Data_Sheet_1_The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila.pdf

IntroductionCilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. We previously isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology.ResultsHere we molecularly characterize the btv gene and demonstrate that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. We also describe morphological changes in Johnston’s organ as flies age to 30 days, and we find that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. We show that NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials.DiscussionThus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs.</p

Complex, polygenic regulation of gestation length is revealed by the B.A CSS panel.

<p>(A) Mean gestation time in total hours for each of 20 CSS. The total number of pregnancies monitored is indicated for each strain and data are presented as the mean +/− S.E.M. (B) Graphical representation of individual CSS with significantly different GLs independent of all other factors. Strains that are not associated by a letter are significantly different from each other (p<0.05).</p

Data summary from individual strain measurements.

<p>Total litter size includes all (live and dead) pups identified. Weight per live pup is the average of all surviving pups for each individual strain. Dead pups were often found desiccated and partially cannibalized and are thus excluded. Survival rate is the percentage of total pups identified that survived until at least postnatal day 3. Maternal weight was measured and recorded following the identification of a copulation plug and at E14.5 to calculate weight gain. At this time point, females were housed in front of the cameras and not disturbed until a birth was recorded. Pregnancy load is the maternal weight gained as a percentage of initial weight following a successful mating.</p

Highly significant differences in gestation length among inbred mouse strains.

<p>(A) Gestational length presented in total hours, measured from the midpoint of the dark cycle prior to the appearance of a copulation plug to the recorded appearance of the first pup. The total number of pregnancies monitored is indicated for each strain and data are presented as the mean +/− S.E.M. A detailed description of the animal husbandry and measurement procedures is provided in the supplementary methods (6). (B) Live litter size (number of pups), maternal weight gain (at E14.5) and maternal load (% weight gain) for each of the 15 inbred strains measured in (A). Complete data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012418#pone-0012418-t001" target="_blank">Table 1</a>. (C) Effects test (analysis of covariance) demonstrating significant differences in GL among inbred strains independent of the effect of total litter size and maternal load. Litter size is also significantly different among strains and is strongly correlated with GL. (D) Graphical representation of strains with significantly different GLs independent of all other factors. Strains that are not associated by a letter are significantly different from each other (p<0.05).</p

Gestation length is primarily dependent upon maternal genotype.

<p>(A) Gestation length presented in hours of C57BL/6J, A/J, B6.CB17-<i>Prkdc^scid</i>/SzJ (B6-<i>scid</i>), and B6-scid with transplanted A/J ovaries (AJ-ov-B6-<i>scid</i>). In order to directly compare the effect of pure B6 and A/J pups in a B6 maternal background, B6-<i>scid</i> mice were sham manipulated and mated to B6-scid males, while AJ-ov-B6-<i>scid</i> females were mated to A/J males following recovery from ovary transplant (see materials and methods for details). (B) Graphical representation of strains with significantly different GLs independent of all other factors. Strains that are not associated by a letter are significantly different from each other (p<0.05), demonstrating that B6 females show no statistical difference in gestation time, regardless of the genotype of the pups.</p

Identification of rare variants associated with ulcerative colitis.

a<p>Positions from Human genome build 36.</p>b<p>Previously reported variant independently identified in the current study.</p>c<p>Minor allele frequencies estimates from combined case:control cohorts; actual allele frequencies can vary between cohorts.</p><p>UC, Ulcerative Colitis; HC, Healthy Controls; NA, data not available.</p

Functional characterization of RNF186.

<p>(A) <i>RNF186</i> encodes a protein with RING domain and two transmembrane domains. E3 ubiquitin-protein ligase activity is intrinsic to the RING domain. This domain contains the disease-coding variant (A64T). (B) <i>RNF186</i> expression response to <i>S. flexneri</i> in young mice (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723.s011" target="_blank">Figure S11</a>). (C) Network building steps. Network is generated by mining multiple sources of interaction databases in Metacore that span human protein-protein, protein-DNA, Protein-RNA and protein-compounds interactions. (D) Transcriptional regulation model for <i>RNF186</i>. IL1-beta and TGF-beta 1 decrease <i>HNF4A</i> mRNA expression <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Caja1" target="_blank">[39]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Wang1" target="_blank">[41]</a>. Knockdown of retinoid X receptor, alpha (<i>RXRA</i>) down-regulates <i>HNF4A</i> gene expression; RXRA interacts with <i>HNF4A</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Tomaru1" target="_blank">[24]</a>. <i>HNF4A</i> is a direct target gene of caudal type homeobox 2 (CDX2); CDX2 increases <i>HNF4A</i> mRNA expression in intestinal epithelial cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Boyd2" target="_blank">[42]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-McKinneyFreeman1" target="_blank">[43]</a>. HNF4A binds promoter region of <i>HNF1A</i> and up-regulates its expression. HNF1A interacts with <i>RNF186</i> and regulates its transcription.</p