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

<p>(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.</p

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

<p>Male (A) and female (B) forelegs with <i>poxn-GAL4</i> driving <i>UAS-mCD8::GFP</i> (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 <i>poxn-GAL4</i> and morphology of the cells or cell clusters: GSO lineage cells (magenta arrows); non-GSO cells that lack <i>poxn-GAL4</i> in T3 (dark blue arrows); non-GSO cells marked by <i>poxn-GAL4</i> 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.</p

<i>dsx</i> regulates axonal morphology independent of GSO number.

<p>(A–F”) <i>fru^GAL4</i> driving <i>UAS-mCD8::GFP</i> (green) labels GRN cell bodies and axons. Forelegs of (A) control male (<i>UAS-mCD8::GFP/SM6; fru^GAL4/+</i> ), (B) male with feminized GRNs (<i>UAS-DSX^F/UAS-mCD8::GFP; fru^GAL4/+</i>), and (C) control female (<i>UAS-mCD8::GFP/SM6; fru^GAL4/+</i>). 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.</p

<em>doublesex</em> Functions Early and Late in Gustatory Sense Organ Development

<div><p>Somatic sexual dimorphisms outside of the nervous system in <em>Drosophila melanogaster</em> 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 <em>doublesex</em> (<em>dsx</em>) 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.</p> </div

<i>dsx</i> regulates the number of foreleg GSOs.

<p>(A) The sex determination hierarchy directs the generation of sex-specific DSX and FRU isoforms. The 2∶2 ratio of <i>X</i> chromosomes to autosomes in females sets off a female-specific alternative RNA splicing cascade in which TRA directs splicing of <i>dsx</i> and <i>fru</i> transcripts into the female forms. The lack of TRA activity in males results in the production of male forms of these transcripts. (B–D) <i>poxn-GAL4</i> driving expression of <i>UAS-mCD8::GFP</i> 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. <i>3XP3DsRed</i> was used to distinguish <i>XY</i> flies from <i>XX</i> flies in a <i>dsx</i>-deficient background where chromosomal sex could not otherwise be distinguished. All <i>XY</i> males had a sex chromosome genotype of <i>w</i>/<i>Y</i>. The genotype of the sex chromosomes of <i>dsx-</i>deficient chromosomal females was <i>w/w, 3XP3DsRed,</i> while all other females were <i>w</i>/<i>y w, 3XP3DsRed.</i> Genotype abbreviations: <i>dsx</i>⁺ (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/TM6B</i>). <i>dsx</i>⁻ (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/dsx^M+R13</i>). <i>dsx^D</i> (<i>UAS-mCD8::GFP</i>; <i>FRT82B dsx¹, poxn-GAL4/dsx^D</i>). <i>dsx</i>⁺ and <i>dsx^D</i> are siblings from the same cross. Error bars indicate SEM. P-values are for comparisons between the indicated <i>dsx</i> mutant and <i>dsx</i>⁺ of the same chromosomal sex. (*p = .07, **p<.0001, † p = .04, ‡ p = .14, Tukey multiple comparisons of means.).</p

DSX is present in SOP daughters of the foreleg disc.

<p>(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 <i>ase-lacZ</i> (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 <i>neur-lacZ</i> 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.</p

Other examples of 12A variants.

<p>(<b>A</b>) An example of the ectopic branch phenotype in T1 (compare with the typical T1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g002" target="_blank">Fig 2B</a> and the ectopic branch phenotype of T2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">Fig 3A</a>). All examples were bilaterally symmetrical with a fully formed commissure (arrowhead), in contrast to 12A in T2, which often exhibits bilateral asymmetry. (<b>B</b>) Example of ventral arch routing of the late-born 12A neurons in T2. Compare the branch on the left (arrowhead) with the unoccupied Neuroglian-stained tract on the right (arrow). (<b>C</b>) Transverse optical section of S3 in which 12A shows the T2 morphology (with unsplit bundles). Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">Fig 6B</a> and the variants of 12A that are missing the intermediate bundle as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g003" target="_blank">3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155957#pone.0155957.g006" target="_blank">6A</a>. (<b>D</b>) Transverse optical section of T1 in which the left bundle fails to split. (<b>E</b>) Two examples of animals with duplicated hemilineages. (<b>F</b>) One of two cases in which one hemilineage ectopically projected all of its neurites to the contralateral side, leaving one T1 hemisegment completely uninnervated by 12A and the other double innervated.</p

