<i>iab-5,6^CI</i> phenotype and rescue.

<p>A. A wild-type adult male cuticle with A4-A6 labeled. Segment A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. For reference, the A6 tergite is indicated by a red arrowhead and the A6 sternite is indicated by a red arrow. B. A wild-type embryonic nerve cord (anterior towards the top) stained with an antibody to <i>Abd-B</i> (brown). Notice the step gradient of <i>Abd-B</i> expression increasing in each parasegment towards the posterior. C. An adult male cuticle of a fly homozygous for the <i>iab-5,6^CI</i> chromosome with A5 and A6 transformed towards A4 (notice the A4-like pigmentation on the tergites and the bristled sternites). D. The embryonic nerve cord of homozygous <i>iab-5,6^CI</i> embryos shows only a transformation of A6 into A5, as seen by the repetition of PS10/A5-like Abd-B levels in PS11/A6, indicating that the inactivation of <i>iab-5</i> is incomplete and not seen in the embryo. E. An adult male cuticle from a fly homozygous for the <i>iab-5,6^rescue</i> chromosome, where the entire 19.3 kb area deleted in <i>iab-5,6^CI</i> is reintegrated into <i>iab-5,6^CI</i>, looks completely wild type. F. The complete rescue is confirmed by the wild-type pattern of <i>Abd-B</i> in the embryonic ventral nerve cord.</p

<i>Fab-6</i> boundary mutations.

<p>The genotypes of the adult male cuticles of A. <i>Fab-6²</i>, and B. <i>Fab-6³.</i> C. (wild type) and D. (<i>Fab-6³</i>) are embryonic nerve cords stained for Abd-B protein. Notice the increased level of Abd-B in PS10 in mutants (D.) relative to wild-type (C.).</p

Phenotypes from initiator mutants.

<p>Genotypes are as follows: A. and D. <i>iab-6⁴</i>. B. and E. wild type. C. and F. <i>Fab-6^IAB5</i>. A.–C. Show the ventral sternite cuticles made from adult males, homozygous for the genotype indicated above. Notice that A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. The opposite homeotic transformations are highlighted by the direction of the arrows on the left and the right of the cuticles. D.–F. Show ventral nerve chords made from homozygous embryos of the genotypes indicated above. Parasegment borders are marked to the left.</p

Oligos used to generate the deletions.

<p>Bold sequences correspond to the FRT-kanamycin-FRT sequences used to prime the amplification of the FRT-kanamycin-FRT cassette. Regular characters correspond to the homology regions used to generate the deletions by recombineering. P1–P7 correspond to the oligos used to generate the proximal breakpoint of the deletions (relative to the <i>Abd-B</i> promoter), while D1–D8 correspond to the oligos used to generate the distal breakpoint.</p

Phenotypes from initiator mutants.

<p>Genotypes are as follows: A.–C. <i>iab-6¹</i>, D.–F. <i>iab-6⁴</i> and G.–I. <i>iab-6⁸</i>. A., D. and G. show adult male cuticles. B., E. and H. show pseudo-darkfield views of the fifth and sixth tergites to visualize the trichome patterns. C., F. and I. show the Abd-B staining pattern in the embryonic nerve cord. In wild-type flies, A5/PS10 differs from A6/PS11 based on the sternite shape, the bristles present on the A5 sternite, the trichome pattern on the fifth and sixth tergites, and the Abd-B staining pattern in the CNS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen-1001260-g003" target="_blank">Figure 3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen-1001260-g004" target="_blank">Figure 4</a>). The <i>iab-6¹</i> and <i>iab-6⁴</i> show transformations of A6 to A5 for all phenotypes monitored. Meanwhile <i>iab-6⁸</i> shows only a partial transformation of A6 to A5 as seen by the sternite shape and trichome pattern on A6, which remain A6-like.</p

Synopsis of the <i>Abd-B</i> locus of the BX-C and diagram of the mutations created for this study.

