Comparison of N-terminal regions of GEI-8-related proteins to NCoR/SMRT.

<p>Sequence alignment of GEI-8 nematode orthologues with their nearest Metazoa/Fungi homologues, both human orthologues NCoR1 and SMRT (NCoR2) are shown. Green bars indicate the position of the alpha-helices in the structure of the upstream DAD domain of human SMRT and homology predicted positions in the second SANT domain. Residues indispensable for regulating HDAC interactions and function are highlighted in blue (needed for the structural integrity), magenta (interaction with HDAC) and red (activation of HDAC). Only the N-terminal part of the sequences is shown. The identical and similar residues are highlighted by different intensity of shading. Sequence identifiers: <i>C. elegans</i>: GEI8_CAEEL, <i>C. brenneri</i>: CN15693, <i>C. briggsae</i>: A8X8F0_CAEBR, <i>C. remanei</i>: RP40355, <i>C. japonica</i>: JA23925 ABLE03010463.1 ABLE03032768.1 ABLE03032771.1 ABLE03032769.1 ABLE03032772.1, <i>Loa loa</i>: E1FVE0_LOALO, <i>Brugia malayi</i>: A8NSC3_BRUMA, <i>Ixodes scapularis</i>: B7PZ26_IXOSC, <i>Saccharomyces cerevisiae</i>: SNT1_YEAST, <i>Drosophila melanogaster</i>: Q9VYK0_DROME, <i>Trichoplax adhaerens</i>: B3SAN1_TRIAD, <i>Branchiostoma floridae</i>: C3XV35_BRAFL, <i>Danio rerio</i>: A8B6H7_DANRE, <i>Xenopus tropicalis</i>: NCOR1_XENTR AAMC01044136.1, <i>Anolis carolinensis</i>: ANOCA15679 2 ENSACAP00000014806; ENSACAT00000015107, <i>Gallus gallus</i>: UPI0000E813A6, <i>Homo sapiens</i>: NCOR2_HUMAN (NCoR2), <i>Homo sapiens</i>: NCOR1_HUMAN (NCoR1). PDB structure 1XC5 was used to determine the position of the helices.</p

Analysis of neuromuscular function of <i>gei-8(ok1671)</i> mutant (VC1213).

<p>Aldicarb and levamisole sensitivity assays revealed increased sensitivity of <i>gei-8</i> mutants towards the acetylcholinesterase inhibitor aldicarb (<b>A</b>) and levamisole (<b>B</b>) suggesting a synaptic defect in cholinergic transmission.</p

Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.

<p>Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.</p

Development of the germline in <i>gei-8(ok1671)</i> mutants and additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (<i>sqrd-2</i>) in homozygous <i>gei-8(ok1671)</i> mutants.

<p>(<b>A</b>) The reproductive structures of a wild-type larva at the L4 stage is shown. The vulva is indicated by an arrowhead and formation of the uterus is visible next to vulval structures. The position of the lead migrating cell for the gonad (distal tip cell) during the larval L4 stage is indicated by arrow. (<b>B</b>) Development of the gonad in a young adult N2 animal. The distal gonad arm continues in growth beyond the position of the vulva (marked by arrowhead) and makes contact with the proximal gonad arm (arrow). (<b>C</b>) <i>gei-8(ok1671)</i> mutant gonadogenesis by Nomarski optics. The arrested gonad arm in a position similar to wild type L4 larva is indicated by arrow. The vulva is marked by an arrowhead. (<b>D</b>) A <i>gei-8(ok1671)</i> mutant with arrested growth of the gonad as visualized by DAPI staining. The distal tip of arrested gonad is marked by an arrow and the vulva by an arrowhead. (<b>E</b>, <b>F</b>, <b>G</b>, <b>H</b>, <b>I</b> and <b>J</b>) Additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (<i>sqrd-2</i>) region including three 21U-RNAs: 21ur-2020, 21ur-11733 and 21ur-9201 in <i>gei-8(ok1671)</i> homozygous mutant animals. (<b>E</b>) A <i>gei-8(ok1671)</i> mutant treated with <i>sqrd-2</i> RNAi shows growth of the gonad beyond the usual arrest point, reaching the position of the vulva (marked by arrow and arrowhead, respectively). (<b>F</b>) Additional phenotypes of <i>gei-8(ok1671)</i> animals treated with <i>sqrd-2</i> RNAi. Nomarski optics view of homozygous <i>gei-8(ok1671)</i> larva treated with <i>sqrd-2</i> RNAi revealing frequent growth defects, including irregular body shapes, (distention of proximal part of the body and thin elongation of the distal part of the body) and extended growth of the distal part of the gonad. The gonad is visualized by DAPI staining in panel <b>G</b> (distal arm of the gonad is marked by right arrow, proximal arm of the gonad is marked by left arrow). Arrowhead indicates the position of vulva in panels E, F and G. (<b>H</b>) Additional growth defects induced by <i>sqrd-2</i> RNAi in homozygous <i>gei-8(ok1671)</i> worms including a Pvul phenotype (arrowhead), accumulation of gonadal cells with a possible incomplete second vulva formation (left arrow) and a distal arm of germline that fails to turn and instead continues to grow in the direction of the thin and elongated tail (right arrow). (<b>I</b>) A mutant animal with germline growth directional changes of both gonad arms induced by <i>sqrd-2</i> RNAi: anterior gonad arm makes an incomplete turn dorsally and continues to grow in the anterior direction (left arrow) while the posterior gonad arm fails to turn and continues in additional growth towards the tail (right arrow). The position of vulva is indicated by arrowhead. (<b>J</b>) A homozygous <i>gei-8(ok1671)</i> mutant developing a convoluted irregular accumulation of cells of distal gonad arm in the position of gonad turn (marked by arrows). The position of vulva is indicated by arrowhead. Scale <b>A</b>, <b>B</b>, <b>D</b>, <b>E</b> and <b>J</b> 50 µm, <b>C</b>, <b>F</b>, <b>G</b>, <b>H</b> an <b>I</b> 100 µm.</p

