<i>acal</i> expression is required in the lateral epidermis during DC.

<p>(A) <i>acal</i> expression in wild type and mutant embryos during DC stages, as determined by qPCR. Means of three independent experiments run twice, +/− SEM. Student’s t test was used to assess significance. (B) Targeted ectodermal (<i>69B-gal 4</i> driver) and lateral epidermis (<i>pnr-gal 4</i> driver) expression of wild type <i>acal</i> in <i>acal</i> mutants. Only dead embryos are classified, mutants surviving embryogenesis constitute the remaining percentage to amount to a hundred percent (open space above bars). (B’-B’’’) are examples of <i>acal^5/5; 69B>acal</i> embryos with no cuticular phenotype, wild type in appearance (B’), with a dorsal open phenotype (B’’), or with an anterior open phenotype (B’’’). Compared with <i>acal^5/5</i> mutants, the cuticular phenotypes are the same, but they differ significantly in the abundance (expression of <i>acal</i> significantly reduces the number of mutant embryos that die and that have cuticular phenotypes). In these and following experiments, cuticular phenotypes do not change, unless otherwise stated. No new cuticular phenotypes are found; thus, examples are not depicted in all figures. Numbers analyzed: <i>acal^5/5,UAS-acal/+</i> = 240, <i>acal^5/5;69B-gal4/+</i> = 441, <i>acal^5/5;69B>acal</i> = 237, <i>acal^5/5;pnr-gal4/+</i> = 435, <i>acal^5/5;pnr>acal</i> = 130. Statistical significance was calculated using chi square tests. (C) Quantification of in situ hybridization shown in (D) in stage 13 embryos (<i>acal^5/5</i>, n = 10; <i>yw</i>, n = 6). <i>acal</i> mutants have significantly lower expression levels in lateral epithelia; chi square test. (D) <i>acal</i> in situ hybridization in embryos throughout embryonic development. Wild type embryos are <i>yw</i>, and mutant is <i>acal^5/5</i>. Arrows indicate expression in the lateral epidermis and arrowheads point to expression in the central nervous system. Sense (negative) controls are also shown. Insets show boxed areas in stages 13, 15, and 17 embryos. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004927#pgen.1004927.s002" target="_blank">S2 Fig.</a></p

<i>acal</i> is a processed long non-coding RNA.

<p>(A) <i>acal</i> full-length transcript sequence conservation plot compared to other dipterans, and pairwise alignments between <i>acal</i> in <i>D. melanogaster</i> and its homologs (adapted from the UCSC genome browser). Location of molecular lesions in <i>acal¹</i> (1) and <i>acal²</i>(2) are marked within brackets after an ‘<b>X’</b> in the transcript (gray arrow). Above the plot is the location of the probes used for small RNA Northern blots in (E) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004927#pgen.1004927.s003" target="_blank">S3 Fig.</a> (B) Protein coding potential of <i>acal</i> and other well known coding (white) and non-coding (black) RNAs. <i>pipsqueak (psq)</i> is a transcription factor and <i>basket (bsk)</i> codes for JNK. <i>polished rice (pri)</i> and <i>pncr003</i> are polycistronic and code for small peptides. <i>roX1</i> (<i>RNA on the X1</i>), <i>iab-4</i> (<i>infra-abdominal 4)</i>, and <i>Heat shock RNA ω</i> (<i>Hsrω)</i> are long non-coding RNAs. <i>bereft (bft)</i> is a microRNA precursor. Negative values correspond to non-coding scores, and positive values are for protein coding RNAs. Asterisks denote significantly coding or non-coding scores. (C) Semi-quantitative RT-PCR of total [T], cytoplasmic [C], and nuclear [N] RNA against <i>acal, Rp49</i> (protein coding gene), and <i>bantam</i> microRNA precursor (<i>pre-ban</i>). [G] is the genomic control. (D) Band intensity of <i>acal</i> cytoplasmic and nuclear amplification, compared to total RNA amplification. Means of 5 independent experiments +/− SEM. Significance was assessed using Student’s t test. (E) Small RNA Northern blots for <i>acal</i> A and B probes, using wild type embryos [E] and pupae [P] RNA. Sizes were estimated from 4 independent experiments.</p

<i>acal</i> is a Long Non-coding RNA in JNK Signaling in Epithelial Shape Changes during Drosophila Dorsal Closure