<p>A. Synopsis of the <i>Abd-B</i> locus of the BX-C. Diagram of the <i>Abd-B</i> gene and its 3′<i>cis-</i>regulatory region. The horizontal line represents the DNA sequence of the BX-C (see scale on top left). The <i>Abd-B</i> expression pattern in the central nervous system of a 10 hours embryo is shown above the DNA line. In parasegment 10 (PS10) <i>Abd-B</i> is present in a few nuclei at a relatively low level. This PS10-specific expression pattern is controlled by the <i>iab-5</i> regulatory domain located 55 kb downstream from the <i>Abd-B</i> promoter. In PS11, PS12 and PS13, <i>Abd-B</i> is present in progressively more nuclei and at higher levels. These patterns are controlled by the <i>iab-6</i>, <i>iab-7</i> and <i>iab-8</i> regulatory domains, respectively. Each regulatory domain functions autonomously from its neighbors due to the presence of the boundaries that flank them (red ovals). B. Diagram of the mutations created for this study. The top line shows the DNA coordinates of <i>iab-6</i>, according to the <i>Drosophila</i> Genome Project. Below this line, and to approximate scale, are the locations of the various elements isolated from the BX-C including the IAB5 initiator<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Busturia1" target="_blank">[12]</a>, DNase hypersentive site 1 (HS1/<i>Fab-6</i> including the CTCF binding sites) and 2 (HS2/PRE) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-PerezLluch1" target="_blank">[43]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Holohan1" target="_blank">[44]</a>, the 2.8 kb <i>iab-6</i> initiator fragment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Mihaly1" target="_blank">[22]</a>, the minimal initiator fragment and the <i>Fab-7</i> boundary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Hagstrom1" target="_blank">[14]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Gyurkovics1" target="_blank">[30]</a>. Below this line are the DNAs reintegrated to make the mutations. The various <i>iab-6</i> alleles are indicated as solid bars, with gaps indicating the areas deleted. These bars are color coded such that blue bars indicate mutants that show no cuticle or CNS phenotypes at 25°C, red bars indicate mutants with <i>Fab-6</i>-type phenotypes, turquoise bars indicate mutants with <i>iab-5,6</i> phenotypes, and green bars indicate mutants with <i>iab-6</i> phenotypes.</p

List of primers used to perform the different constructs described in Materials and Methods.

<p>List of primers used to perform the different constructs described in Materials and Methods.</p

Post translational modification, stability, and abundance of seminal fluid proteins in mates of <i>iab-6^cocu</i> or control males.

<p>Western blots using antibodies of known LTR-associated Acps CG9997, CG1656, CG1652, and CG17575 as well as STR Acp ovulin (Acp26Aa). Accessory gland extracts from a single control (lane 1) and <i>iab-6^cocu</i> male (lane 2) were used as positive controls and reproductive tract extracts from 4 virgin females (lane 9) were used as a negative control. Extracts from the reproductive tracts of females mated to control (+) or <i>iab-6^cocu</i> (Δ) were collected at 15′ (lanes 3–4, 2 RTs per), 30′ (lane 5–6 s, 3 RTs per), and 1 h ASM (lanes 7–8, 6 RTs per). A) Full length CG9997 is produced by <i>iab-6^cocu</i> males but is not present in the reproductive tracts of their mates. The smaller processed form of CG9997 is present in mates of <i>iab-6^cocu</i> suggesting that CG9997 is transferred. Both CG1656 and CG1652 are transferred to females normally by <i>iab-6^cocu</i> males, but both of these proteins run at a lower apparent molecular weight than in control males. B) <i>iab-6^cocu</i> males transfer more CG17575 to their mates than control males. Tubulin was used as a loading control for the female reproductive tracts. C) Both mates of control and <i>iab-6^cocu</i> males receive ovulin. However, the ovulin produced by <i>iab-6^cocu</i> males also runs at a lower apparent molecular weight than in controls.</p

Mutants affecting <i>Abd-B</i> expression in the accessory gland.

