Yeast Communities of Diverse Drosophila Species: Comparison of Two Symbiont Groups in the Same Hosts

<p>Supplemental data for</p> <p>Yeast Communities of Diverse Drosophila Species: Comparison of Two Symbiont Groups in the Same Hosts</p

Dsx expression in distantly related lineages.

<p>Dsx immunostaining is in green. (A) Simplified phylogeny of the species shown in this figure and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001131#pbio-1001131-g006" target="_blank">Figure 6</a>. (B) Male T1 leg disc of <i>S. lebanonensis</i>. (C) Male T1 leg disc of <i>D. busckii</i>. (D) Adult male T1 leg of <i>D. pseudoobscura</i> carries sex combs on the ta1 and ta2 segments. (E) Dsx expression in the corresponding segments of the male T1 leg at the early pupal stage. (F–L) <i>D. willistoni</i>. (F) Adult male T1 leg. Note the absence of sex combs and the very small number of long and curved chemosensory bristles (compare to M). (G) Male T1 leg disc stained with the DsxC antibody. Arrowhead, an expression domain unique to the male T1 disc. (H) Male T2 leg disc stained with the DsxC antibody. (I) Female T1 leg disc stained with the DsxC antibody. (J) Adult male brain stained with the DsxC antibody, showing the PC1 (arrow) and PC2 (arrowhead) neuronal clusters. (K) Male T1 prepupal leg at 5 h AP stained with the DsxC antibody. (L) Male T1 pupal leg at 24 h AP. (M–Q) <i>D. hydei</i>. (M) Adult male T1 leg. (N) Male T1 leg disc. Arrow and arrowhead point to the dorsal and ventral expression domains respectively. (O) Male T2 leg disc. (<b>P</b>) Male T1 prepupal leg at 8 h AP. The two domains are still visible (arrow, arrowhead). (Q) Male T1 pupal leg at 40 h AP.</p

<i>dsx</i> and <i>Scr</i> control sex comb development.

<p>(A) Wild-type male adult T1 leg. ta, tarsus; bracket, TBRs; arrow, sex combs. (B) <i>tub-Gal80^ts</i>; <i>neur-Gal4/UAS-dsxM</i> male. The bristles in TBRs are transformed into ectopic sex comb teeth (bracket). Arrow points to the normal sex comb. (C) <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-dsxRNAi</i> male. The sex comb is only partially rotated and has fewer and thinner teeth (arrow). (D) Scr expression in <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-dsxRNAi</i> male at 24 h AP. Scr is down-regulated except in the cells distal to the sex comb (arrow). (E) Wild-type female adult T1 leg. Bracket, TBRs. (F) <i>tub-Gal80^ts</i>; <i>neur-Gal4/UAS-dsxM</i> female. As in the male of the same genotype (B), TBR bristles assume sex comb-like morphology (bracket). (G) <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-dsxM</i> female. The two most distal TBRs develop into partially rotated sex combs (arrows). (H) Scr expression in <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-dsxM</i> female T1 leg at 24 h AP. Scr is up-regulated in cells distal to the ectopic sex comb (arrow); compare to (D). (I) <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-Scr</i> male. Ectopic sex combs are formed on distal tarsal segments (arrow). (J) <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-ScrRNAi</i> male. The sex comb and TBR are lost from the distal part of ta1, where <i>rn</i> is expressed (bracket). (K) <i>tub-Gal80^ts</i>; <i>neur-Gal4/UAS-ScrRNAi</i> male. The number of teeth is reduced, but tooth morphology is normal (arrow). (L) T1 leg disc of <i>tub-Gal80^ts/UAS-Gal4</i>; <i>rn-Gal4/UAS-ScrRNAi</i> male. No Dsx is detectable. (M) T2 leg disc of <i>tub-Gal80^ts</i>; <i>rn-Gal4/UAS-Scr</i> male at the wandering stage. Ectopic Dsx expression is detected throughout the <i>rn</i> expression domain.</p

Dsx expression in species that evolved lineage-specific sexually dimorphic structures.

<p>Dsx immunostaining is in green. (A–G) <i>D. immigrans</i>. (A, B) Adult male and female T1 legs, respectively. (C) Male T1 leg disc. (D) Male T2 leg disc. (E) Male T1 prepupal leg. (F, G) Male and female T1 pupal legs, respectively, at 48 h AP. (H–L) <i>Zaprionus tuberculatus</i>. (H, I) Adult male and female T1 legs, respectively. (J) Male-specific brush structure shown at higher magnification. (K) Male T1 leg disc. Arrow, dsx expression domain. (L) Male T1 pupal leg at 48 h AP.</p

Transcriptional regulation of <i>dsx</i>.

<p>(A–C) Localization of <i>dsx</i> transcripts by in situ hybridization. Anterior is to the left, dorsal is up. (A) In the wandering L3 male T1 leg disc, <i>dsxM</i> is expressed in an anterior proximal (arrowhead) and a distal (arrow) crescent domain. (B) No detectable signal is seen in male T2. (C) <i>dsxM</i> expression in the 24 h AP male T1 leg. The only epidermal expression is in the presumptive sex comb region (arrow). Strong staining in the center of the leg is non-specific. (D, E) DsxM immunostaining in <i>rn-Gal4/UAS-dsxM</i> male (D) and female (E) T1 leg discs is seen throughout the <i>rn</i>-expression domain.</p

A model for the origin of a new sex-specific developmental pathway.