Induction of additional gonad and body shape phenotypes in homozygous <i>gei-8</i>(<i>ok1671</i>) mutant worms by RNAi directed against <i>sqrd-2</i> or <i>sqrd-1</i>.

<p>Induction of additional gonad and body shape phenotypes in homozygous <i>gei-8</i>(<i>ok1671</i>) mutant worms by RNAi directed against <i>sqrd-2</i> or <i>sqrd-1</i>.</p

GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in <em>Caenorhabditis elegans</em>

<div><p>NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although <i>C. elegans</i> has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in <i>Drosophila</i> or <i>C. elegans</i>. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in <i>C. elegans</i> and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the <i>gei-8</i> coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous <i>gei-8</i> mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.</p> </div

Analysis of the pharyngeal pumping rate of <i>gei-8(ok1671)</i> mutant animals and controls.

<p>Pharyngeal pumping rate is regulated by cholinergic transmission. In <i>gei-8</i> mutants the pumping rate is low compared to wild-type animals and decreases with age (n = 10 for each category).</p

Analysis of <i>gei-8</i> expression using transgenic lines.

<p>The expression of <i>gei-8</i> was studied using transgenic lines carrying three different predicted promoters (#1, #2 and #3) fused with gene coding for GFP (indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058462#pone-0058462-g002" target="_blank">Figure 2C</a>) <i>gei-8::GFP</i>. Panels B and D show the expression from promoter #1 and panels F, H, I, J and K show the expression from promoter #3. Expression from promoter #2 construct was identical with that from promoter #3 and is not shown. (<b>A</b> and <b>B</b>) Embryonic GFP expression is ubiquitously present since comma stage. (<b>C</b> and <b>D</b>) L2 larva expressing <i>gei-8::GFP</i> ubiquitously with the highest expression in the head neurons and in the neuronal ring (arrowheads) and intestinal cells (arrows). (<b>E</b> and <b>F</b>) Expression of GEI-8::GFP in pharyngeal neurons (arrowheads), ventral nerve cord (arrows), anal sphincter (arrow - as) and tail neurons (arrow - tn) of an L4 larva. (<b>G</b> and <b>H</b>) Expression of GEI-8::GFP in L4 male larva. Additional expression is seen in male specific neurons (arrowheads). (<b>I</b>) L4 larva expressing GEI-8::GFP in egg laying structures, vulval and uterine muscles (arrows), egg laying neurons (arrowheads). (<b>J</b>) GEI-8::GFP expression in somatic muscles (arrows) and nerve cord (arrowheads). (<b>K</b>) Detail of expression of GEI-8::GFP in hermaphrodite tail neuron (arrowhead) and anal sphincter (arrows - as). (Figs. <b>A</b>, <b>C</b>, <b>E</b>, <b>G</b> in Nomarski optics and <b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>, <b>I</b>, <b>J</b>, <b>K</b> in fluorescence microscopy). Scale: <b>A</b>, <b>B</b>, <b>I</b>, <b>J</b> 20 µm; <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>, <b>G</b>, <b>H</b> 100 µm; <b>K</b> 50 µm.</p