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

BBS-7::GFP restores phasmid dye-filling in <i>bbs-7(my13)</i> mutant animals.

<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 wild-type (<i>him-5(e1490)</i> animals take up dye in both the amphids and phasmids. Some wild-type and <i>bbs-7(my13)</i> animals carrying the BBS-7::GFP extrachromosomal array are tailDyf.</p><p>BBS-7::GFP restores phasmid dye-filling in <i>bbs-7(my13)</i> mutant animals.</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

<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

<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

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 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

Amplification of <i>hecate/grip2a</i>-dependent symmetry breaking event by a general animal-directed long-range transport system.

<p>A) Cortical shifts of various vegetally localized components, including <i>wnt8a</i> mRNA, Sybu protein and <i>grip2a</i> mRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Lu1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Tran1" target="_blank">[8]</a>; this report) are short-range and dependent on microtubule bundling and alignment, itself dependent on <i>hec</i> function. In wild-type embryos, such a short-range shift generates a symmetry breaking event that is subsequently amplified by long-range, animally-directed transport mechanism independent of <i>hec</i> function and not restricted to the prospective dorsal axis. B) In <i>hec</i> mutant embryos, neither reorganization of vegetal microtubules into aligned bundles nor a short-range shift occur, so that, even though long-range transport remains intact, vegetal determinant transport to the animal pole is affected. The mechanistic basis for the long-range transport, occurring in the region of a loosely organized mediolateral microtubule cortical network remains to be determined (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#s3" target="_blank">Discussion</a>).</p

Molecular identification of the <i>hecate</i> locus.

<p>A) Linkage map of the <i>hec</i> locus. The number of recombinants over the total number of analyzed meiosis is indicated. <i>hec</i> linkage was initially identified between SSLP markers z59658 and z24511 on chromosome 8. Fine mapping analysis with newly identified RFLP markers further narrowed the region between the gene <i>gpd1a-1</i> and the RFLP zC150E8y. B) Contig of five BAC clones covering the <i>hec</i> critical region. CH73-233M11, CH73-272M14, CH73-250D21, DKEY-43H14 and CH211-150E8 are five sequenced and overlapping BAC clones in this interval. C) Exon-intron structure of the <i>hec/grip2a</i> gene, which contains 16 exons. The <i>hec^p06ucal</i>, <i>hec^t2800</i> and <i>hec^p08ajug</i> alleles each cause a premature stop-codon in exon 4, exon 10 and exon 12, respectively. D) Sequence traces of the cDNA products from wild-type and the three mutant <i>hec</i> alleles. Nucleotide substitutions are indicated by the red box. Mutant cDNAs show a C-A transversion in codon 118 (<i>hec^p06ucal</i>), a C-T transversion in codon 414 (<i>hec^t2800</i>), or a C-T transversion in codon 499 (<i>hec^p08ajug</i>), all creating premature STOP codons. E) Schematic diagram showing the protein domain structures of Grip2a in the wild-type and mutant alleles. Red boxes represent conserved PDZ domains.</p

Defects in the vegetal localization of <i>wnt8a</i> mRNA and Sybu protein.

<p>A–D) Off-center shift of <i>wnt8a</i> mRNA is affected in <i>hec</i> mutants. Whole mount in situ hybridization of wild-type embryos (A,C) and <i>hec</i> mutant embryos (B,D) at the 1- (A,B, 30 mpf) and 4- (C,D, 60 mpf) cell stages. Images show representative embryos. A majority of wild-type embryos showed a clear off-center shift (85%, n = 27 at 30 mpf and 74%, n = 47 at 60 mpf). A majority of <i>hec</i> mutant embryos showed vegetal localization without a shift at 30 mpf (79%, n = 33, remaining embryos show no localization) and absence of localization at 60 mpf (89%, n = 38, remaining embryos show reduced vegetal localization without a shift). The apparent label at the base of the blastodisc is observed in a majority of mutant embryos (71%, n = 38) but not in wild-type (C) or control embryos labeled with other probes (not shown) and may reflect remaining <i>wnt8a</i> mRNA that has lost anchoring at the vegetal pole and has moved animally through the action of axial streamers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Fuentes1" target="_blank">[83]</a>. E–I) Localization of Sybu protein is affected in <i>hec</i> mutants. Whole mount immunofluorescence to detect Sybu protein of untreated wild-type (E,G) and <i>hec</i> mutant (F,H,) embryos and nocodazole-treated wild-type embryos (I) at the indicated stages. In wild-type embryos, an off-center shift in Sybu protein can be observed starting at 30 mpf (G). In <i>hec</i> mutants, Sybu protein becomes undetectable levels by this same time point (H). Patterns of localization of Sybu protein at 10 mpf and 20 mpf time points (combined n: 32 WT, 19 mutant for 10–20 mpf), and 30 mpf and 40 mpf time points were similar and have been combined. 59% (n = 32) of wild-type and 63% (n = 19) of <i>hec</i> mutant embryos showed centered vegetal localization during 10–20 mpf. At 30–40 mpf, the percent of embryos that showed vegetal localization, now with an off-center shift, was reduced to 25% (n = 28) in wild-type, and 0% (n = 25) of <i>hec</i> mutants showed any localization at these time points. Treatment of wild-type embryos with nocodazole inhibits the shift but does not result in delocalization from the vegetal cortex (I, embryo at 40 mpf), as previously shown <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>. Magnification bars in (D) and (I) correspond to 100 µm for panels sets (A–D) and (E–I), respectively.</p