<p>Ancestral regulatory interactions are indicated in black, and newly evolved interactions in red. In the ancestral condition (left), leg patterning genes lay down the basic bristle pattern and establish a domain of high <i>Scr</i> expression on the ventral-anterior surface of the distal Ti and ta1. High levels of <i>Scr</i> organize the ventral-anterior bristles into TBRs. <i>dsx</i> is not expressed in the TBRs so they develop in a sexually monomorphic manner. In the <i>melanogaster</i>-<i>obscura</i> clade (right), <i>dsx</i> was recruited into the TBR development pathway under the control of both <i>Scr</i> and leg patterning genes. <i>Scr</i> activates <i>dsx</i> in T1 at the late larval stage, while <i>dsx</i> modulates <i>Scr</i> at the pupal stage to make its expression sexually dimorphic in some species. Both genes have acquired new downstream targets involved in bristle patterning and morphogenesis.</p

Dsx and Scr expression in the <i>melanogaster</i> species group.

<p>ta1–2 of adult male T1 legs are shown on the left. Scr (red) and Dsx (green) immunostaining of the same segments in mid-pupal male T1 legs are shown in the right panels. Developing sex combs are indicated by arrows (longitudinal combs) or arrowheads (small and transverse combs). In all species, Dsx expression is highest in the sex comb teeth, while Scr is low in the sex comb teeth but high in the surrounding cells. (A) <i>D. ficusphila</i>. (B) <i>D. biarmipes</i>. (C) <i>D. takahashii</i>. (D) <i>D. nikananu</i>. (E) <i>D. kikkawai</i>. (F) <i>D. bipectinata</i>. (G) <i>D. malerkotliana</i>. (H) Phylogenetic relationships among the species shown in this figure. The latest common ancestor of <i>D. kikkawai</i> and <i>D. nikananu</i> had a sex comb similar to that of <i>D. kikkawai</i>; the latest common ancestor of <i>D. malerkotliana</i> and <i>D. bipectinata</i> had a sex comb similar to <i>D. malerkotliana</i> (Barmina and Kopp 2007) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001131#pbio.1001131-Barmina1" target="_blank">[10]</a>.</p

Dsx and Scr expression during sex comb development in <i>D. melanogaster</i>.

<p>Immunostaining with anti-Scr (red) and anti-DsxC (green) antibodies. Ti, tibia; ta, tarsus; AP, after pupariation. All panels except (E) and (F) are merged images. (A) L3 male T1 leg disc. Anterior is to the left, dorsal is up. Scr is expressed at a high level in the anterior part of distal Ti and ta1 (arrow) and in a more proximal region corresponding to the presumptive body wall (arrowhead). Low expression is present in the rest of the disc. (B) Wandering male T1 leg disc. Dsx is expressed in the distal part of the Scr domain (overlap in yellow) and in the more central region (arrow). Inset shows a magnified view of the boxed area. (C) Wandering female T1 leg disc. (D) Wandering male T2 leg disc. The only detectable Scr expression is subepidermal. (E–H) 5 h AP T1 leg (E–G, male; H, female). The tarsal segments are numbered. Dsx is strongly expressed on the ventral-anterior side of distal ta1 in both sexes (arrow). (F) In the male, Dsx is expressed in the distal ta1 and in small dorsal and ventral patches in ta2–4 (asterisks). (H) In the female, Dsx expression is only in the distal ta1 and is weaker than in the male. (I–K) 16 h AP T1 leg (I, J, male; K, female). Arrows point to the rotating sex comb. Note the absence of Dsx expression in ta2. High background staining is caused by the pupal cuticle, which is still attached to the epidermis at this stage. (L–N) 24 h AP T1 leg. (L, M, male; N, female). (O) 36 h AP male T1 leg. (P, Q) Scanning electron micrographs of the distal ta1 in the adult male (P) and female (Q). Ventral is to the right and anterior is facing out of the page.</p

Bacterial Communities of Diverse Drosophila Species: Ecological context of a host-microbe system

<p>Supplemental data for</p> <p>Bacterial Communities of Diverse Drosophila Species: Ecological context of a host-microbe system</p

A Distalless-responsive enhancer of the Hox gene <i>Sex combs reduced</i> is required for segment- and sex-specific sensory organ development in <i>Drosophila</i>

<div><p>Hox genes are involved in the patterning of animal body parts at multiple levels of regulatory hierarchies. Early expression of Hox genes in different domains along the embryonic anterior-posterior (A/P) axis in insects, vertebrates, and other animals establishes segmental or regional identity. However, Hox gene function is also required later in development for the patterning and morphogenesis of limbs and other organs. In <i>Drosophila</i>, spatiotemporal modulation of <i>Sex combs reduced</i> (<i>Scr</i>) expression within the first thoracic (T1) leg underlies the generation of segment- and sex-specific sense organ patterns. High <i>Scr</i> expression in defined domains of the T1 leg is required for the development of T1-specific transverse bristle rows in both sexes and sex combs in males, implying that the patterning of segment-specific sense organs involves incorporation of <i>Scr</i> into the leg development and sex determination gene networks. We sought to gain insight into this process by identifying the <i>cis</i>-and <i>trans</i>-regulatory factors that direct <i>Scr</i> expression during leg development. We have identified two <i>cis</i>-regulatory elements that control spatially modulated <i>Scr</i> expression within T1 legs. One of these enhancers directs sexually dimorphic expression and is required for the formation of T1-specific bristle patterns. We show that the Distalless and Engrailed homeodomain transcription factors act through sequences in this enhancer to establish elevated <i>Scr</i> expression in spatially defined domains. This enhancer functions to integrate <i>Scr</i> into the intrasegmental gene regulatory network, such that <i>Scr</i> serves as a link between leg patterning, sex determination, and sensory organ development.</p></div