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Results: 7

1.
Figure 7

Figure 7. Analysis of neuromuscular function of gei-8(ok1671) mutant (VC1213).. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

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

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
2.
Figure 5

Figure 5. Analysis of the pharyngeal pumping rate of gei-8(ok1671) mutant animals and controls.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

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

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
3.
Figure 3

Figure 3. Normalized expression of gei-8a.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

The expression of gei-8a was measured for two regions (with primers 6168 and 01/042 and 6200 and 01/153) and quantitated relative to the constitutive gene ama-1. The expression of gei-8a peaks in the L4 stage. Relative expression was determined as proportion of lowest expression found in the embryonic stage and indicated as arbitrary units.

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
4.
Figure 2

Figure 2. Expression analysis of gei-8.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

(A) Schematic representation of the predicted gei-8a isoform consisting of 17 exons compared with detected expression. cDNA clones 7320, 7323 and 7324 are indicated with their exons (open rectangles). Expression of exon 12 and 16 is not constant. Exon 12 in cDNA clone 7323 and 45 bp from exon 16 in cDNAs 7323 and 7324 were removed by alternative splicing (bottom two lines). The location of predicted SANT and glutamine-rich interaction domains is marked by lines above the gei-8a diagram. Location of gei-8(ok1671) mutation used in expression analysis is marked by a line below the gei-8a diagram. Two regions identified as putative CoRNR nuclear receptor binding motifs are indicated in the exon 8 and 15 (NR). (B) Schematic representation of predicted gei-8 isoforms a, b and c. (C) GFP reporter gene constructs #1, #2 and #3 used for expression analysis. (D) Overlapping regions of gei-8 gDNA used for rescue. The size of the overlapping region is 191 bp.

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
5.
Figure 1

Figure 1. Comparison of N-terminal regions of GEI-8-related proteins to NCoR/SMRT.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

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: C. elegans: GEI8_CAEEL, C. brenneri: CN15693, C. briggsae: A8X8F0_CAEBR, C. remanei: RP40355, C. japonica: JA23925 ABLE03010463.1 ABLE03032768.1 ABLE03032771.1 ABLE03032769.1 ABLE03032772.1, Loa loa: E1FVE0_LOALO, Brugia malayi: A8NSC3_BRUMA, Ixodes scapularis: B7PZ26_IXOSC, Saccharomyces cerevisiae: SNT1_YEAST, Drosophila melanogaster: Q9VYK0_DROME, Trichoplax adhaerens: B3SAN1_TRIAD, Branchiostoma floridae: C3XV35_BRAFL, Danio rerio: A8B6H7_DANRE, Xenopus tropicalis: NCOR1_XENTR AAMC01044136.1, Anolis carolinensis: ANOCA15679 2 ENSACAP00000014806; ENSACAT00000015107, Gallus gallus: UPI0000E813A6, Homo sapiens: NCOR2_HUMAN (NCoR2), Homo sapiens: NCOR1_HUMAN (NCoR1). PDB structure 1XC5 was used to determine the position of the helices.

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
6.
Figure 4

Figure 4. Analysis of gei-8 expression using transgenic lines.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

The expression of gei-8 was studied using transgenic lines carrying three different predicted promoters (#1, #2 and #3) fused with gene coding for GFP (indicated in Figure 2C) gei-8::GFP. 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. (A and B) Embryonic GFP expression is ubiquitously present since comma stage. (C and D) L2 larva expressing gei-8::GFP ubiquitously with the highest expression in the head neurons and in the neuronal ring (arrowheads) and intestinal cells (arrows). (E and F) 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. (G and H) Expression of GEI-8::GFP in L4 male larva. Additional expression is seen in male specific neurons (arrowheads). (I) L4 larva expressing GEI-8::GFP in egg laying structures, vulval and uterine muscles (arrows), egg laying neurons (arrowheads). (J) GEI-8::GFP expression in somatic muscles (arrows) and nerve cord (arrowheads). (K) Detail of expression of GEI-8::GFP in hermaphrodite tail neuron (arrowhead) and anal sphincter (arrows - as). (Figs. A, C, E, G in Nomarski optics and B, D, F, H, I, J, K in fluorescence microscopy). Scale: A, B, I, J 20 µm; C, D, E, F, G, H 100 µm; K 50 µm.

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.
7.
Figure 6

Figure 6. Development of the germline in gei-8(ok1671) mutants and additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (sqrd-2) in homozygous gei-8(ok1671) mutants.. From: GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans.

(A) 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) 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). (C) gei-8(ok1671) 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. (D) A gei-8(ok1671) 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. (E, F, G, H, I and J) Additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (sqrd-2) region including three 21U-RNAs: 21ur-2020, 21ur-11733 and 21ur-9201 in gei-8(ok1671) homozygous mutant animals. (E) A gei-8(ok1671) mutant treated with sqrd-2 RNAi shows growth of the gonad beyond the usual arrest point, reaching the position of the vulva (marked by arrow and arrowhead, respectively). (F) Additional phenotypes of gei-8(ok1671) animals treated with sqrd-2 RNAi. Nomarski optics view of homozygous gei-8(ok1671) larva treated with sqrd-2 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 G (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. (H) Additional growth defects induced by sqrd-2 RNAi in homozygous gei-8(ok1671) 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). (I) A mutant animal with germline growth directional changes of both gonad arms induced by sqrd-2 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. (J) A homozygous gei-8(ok1671) 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 A, B, D, E and J 50 µm, C, F, G, H an I 100 µm.

Pavol Mikoláš, et al. PLoS One. 2013;8(3):e58462.

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