Differences in the amount of different Hox genes cause accumulation of <i>sqh</i>-GFP in imaginal discs.

<p>(A–A′′) Z-stack of an <i>Abd-B</i> mutant clone induced in the genital disc and marked by the absence of lacZ expression (in blue in A′) showing increased <i>sqh</i>-GFP expression around it (in green, A, A′). (B, B′) Z-stack of a control <i>Abd-B</i> clone similarly marked but induced in the wing disc, showing there is no increase in <i>sqh</i> levels. (C) In <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; UAS-<i>Abd-B</i>/<i>tub-</i>Gal80^ts wing discs, the D/V boundary is smooth (compare with Fig. 3C). (D) A similar result is obtained in <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; UAS-O<i>Ubx</i><b>/</b><i>tub-</i>Gal80^ts.</p

Differences in amount of <i>Ultrabithorax</i> between adjacent cells induce accumulation of <i>spaghetti-squash, zipper</i> and <i>bazooka.</i>

<p>(A–C′′′) Z-stacks of <i>Ubx</i> clones induced in the haltere disc, marked by the absence of <i>arm</i>-lacZ (in grey in A, B and C, in blue in A′′′, B′′′ and C′′′), showing a ring of <i>sqh</i>-GFP, <i>zip</i>-GFP or <i>baz</i>-GFP (in green in A′, B′ and C′, respectively), and higher levels of F-actin (in red in A′′, B′′ and C′′) around the clones. Merged images in A′′′, B′′′ and C′′′. In D–D′′′ we show a sagital section of the clone shown in C–C′′′. Note the invagination of the clone and the accumulation of <i>baz</i>-GFP and F-actin in the border of the clone (arrowheads). (E–E′′′) Haltere disc of the <i>bx³ hh</i>-lacZ/<i>TM2, Ubx¹³⁰</i> genotype, showing accumulation of <i>sqh</i>-GFP (in green in E′, arrowhead) and F-actin (in red in E′′) at the A-P boundary, where compartments with (P compartment) and without (A compartment) <i>Ubx</i> abut (<i>Ubx</i> expression in grey in E and in blue in E′′′). Merged image in E′′′. (F–F′′) In <i>bx³ hh</i>-lacZ/<i>TM6B</i> haltere discs, by contrast, there is no accumulation of either <i>sqh</i>-GFP (in green in F′) or F-actin (in red in F′′) at the A-P boundary; <i>ß-galactosidase</i> expression is in grey in F. (G) <i>abx bx³ pbx</i>/<i>TM2, Ubx¹³⁰</i> adult showing a fusion of the T2 and T3 (transformed into the T2) segments (arrow). Scale bars are 10 µm in A′′′, B′′′ and C′′′, and 30 µm in E′′′ and F′′.</p

<i>Ultrabithorax</i> can maintain the A/P boundary in the absence of Hedgehog signaling.

<p> In all the panels of this Figure, the clones are marked by the lack of GFP in green, and the posterior compartment (to the right) is marked by either <i>hh</i>-lacZ or <i>en</i>-lacZ reporters in red. (A–B′′) Wing (A–A′′) and haltere (B–B′′) discs showing anterior clones double mutant for <i>smo</i> and <i>Ubx</i> that invade the posterior compartment. (C–C′′) <i>smo</i> clone induced in the anterior compartment of a <i>bx³/TM2, Ubx¹³⁰</i> haltere disc. See that it does not cross the compartment boundary. Note that a few cells in the A compartment weakly express <i>hh</i>-lacZ. (D–D′′) An anterior <i>smo</i> clone induced in the third leg disc cross the A/P compartment boundary. (E–E′′) A similar clone induced in the second leg disc does not cross the boundary. Scale bars are 30 µm except in A′′ (60 µm).</p

Hedgehog signaling and <i>Ultrabithorax</i> provide specific cell affinities to the cells.

