SM6; fru^GAL4/+ ), (B) male with feminized GRNs (UAS-DSX^F/UAS-mCD8::GFP; fru^GAL4/+), and (C) control female (UAS-mCD8::GFP/SM6; fru^GAL4/+). Cuticular autofluorescence (magenta). There is no difference in the number of FRU^M-positive GRN clusters between forelegs of the two male genotypes, while both have more than the female. (D–F”) VNC prothoracic neuromeres with labeled GRN projections (D, E, F; green in D”, E”, F”) and counterstained for DN-cadherin (D’, E’, F’; magenta in D”, E”, F”). Arrowheads indicate the VNC midline. (D–D”) GRNs cross the midline in control males, but feminized male GRNs do not cross (E–E”). (F–F”) GRNs also do not cross the midline in control females.