<p>A) The Molecular map of the <i>Abd-B</i> gene region is shown with its extensive 3′ <i>cis</i>-regulatory domains <i>iab-5</i> through <i>iab-8</i> (the <i>iab-4</i> domain regulates <i>abd-A</i>). The extents of the various deficiencies that were used to map the enhancer responsible for <i>Abd-B</i> expression in the secondary cells are shown below the molecular map. The location of DNA sequence used to make the 2.8 kb-long D5rsG4rs driver (thereby refereed as D5 Gal4 driver).is shown under the map. The red circles on the map represent the boundaries separating the parasegment-specific <i>cis-</i>regulatory domains of <i>Abd-B</i>. The green triangle above the <i>iab-6</i> domain marks the <i>iab-6</i> initiator. B) UAS-GFP expression driven by <i>Abd-B</i>-Gal4 in a WT for the BX-C. C) same as B, but in an <i>iab-6</i>, <i>7^IH</i> homozygous male or in an <i>iab-5,6^J82</i> homozygous male(D). Note that in the <i>iab-6, 7^IH</i> and <i>iab-5,6^J82</i> background, the numerous vacuoles, characteristic of the secondary cells (visible by black holes in the GFP background), are lost. However, the vacuoles are not affected in <i>iab-4,5,6^DB</i>. Thus, the critical region required for proper secondary cell specification based on these 3 deficiencies is indicated by the dotted-line box in panel A. E) UAS-GFP expression driven by <i>Abd-B</i>-Gal4 in secondary cells of <i>iab-6⁴</i> (initiator deletion) and of <i>iab-6^cocu</i> males (F). Note the normal aspect of GFP staining in <i>iab-6⁴</i> (E) relative to the WT shown in B). In <i>iab-6^cocu</i> however (F), the vacuoles are lost, giving rise to staining comparable to panels C and D. Panels G) and H) show <i>iab-6⁴</i> (G) and <i>iab-6^cocu</i> (H) accessory glands stained with an <i>Abd-B</i> antibody. While <i>Abd-B</i> expression appears normal in <i>iab-6⁴</i> (G), the signal is absent in <i>iab-6^cocu</i> (H). I) shows the tip of an accessory gland from a fly carrying the D5-Gal4 driver driving GFP expression in the secondary cells (the staining is shown in yellow to distinguish it from panels B–F depicting GFP driven by the Gal4 Bac. The white horizontal scale bars in each of the panels represents 50 µm.</p

Egg-laying and receptivity in mates of <i>iab-6^cocu</i> or control males.

<p>A) The mean number of eggs laid per female mated to either control males (dashed line), <i>iab-6^cocu</i> males (solid line), or DTA-E males (grey dot dashed line) over a 10 day period. Mates of <i>iab-6^cocu</i> males lay normal numbers of eggs during the first day after mating (WRST p = 0.300) but lay significantly fewer eggs over 10 days when compared to mates of control males (rmANOVA p = <0.0001*, Control N = 51, <i>iab-6^cocu</i> N = 45, DTA-E N = 17). The drop in egg laying for controls on day 5 is atypical and was likely a response to food quality. B) The mean hatchability (#progeny/#eggs) per female for mates of control, <i>iab-6^cocu</i>, and DTA-E males for days 1–4 of the egg laying results reported in (A). Days 5–10 were omitted because <i>iab-6^cocu</i> mated females do not lay enough eggs on these days for analysis. Mates of <i>iab-6^cocu</i> males have comparable hatching totals for the eggs that they do lay when compared to mates of control males (WRST p = 0.37). Because DTA-E males do not produce sperm, their mates are expected to show zero hatchability. (Values greater than 1 represent instances where the number of progeny produced exceeded the number of eggs counted; this under-counting can result when females lay eggs under bubbles in the medium or directly on top of previously laid eggs. Hatchability values were not normalized to 1 so as to accurately report counter error.) C) The percentage of mated females willing to mate within 1 hour of exposure to a wild type male at 1, 4, and 10 days after an initial mating. Both groups of females initially mated to <i>iab-6^cocu</i> or control males are unreceptive (WRST p = 0.21, control n = 20, <i>iab-6^cocu</i> n = 26). At 4 d ASM females initially mated to <i>iab-6^cocu</i> males are significantly more receptive to courting males compared to mates of control males (WRST p = <0.0001*, control n = 21, <i>iab-6^cocu</i> n = 26). By 10 d ASM there is no difference between mates of control or <i>iab-6^cocu</i> males (WRST p = 0.84, control n = 19, <i>iab-6^cocu</i> n = 22).</p