<div><p>Dorsal closure is an epithelial remodeling process taking place during Drosophila embryogenesis. JNK signaling coordinates dorsal closure. We identify and characterize <i>acal</i> as a novel negative dorsal closure regulator. <i>acal</i> represents a new level of JNK regulation. The <i>acal</i> locus codes for a conserved, long, non-coding, nuclear RNA. Long non-coding RNAs are an abundant and diverse class of gene regulators. Mutations in <i>acal</i> are lethal. <i>acal</i> mRNA expression is dynamic and is processed into a collection of 50 to 120 bp fragments. We show that <i>acal</i> lies downstream of raw, a pioneer protein, helping explain part of raw functions, and interacts genetically with <i>Polycomb. acal</i> functions in trans regulating mRNA expression of two genes involved in JNK signaling and dorsal closure: <i>Connector of kinase to AP1 (Cka)</i> and <i>anterior open (aop). Cka</i> is a conserved scaffold protein that brings together JNK and Jun, and <i>aop</i> is a transcription factor. Misregulation of <i>Cka</i> and <i>aop</i> can account for dorsal closure phenotypes in <i>acal</i> mutants.</p></div

Molecular and genetic characterization of <i>acal</i> mutants.

<p>(A-L) Cuticular analysis of DC defects. In all figures, embryos are shown in a lateral view, with dorsal up and anterior left. Arrowheads show extent of cuticular holes. (A) Wild type cuticle. (B-D) <i>bsk¹</i> (JNK), <i>Cka¹</i>, and <i>aop¹</i> mutant phenotypes. (E-L) <i>acal</i> mutants. (M-M’’) Schematic representation of the <i>acal</i> locus. (M) Deficiencies used to map <i>acal</i> mutants to the intergenic region between <i>lola</i> and <i>psq</i> (for simplicity, other genes are not depicted). Boxed area in (M) is amplified in (M’). (M’) The genomic rescue construct,<i>178D09</i>, spans the full <i>acal</i> genomic region, <i>CR45135</i>, and a small part of <i>lola</i> 5’. Boxed area in (M’) is amplified in (M’’). (M’’) Parental insertion <i>P{KG09113}</i> for <i>acal¹</i> and <i>acal²</i> and insertion site of <i>acal⁶</i> are shown. Molecular lesions of <i>acal¹</i> and <i>acal²</i> are highlighted in bold. A putative poly-adenylation site is also depicted. Primer pairs numbers and amplicons used to sequence mutant alleles are also depicted. (N) A genomic rescue transgene significantly suppresses <i>acal</i> DC mutant phenotypes. Only mutant embryos with cuticle defects are shown, mutants surviving embryogenesis are not depicted (and constitute the open space above bars to amount to a hundred percent). In this and all figures, unless noted, mutant embryos were selected by lack of GFP expression, present in control embryos (possessing balancer chromosomes that express GFP). Number of animals analyzed: <i>acal^1/1</i> = 217, <i>acal^1/1;178D09</i> = 314, <i>acal^2/2</i> = 192, <i>acal^2/2;178D09</i> = 159. Chi square tests were used to assess significance. (O) Similarity tree of <i>acal</i> homologs among some sequenced Drosophilids. Bootstrap values are shown. Further genetic characterization is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004927#pgen.1004927.s001" target="_blank">S1 Fig.</a></p

<i>raw</i> and <i>acal</i> act together to counteract JNK signaling.

<p>(A) Wild type cuticle. (B) Cuticle phenotype of <i>raw</i> mutant embryo. (C) Genetic interaction between <i>raw²</i> and <i>acal⁵</i> mutants. <i>raw</i>-like phenotype is depicted in (B). In <i>raw^+/+; acal^5/5</i> mutants a small percentage survives embryogenesis, and constitute the open space above the bar to amount to a hundred percent total. Number of animals analyzed: <i>raw^+/+,acal^5/5</i> = 391, <i>raw^2/+,acal^5/5</i> = 139, <i>raw^2/2,acal^+/+</i> = 366, <i>raw^2/2,acal^5/+</i> = 152, <i>raw^2/2,acal^5/5</i> = 208. Significance was assessed with chi square tests. (D-E) <i>acal</i> in situ hybridization in <i>raw²</i> mutants (n = 45; D) and heterozygous siblings (n = 106; E). Arrow in (E) points to decreased <i>acal</i> expression in the lateral epidermis. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004927#pgen.1004927.s005" target="_blank">S5 Fig.</a> (F-I) Scanning electron micrographs of dorsal views of adult thoraces, anterior is up. Scale bars are 100 μm. (F) <i>UAS-acal/+</i> control, (G) <i>pnr-gal4/+</i> control, and (H) over-expression of two <i>UAS-acal</i> copies. The white box in (H) is amplified in (I), depicting distances (red lines) measured to determine the thoracic cleft index, using anterior dorso-central (ADC) and posterior dorso-central (PDC) bristles as references (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004927#sec004" target="_blank">Materials and Methods</a>). (J) Percentage change of thoracic cleft index for different experimental conditions. Mean of 15 flies +/− SEM. Significance was calculated using ANOVA and Bonferroni correction.</p