Genetic and Environmental Control of Neurodevelopmental Robustness in <i>Drosophila</i>

<div><p>Interindividual differences in neuronal wiring may contribute to behavioral individuality and affect susceptibility to neurological disorders. To investigate the causes and potential consequences of wiring variation in <i>Drosophila melanogaster</i>, we focused on a hemilineage of ventral nerve cord interneurons that exhibits morphological variability. We find that late-born subclasses of the 12A hemilineage are highly sensitive to genetic and environmental variation. Neurons in the second thoracic segment are particularly variable with regard to two developmental decisions, whereas its segmental homologs are more robust. This variability “hotspot” depends on Ultrabithorax expression in the 12A neurons, indicating variability is cell-intrinsic and under genetic control. 12A development is more variable and sensitive to temperature in long-established laboratory strains than in strains recently derived from the wild. Strains with a high frequency of one of the 12A variants also showed a high frequency of animals with delayed spontaneous flight initiation, whereas other wing-related behaviors did not show such a correlation and were thus not overtly affected by 12A variation. These results show that neurodevelopmental robustness is variable and under genetic control in Drosophila and suggest that the fly may serve as a model for identifying conserved gene pathways that stabilize wiring in stressful developmental environments. Moreover, some neuronal lineages are variation hotspots and thus may be more amenable to evolutionary change.</p></div

12A in T2 is a variability “hot-spot.”

<p>(<b>A</b>) Two examples of transverse projections through the T2 neuromere showing the neurite bundles of hemilineages 11A and 11B (magenta) relative to those of 12A (green). The right 12A hemilineages failed to split but the lineage 11 projections were invariant. Lineages from the genotype: <i>w; R26B05-LexA(attP40)/LexAop2-myr</i>::<i>tdTomato-P10; R24B02-GAL4(attP2)/UAS-myr</i>::<i>GFP-P10</i>. (<b>B</b>) Single examples of S3, T1, and T2, showing the effects of expressing UAS-Ubx.Ia. The immature neurite bundles in S3 are consistently transformed toward the unsplit T2 morphology. The 12A neurons in T1 become T2-like with respect to variability in the failure to split and in forming ectopic branches. Percentages shown are for the fraction of hemilineages that split into a dorsal and intermediate bundle and the fraction of split hemilineages that have an ectopic branch. (<b>C</b>) Quantification of anti-Ubx staining for four individuals that showed bilateral asymmetry. A difference in staining intensity was only observed in one of the four individuals. (<b>D</b>) An image stack from one of the four individuals (Sample1), with GFP in green and anti-Ubx in magenta. (<b>E</b>) A single optical section of the sample in D (showing area in the dashed box). Note the Ubx expression in T2 and lack of expression in T1. (<b>F</b>) When Ubx was expressed in 12A neurons in T1 using R24B02-GAL4 with UAS-Ubx.1a, Ubx was detectable (arrows) in the T1 neurons but still very low when compared to the endogenous levels in T2.</p

12A variability is sensitive to genetic background and temperature.

<p>(<b>A-B</b>) Proportions of unsplit hemilineages and ectopic branching in various genotypes at 25°C. (<b>A</b>) T/CS proportions were significantly different from all other groups (p < 0.05), but only the comparison with T/OR is shown. (<b>B</b>) T/OR had the largest proportion of ectopic branches, followed by T/CS. (<b>C</b>) Proportion of hemilineages with the typical morphology. (<b>D-E</b>) Effect of temperature on bundle splitting and ectopic branching. Data at 25°C are from 7A and 7B. p values are for comparisons between 16°C and 29°C for each genotype. (<b>F</b>) Effect of temperature on typical morphology. For all panels, vertical error bars represent 95% confidence intervals. Chi-squared tests were used to obtain p values and 95% CI. Holm correction for multiple comparisons (21 pairwise comparisons) was applied to p values in Fig 7A and 7B. T/CS: progeny of tester strains crossed to Canton S; T/OR: tester/Oregon R; T/yw: tester/<i>yw</i>; HI: Recently derived strain from Hawaii; CT: Recently derived strain from Connecticut.</p