<p>In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057159#pone-0057159-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057159#pone-0057159-g002" target="_blank">2</a> anterior compartments (A) of the imaginal discs are to the left and posterior ones (P) to the right. (A) <i>Ubx</i> mutant clones, marked by the absence of <i>arm</i>-lacZ expression (in green), are round and tend to segregate from the surrounding tissue. (B) An <i>Ubx</i>-expressing clone (arrow), marked with <i>yellow</i> and induced in the second thoracic segment also segregates from the rest of the notum. (C, C′) A <i>smo</i> clone in the anterior compartment of the wing pouch, marked by the absence of GFP signal (in green), penetrates into the posterior compartment, which is marked by <i>en</i>-lacZ expression (in red). (D) <i>hh</i>-lacZ expression in the haltere disc. (E–E′′) A <i>smo</i> clone in the anterior compartment of the haltere pouch, marked as in C also penetrates into the posterior compartment, marked with <i>hh</i>-lacZ (in red, E′). Merged image in E′′. Scale bars are 30 µm in A, D, E′′ and 60 µm in C′.</p

<i>Ultrabithorax</i> can maintain a smooth D/V boundary in the absence of <i>Notch</i> signaling.

<p>(A, B) <i>ap</i>-Gal4 UAS-GFP wing (A) and haltere (B) discs, showing the smooth boundary between dorsal (D, in green) and ventral (V) compartments. (C, D) In <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i> wing (C) and haltere (D) discs, this boundary is uneven. Note in D a group of dorsal cells in the ventral compartment (arrow). (E, F) In <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; UAS-<i>Ubx</i>/<i>tub-</i>Gal80^ts wing discs (E), or in haltere discs of <i>ap</i>-Gal4 UAS-GFP/<i>ap^UGO35</i>; <i>Df109</i> UAS-<i>dsUbx</i>/<i>+</i> larvae (F), the straight D/V boundary is restored. See that the dorsal compartment in E is slightly reduced and that in F slightly enlarged. Scale bars are 40 µm in A, C and 30 µm in B, D, E and F.</p

Summary of the results obtained with clones of different genotypes in the haltere disc.

<p>Anterior compartments are to the left, and the red line marks the A/P compartment boundary. <i>Ubx</i> expression is marked in blue and hatching indicates absence of Hh signaling. The red arrows indicate rejection of the cells of the clone due to different <i>Ubx</i> expression and the green arrows rejection due to different Hh signaling. (A) <i>Ubx⁻</i> clones are segregated from the rest of the tissue. (B) Anterior <i>smo</i> clones cross from the A to the P compartment. (C) Anterior <i>smo Ubx</i> clones undergo rejection from both A and P cells (because of their lack of <i>Ubx</i>) and rejection from A cells due to the absence of <i>smo</i>. The end result is the crossing of the boundary. (D) Clones like those in B, but induced in a <i>bx</i> background are rejected by both A and P cells and do not cross the boundary.</p

Number of clones crossing or respecting the A/P boundary in haltere, second and third leg disc in wildtype and mutant combinations.

*<p>Small clones or clones where the crossing or not crossing was not evident.</p

Comparison of gain-of-function activities of miR-279 and miR-996.

<p>(A) Luciferase sensor assays in S2 cells indicated that 3' UTRs of multiple miR-279 targets are all additionally responsive to miR-996. The control <i>Hairless</i> 3' UTR has no miR-279/996 seed match and was not repressed by these miRNAs. Error bars represent standard deviation from quadruplicate assays. (B) Northern confirmation of ectopic miR-279 and miR-996 in S2 cell experiments. pre = pre-miRNA hairpin, mature = mature miRNA product. Overexpressed miRNAs were calculated relative to endogenous mature miRNAs, normalized to 2S loading control. (C-D) Averaged activity profiles for control and miRNA overexpressing flies for 7 days in constant darkness since the second day after transferring from LD to DD. Some of these experiments utilized the amplifier driver <i>tim-UAS-Gal4</i>, as schematized in (E). (C) Overexpression of miR-279 by <i>tim-Gal4</i> induced strong arrhythmia. (D) Ectopic expression of miR-996 by <i>tim-Gal4</i> had no significant affect on circadian behavior, but further induction by <i>tim-UAS-Gal4</i> led to a complete arrhythmia. n = ~32 for each genotype; the number of flies assayed for each genotype is indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005245#pgen.1005245.t002" target="_blank">Table 2</a>. (F) Validation that higher levels of mature miR-996 are induced by <i>tim-UAS-Gal4</i>, compared to <i>tim-Gal4</i>. Overexpressed miRNAs were quantified as in (B) using Northern blotting, and normalized to 2S loading control. These tests also confirm that <i>tim-Gal4</i>><i>UAS-mir-996</i> flies effectively misexpressed miR-996, even though they lacked circadian defects.</p

