Identification of Factors Affecting Proper Localization of C. elegans PKD-2

Color poster with text, images, diagrams, charts, and tables.Primary cilia are cellular antennae that mediate responses to the external environment. Response requires perception of a cue and transmission of that signal to the cell. The purpose of this study was to understand how primary cilia, senory antennae, sense and respond to cues from the cellular environment.National Institute of Health; University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

RNAinterference Identifies Genes Important for Cilia Structure and Function

Color poster with text, images, and charts.Primary cilia are cellular antennae that mediate responses to the external environment. Response requires perception of a cue and transmission of that signal to the cell. This study employed a reverse genetic strategy known as RNAinterference to systematically reduce the function of each gene chromosome I and examine the effects on PKD-2 localization in C. elegans. Once the genes have been identified that affect PKD-2 localization, the role of these genes in cilia structure and function will be examined.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs; National Institutes of Health grant; Kell Container award

Identification and characterization of a novel allele of Caenorhabditis elegans bbs-7.

Primary cilia play a role in the sensation of and response to the surrounding environment. Caenorhabditis elegans (C. elegans) have primary cilia only on the distal tips of some dendrites. In order to better understand the relationship between receptor localization to cilia, cilia structure and cilia function, we have characterized a mutation originally identified in a forward genetic screen for mutants with defective PKD-2 ciliary localization. Through behavioral assays and examination of the structure of cilia in the cil-5 (my13) mutant animals, we have found that my13 disrupts not only receptor localization, but also some cilia-mediated sensory behaviors and cilia structural integrity. We have identified the my13 lesion and found that it is a missense mutation in bbs-7, an ortholog of human BBS-7, a gene known to affect human cilia and to be involved in Bardet-Biedl syndrome. Finally, we show that bbs-7(my13) also affects the glia cells which support the cilia

<i>bbs-7(my13)</i> animals have vacuoles present in amphid sheath cells.

<p>The sheath glia surround the amphid neurons (A). Wild-type worms express F16F9.3pro: mCherry in the amphid sheath cells (B). <i>bbs-7(my13)</i> also express F16F9.3pro: mCherry in the amphid sheath cells but the sheath cells have round areas which lack expression (C, D and E). Enlarged views of boxed areas in C, D and E are shown in C′, D′, and E′, respectively. (Asterisks indicate vacuoles). Scale  = 20 microns. Anterior to left. The percent of animals with vacuoles in the amphid sheath cells differs between wild-type and mutant animals at the L4, day 1 adult, and day 2 adult stages but not at the day 4 adult or day 6 adult stage (F). (Bars indicate the 95% confidence interval calculated using a 1-sample proportions test with continuity correction.)</p

Distance traveled in response to different odorants.

<p>Percentage of animals in the sections closest to the point sources of chemicals or diluent (A and E) in the chemotaxis assays. Wild-type animals traveled further on the plate than <i>bbs-7(my13)</i> animals when only ethanol, the diluent, was present on the plate (p-values: at 10 minutes  = 0.00158, at 20minutes  = 0.000525, at 30minutes  = 0.00873). In the presence of a strong repellent (100% benzaldehyde) or a strong attractant (1% diacetyl), both wild-type and <i>bbs-7(my13)</i> animals travel similar distances on the assay plate (p-values for 100% benzaldehyde: at 10 minutes  = 0.150, at 20 minutes  = 0.375, at 30 minutes  = 0.08501; p-values for 1% Diacetyl: at 10 minutes  = 0.589, at 20 minutes  = 0.322, at 30 minutes  = 0.0681). Value in parentheses indicates standard error of the mean.</p><p>Distance traveled in response to different odorants.</p

Complementation tests involving three alleles of <i>bbs-7</i> reveal complexity in interactions between alleles.

<p>Animals homozygous for the <i>my13</i> allele do not take up fluorescent dye in their phasmids and exhibit abnormal dye-filling in the amphids (tailDyf) while <i>ok1351</i> homozygotes and <i>n1606</i> homozygotes do not take up dye in either the amphids or phasmids (Dyf). Trans-heterozygotes involving the <i>my13</i> allele (<i>my13/ok1351</i> and <i>my13/n1606)</i> have a similar phenotype to the <i>my13</i> homozygotes. <i>ok1351/n1606</i> trans-heterozygotes are pre-dominantly dye-filling defective. Wild-type animals (<i>him-5(e1490))</i> were 100% nonDyf (n = 46). The percentage of animals with PKD-2::GFP mislocalization (the Cil phenotype) is less in the trans-heterozygotes compared to animals homozygous for <i>my13</i> allele.</p><p>Complementation tests involving three alleles of <i>bbs-7</i> reveal complexity in interactions between alleles.</p

The <i>my13</i> mutation affects <i>bbs-7</i>.

<p>Structure of the <i>bbs-7</i> gene (A). Exons are numbered and introns are lettered. The <i>my13</i> mutation changes the first nucleotide of exon 6 from a G to an A. RT-PCR of mRNA isolated from wild-type and homozygous <i>my13</i> animals results in products that are the same size (B). Chromatograms showing partial wild-type and mutant <i>bbs-7</i> sequence (C). Alignment of the region of BBS-7 affected by the mutation in <i>my13</i> (D). Alignments of BBS-7 proteins from six species were generated in ClustalOmega and displayed with Boxshade using a threshold of 50% sequence identity. Conserved and similar amino acids are shown in black and gray boxes, respectively. Asterisk denotes the <i>C. elegans</i> glycine (amino acid 314) affected by the <i>my13</i> mutation.</p

<i>bbs-7(my13)</i> animals exhibit altered responses to some volatile chemicals.

<p>Wild-type worms approach a point source of 100% isoamyl alcohol more than <i>bbs-7(my13)</i> mutant animals (A). Wild-type and <i>bbs-7(my13)</i> mutant animals respond in a similar fashion to 1% isoamyl alcohol, 100% benzaldehyde and 100% diacetyl (B, C and D) when assessed using the Worm Chemotaxis Index. <i>bbs-7(my13)</i> animals do not show the same strength of attraction as wild-type animals to 1% isoamyl alcohol (E) but show the same degree of avoidance of 100% benzaldehyde (F). Error bars indicate standard error of the mean. P-values calculated using a standard t-test. ***<.001 **<.005 *<.05</p

<i>bbs-7(my13)</i> animals exhibit defects in PKD-2::GFP localization and the ability of sensory neurons to take up lipophilic dye.

<p>PKD-2::GFP localizes to the cilium proper and base of the male-specific CEM neurons in wild-type worms (bracket and asterisk, respectively, in A). The <i>bbs-7(my13)</i> male CEM neurons also have PKD-2::GFP in the cilium proper and base but the CEM cilia curve inward and there is additional accumulation of PKD-2::GFP in the base (bracket and asterisk, respectively, in B). Wild-type worms take up DiI in both the dendrites and cell bodies of the amphids (C and G) and the phasmids (D). <i>bbs-7(my13)</i> animals take up dye in the amphid neurons (D and H) but not the phasmid neurons (F) and the quality of the dye-filling in the amphid neurons is not equal to wild-type. Different worms are shown in panels C and G, and D and H. Scale  = 100 microns in A and B. Scale  = 10 microns in E and F. Anterior to left.</p