abd-A Regulation by the iab-8 Noncoding RNA

The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This “iab-8 RNA” produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only “in cis.” Transcriptional interference with the abd-A promoter is the most likely mechanism

Initiator Elements Function to Determine the Activity State of BX-C Enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain

Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1

AbstractThe actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps

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

<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

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