<i>acal</i> regulates <i>aop</i> expression.

<p>(A) Genetic interaction between <i>aop</i> and <i>acal</i> mutants. Embryos surviving embryogenesis represent the open space above bars to amount to a hundred percent total embryos analyzed. Number of animals analyzed: <i>aop^+/+,acal^5/5</i> = 391, <i>aop^1/+,acal^5/5</i> = 253, <i>aop^1/1,acal^+/+</i> = 269, <i>aop^1/1,acal^5/+</i> = 185. (B) Relative expression of <i>aop</i> in wild type and mutant embryos, as determined by qPCR. (C) Expression of a constitutive active <i>aop</i> transgene (UAS-<i>aop^ACT</i>) in the ectoderm (<i>69B-gal4</i>) or in the lateral epidermis (<i>pnr-gal4</i>), of wild type or <i>acal</i> mutant embryos. Number of animals analyzed: <i>69B>aop^ACT</i> = 642, <i>69B>aop^ACT;acal^5/5</i> = 210, <i>pnr>aop^ACT</i> = 310, <i>pnr>aop^ACT;acal^5/5</i> = 180. (D) <i>acal</i> over-expression under the <i>69B-gal4</i> driver results in significantly decreased <i>Cka</i> and <i>aop</i> expression. In (B) and (D), graphs show mean values +/− SEM of at least three independent experiments run twice.</p

Model for <i>acal</i> function during DC.

<p><i>acal</i>, expressed in the lateral epidermis (blue), restrains JNK activation to the leading edge cells. Raw, also expressed in the lateral epidermis, regulates <i>acal</i> expression. In turn, <i>acal</i> regulates the expression of <i>Cka</i> and <i>aop</i> to fine-tune JNK signaling. Not depicted is <i>aop</i> positive role during DC.</p

<i>Cka</i> is downstream of <i>raw</i> and <i>acal</i>.

<p>(A) Genetic interaction between <i>Cka</i> and <i>acal</i> mutants. Number of animals analyzed: <i>Cka^+/+,acal^2/2</i> = 192, <i>Cka^1/+,acal^2/2</i> = 219, <i>Cka^+/+,acal^5/5</i> = 391, <i>Cka^1/+,acal^5/5</i> = 171. (B) <i>Cka</i> relative expression in wild type and mutant embryos, as determined by qPCR. (C) Expression of a heat-shock inducible <i>Cka</i> transgene results in DC defects. Number of animals analyzed: <i>yw</i>, 25°C = 526, <i>yw</i>, 29°C = 591, <i>HS-Cka</i>, 25°C = 2006, <i>HS-Cka</i>, 29°C = 3227. (D) <i>Cka</i> expression increase due to heat shock in <i>hs</i>-Cka flies was confirmed by qPCR. For (A,C) embryos surviving embryogenesis represent the open space above the bar to amount to one hundred percent total of embryos analyzed. Chi square tests were used to calculate significance. For (B,D) represents the means of three independent experiments run twice +/− SEM. Significance was assessed using Student’s t test. (E-H) <i>dpp</i> in situ hybridization experiments, showing JNK-induced <i>dpp</i> expression (arrows). (E) Wild type embryo. (F) <i>raw^1/1,en-gal4/+</i> control, showing <i>dpp</i> ectopic activation (arrow). (G) Expression of <i>UAS-rawRA</i> with <i>en-gal 4</i>, which expresses <i>gal 4</i> at posterior compartments of each segment. Arrowheads show cell-autonomous suppression of <i>dpp</i> ectopic expression. (H) Silencing of <i>Cka</i> with an RNAi construct (<i>UAS-Cka-IR</i>) under <i>en-gal 4</i> also suppresses <i>dpp</i> ectopic expression in posterior compartments of <i>raw¹</i> mutants (arrowheads).</p