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

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. 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

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

<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

Map of the abdominal half of the bithorax complex.

<p>The horizontal bar indicates the DNA sequence map, numbered in kb according to Martin et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Martin1" target="_blank">[4]</a> (Genbank U31961). Base #1 corresponds to base 12,809,162 on chromosome 3R in release 5.37 of the <i>Drosophila</i> genome. The coordinates proceed distal to proximal on chromosome 3R, which is opposite in orientation to the whole genome numbering. The regulatory domains <i>iab-2</i> through <i>iab-8</i> are color coded; the domain borders are defined by deletion mutations (<i>Fab8 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Barges1" target="_blank">[<i>22</i>]</a>, ; <i>Fab7</i>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Gyurkovics1" target="_blank">[41]</a>; <i>Mcp</i>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Karch3" target="_blank">[44]</a>; <i>iab-3/iab-4</i>, L. Sipos personal communication), or inferred from the binding sites of the CTCF factor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Holohan1" target="_blank">[45]</a>. Below the DNA bar are shown the splicing patterns of <i>abd-A</i> and <i>Abd-B</i> (in black), a cDNA derived from the <i>Mi(Hto-WP)LNP</i> insertion (red), and the MIP06894 cDNA (green). At the bottom, the splicing pattern for the iab-8 ncRNA is shown in dark blue, with numbered exons, and alternate 5′ or 3′ extensions indicated with light blue lines. Mutant lesions are indicated above the DNA bar. The rearrangement breakpoints are color coded according to their phenotypes when heterozygous with the <i>mfs5649</i> insertion.</p

ABD-A expression in rearrangements truncating the iab-8 ncRNA.

<p>Embryos homozygous for the indicated mutations were doubly stained for ENGRAILED (green) and ABD-A (red), and the CNS's were dissected and photographed. The posterior end of each CNS is shown; the ENGRAILED stripes mark the parasegmental boundaries. The <i>iab-4</i> and <i>iab-7</i> breaks cause widespread misexpression of ABD-A in PS13, but <i>iab-3</i> breaks show only subtle misexpression in a few nuclei. Embryos homozygous for a deletion of the iab-8 miRNA also show misexpression in only a few nuclei.</p

ABD-A expression in <i>Abd-B</i> mutant embryos.

<p>Stage 14 embryos in A–C were stained with antibody to ABD-A, opened along the dorsal midline and flattened for photography. ABD-A is absent from PS13 in wild type (A), but appears throughout PS13 in <i>Df(3R)C4</i> homozygotes (B). In <i>Abd-B^D16</i> homozygotes, ABD-A is only in the lateral and dorsal epidermis of PS13 (C). Dissected CNS's from stage15 embryos in D were doubly stained for ABD-A (red) and ABD-B (green). In wild type, the expression domains overlap through PS10-12, with some nuclei expressing both proteins. In <i>Abd-B^D14</i> homozygotes, ABD-B expression is absent from PS10-13, but the ABD-A pattern is unchanged, leaving PS13 without either protein.</p

Evolutionary conservation.

<p>A. A comparison of exon 3 and neighboring bases with the homologous regions from the genomes of three other <i>Drosophila</i> species. B. Potential nine amino acid peptide within exon 8 of the iab-8 ncRNA. The <i>D. melanogaster</i> sequence is compared to that of <i>D. ananassae</i>. The initial methionine codon is preceded by a perfect translation start consensus sequence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Cavener1" target="_blank">[46]</a>, and there are two stop codons after the 9th amino acid. The three bases altered in <i>D. ananassae</i> are highlighted in red; only one changes the predicted amino acid.</p