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First published online 19 October 2005
doi: 10.1242/dev.02069

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Specification of muscle neurotransmitter sensitivity by a Paired-like homeodomain protein in Caenorhabditis elegans

Department of Biology, McGill University, 1205 Avenue Dr Penfield, Montréal, Québec H3A 1B1, Canada

View larger version (17K): [in a new window]   Fig. 1. The defecation phenotypes of dsc-1 mutants. (A) dsc-1 mutants have a shortened defecation cycle length. The bars represent the mean of the mean cycle lengths of n animals that had each been scored for five consecutive defecation cycles; the error bars represent the standard deviation of the animal means (n=20-30 animals). (B) dsc-1 mutants have an expulsion defective (Exp) phenotype. The bars represent the mean number of expulsions per six pBocs; the error bars represent the range (of number of expulsions/six pBocs) observed (n=20 animals). For both A and B, the asterisks indicate that the data are significantly different from that of N2. All significant differences detected were at a level of P<0.0001.

View larger version (124K): [in a new window]   Fig. 2. The Con phenotype of Exp mutants. Nomarski images of the tail region of wild-type and Exp mutant hermaphrodites (A-D) and their male counterparts (E-H). Arrows indicate the width of the intestinal lumen. N2 hermaphrodites and males are neither expulsion defective (Exp) nor Constipated (Con). dsc-1(qm133) (B) exp-1(sa6) (C) and unc-25(e156) (D) hermaphrodites are Exp and, as a result, exhibit the Con phenotype, as the gut lumen fills with bacterial debris and becomes distended. Neither dsc-1 (F) nor exp-1 (G) males are Con, indicating that they are not expulsion defective. By contrast, unc-25(e156) males are Exp, and as a result are severely Con (H). Scale bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 3. Positional cloning of dsc-1. (A) Mutations, deficiencies and cosmids used for the positional cloning of dsc-1. (B) Genomic structure of dsc-1 and the lesions of the two dsc-1 alleles. The dark gray box indicates the homeobox, and the light gray box indicates the 3' UTR. (C) Comparison between the homeodomain of DSC-1 with the consensus Q50 Paired-like homeodomain (Prd HD) (see Galliot et al., 1999), and the homeodomains of the six other Q50 Paired-like homeodomain proteins in C. elegans. The residues shaded in gray indicate the six residues that are diagnostic of the Paired class; the residues in bold indicate invariant or highly conserved residues that are not specific to the Paired class; the box indicates the Q50. The qm133 mutation results in an R-to-H substitution in an absolutely conserved residue at position 53 of the homeodomain (indicated by an asterisk).

View larger version (67K): [in a new window]   Fig. 4. The expression patterns observed for dsc-1 transcriptional and translational reporter gfp fusions. (A-C) The pattern of expression observed with a transcriptional gfp reporter. dsc-1 is expressed in four pairs of sensory neurons in the head (A), the PVD neuron (B) and the sphincter muscle (C). (D-F) The pattern of expression observed with a fully functional translational gfp reporter. Expression of a translational fusion of dsc-1 can be detected in the nuclei of the same sensory neurons that express the transcription fusion (D,E); however, by contrast to the transcriptional fusion, the translational fusion is expressed in the intestinal and anal depressor muscles, but not in the sphincter (F). All animals were adults, albeit of different ages and sizes. Scale bar: 10 µm.

View larger version (28K): [in a new window]   Fig. 5. Expression of dsc-1(+) in two enteric muscles in sufficient to rescue the defecation defects of dsc-1 mutants. (A) Schematic of construct used to express dsc-1 specifically in the AD and IM. (B) Expression of the construct depicted in (A) in dsc-1(qm133) animals. The dotted line represents the anus. (C) Comparison of the degree of rescue of dsc-1(qm133) mutants obtained by expression of the dsc-1 cDNA specifically in the IM and AD under the NdE-box promoter in comparison to the expression of the dsc-1 genomic region under its own promoter, which expresses dsc-1 in sensory neurons as well as in the two enteric muscles (shown in Fig. 3). Dark gray bars represent transgenic worms, and light gray bars represent the non-transgenic siblings from the transgenic lines. For the left panel, the bars represent the average number of expulsions per six pBocs observed for three independent transgenic lines, and the error bars represent the standard deviations of the means obtained for each transgenic line. For the right panel, the bars represent that average defecation cycle length observed for three independent transgenic lines, and the error bars represent the standard deviations of the means obtained for each transgenic line. Twelve transgenic and 12 non-transgenic animals were scored per line. Scale bar: 10 µm.

View larger version (58K): [in a new window]   Fig. 6. The Exp defect of dsc-1 is due to the lack of exp-1 expression. (A,B) Expression of an exp-1::gfp transcriptional reporter clone (pAB04) (see Beg and Jorgensen, 2003) in wild-type and dsc-1 mutant backgrounds. (The same extra-chromosomal array was expressed in all three backgrounds; the same results were obtained with >4 independent extra-chromosomal arrays.) (C,D) Expression of an integrated unc-49B::gfp translational reporter clone in wild-type and dsc-1 mutant backgrounds. The same integrated array (oxIs22) (see Bamber et al., 1999) was expressed in all three backgrounds. Scale bar: 10 µm. NC, nerve chord; Sph, sphincter. (E) Expression of an Nde-box::exp-1 fusion in exp-1(sa6) and dsc-1(qm133) backgrounds. For the left panel, the bars represent the average number of expulsions per six pBocs observed for three independent transgenic lines, and the error bars represent the standard deviations of the means obtained for each transgenic line. For the right panel, the bars represent that average defecation cycle length observed for three independent transgenic lines, and the error bars represent the standard deviations of the means obtained for each transgenic line. Twelve transgenic and 10 non-transgenic animals were scored per line.

View larger version (21K): [in a new window]   Fig. 7. The effect of Exp mutations on the defecation cycle length in wild-type and clk-1 mutant backgrounds. The bars represent the mean of the mean cycle lengths of n animals that had each been scored for five consecutive defecation cycles; the error bars represent the standard deviation of the animal means (n>10 animals). (A) In a wild-type (N2) background, both dsc-1(qm133) and exp-1(sa6) significantly shorten the defecation cycle; unc-25(e156) and hlh-8(nr2061) significantly lengthen the cycle. All the Exp mutants show an oscillation in cycle length, with the cycles getting progressively shorter as the worm becomes increasingly constipated (Con), which eventually produces a forceful expulsion, and is then followed by one or two longer cycles. This phenotype is revealed by the variability of the cycle length in individual animals scored for five cycles. This can be expressed as a per animal standard deviation. The average per animal standard deviations are as follows: N2: 0.89, dsc-1: 1.90, exp-1: 3.43, unc-25: 3.92 and hlh-8: 11.68. (B) Of the four Exp mutants surveyed, only the dsc-1(qm133) mutation can significantly suppress the lengthened defecation cycle of clk-1(qm30) mutants. The asterisks indicate the data that are significantly different from N2 (A) or clk-1 (B). All significant differences detected were at a level of P<0.0001.