Severe loss of mature miR-996 expression in <i>mir-279</i> deletion alleles.

<p>(A) Northern blots of miR-279 and miR-996 in various <i>mir-279</i> and <i>mir-996</i> homozygous or trans-heterozygous allele combinations. In <i>mir-279</i> alleles <i>[ex117]</i> and <i>[ex36]</i> that retain the <i>mir-996</i> genomic DNA, the levels of mature miR-996 are strongly diminished (<i>[ex117]</i>) or nearly undetectable (<i>[ex36]</i>). <i>mir-996[ex310]</i> is a deletion of the <i>mir-996</i> region that does not affect <i>mir-279</i> and <i>mir-279/996[ex15C]</i> deletes both miRNAs. (B) Quantifications of mature miR-279 and miR-996 levels. Homozygous <i>[ex117]</i> mutants expressed ~10% of the wild type level of miR-996 and <i>[ex117/ex36]</i> transheterozygous mutants expressed ~5% of miR-996. Note that the expression level for both miRNAs is copy-number dependent, since heterozygous <i>mir-279/996[ex15C]</i> flies expressed roughly half of both miRNAs compared to wild type animals.</p

Both miR-279 and miR-996 contribute to maintenance of circadian rhythm.

<p>(A-F) Typical activity profiles of individual flies of various <i>mir-279/996</i> genotypes. Animals were entrained in 12hr-light/12hr-dark (LD) cycles for four days, and then kept in constant darkness (DD) for seven more days. In LD cycles, white bars represent the light phase (day) and black bars represent the dark phase (night). During DD cycles, grey and black bars represent the subjective day and night time, respectively. (A) <i>mir-279[ex117]</i> heterozygotes behave normally in that they maintain circadian activity in the dark, although the strong morning and evening activity peaks and mid-day siesta are not as well maintained. (B-C) In <i>mir-279[ex117]</i> homozygotes, the majority of animals gradually lost behavioral rhythmicity after transferring to constant darkness (B), but about 1/3 of animals could maintain circadian activity in constant darkness (C). Note that all <i>mir-279[ex117/ex117]</i> animals exhibited generally less activity than heterozygotes. The activity and circadian defects in <i>mir-279[ex117/ex117]</i> animals were rescued by single copies of the wild-type 16.6kb <i>mir-279/996</i> transgene (D) or the <i>2x-mir-279</i>-only (E) or <i>2x-mir-996</i>-only (F) transgenes. (G-J) Averaged activity profiles of various <i>mir-279/996</i> genotypes. (G) <i>mir-279/996[ex15C]</i> heterozygotes exhibit robust behavioral rhythmicity after transferring to constant darkness, but <i>mir-279[ex117]</i> homozygotes do not. In the <i>mir-279/996[ex15C]</i> homozygous background (which is normally mostly lethal by ~4 days), expression of only a single <i>1x-mir-279</i> (I) or single <i>1x-mir-996</i> (J) transgene can restore normal rhythmic behavior in constant darkness. n = ~32 for each genotype; the number of flies assayed for each genotype are indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005245#pgen.1005245.t001" target="_blank">Table 1</a>.</p