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Figures
- Fig. 1.
An overview of selected mutants isolated from our screen. (A) Schematic of the right-hand side of an adult C. elegans hermaphrodite. The body wall muscles are depicted as rhomboids and those that express him-4 at high levels are indicated in red. The box indicates the region shown in B. Anterior is to the right in all panels. In A and B, dorsal is up. (B) Select details of C. elegans neuromuscular anatomy. (C-F′) Fluorescent micrographs of the four distal muscles of dorsal left and right quadrants (left column) and the four distal ventral left and right muscles (right column) that express the him-4p::membrane-anchored YFP muscle arm reporter from the trIs30 integrated transgenic array. The genotype is indicated. Motoneuron cell bodies and/or axons are false-colored blue. The nerve cord is indicated with a yellow arrowhead. In micrographs of dorsal muscles, left is up. In micrographs of the ventral muscles, left is down. Dorsal right muscle 15 (DR15) is indicated with a white arrow in the left-hand column, and ventral left muscle 11 (VL11) is indicated with a white arrow in the right-hand column (Dixon and Roy, 2005). The muscle arms of DR15 and VL11 were counted for all analyses herein and are indicated with red arrowheads. Scale bars: 50 μm. (G) Summary of the number of DR15 (dark gray) and VL11 (light gray) muscle arms per muscle for the indicated genotypes. The unc-93 alleles tr120 and e1500 are semi-dominant (sd) with respect to uncoordinated movement, but not muscle arm extension (see Table S1 in the supplementary material), and are likely to be gain-of-function alleles (Levin and Horvitz, 1992). unc-93(e1500n234) is a loss-of-function allele. For muscle arm numbers for heterozygous controls, see Table S1 in the supplementary material. ND (not determined) indicates that only VL11 muscle arms were counted. Error bars indicate s.e.m.
- Fig. 2.
unc-40 functions cell-autonomously to regulate muscle arm extension. (A-E′) Dorsal (A-E) and ventral (A′-E′) views of muscle arms of C. elegans animals of the indicated genotypes. The annotation is the same as in Fig. 1. (D,D′) UNC-40::YFP was expressed in the nervous system using the unc-119 promoter (neuro-p in F). The white arrow in D points to the area where the DR15 muscle is normally positioned, but is not fluorescing in this animal. (E,E′). UNC-40::YFP was expressed in the distal muscles using the him-4 promoter (muscle-p in F). (F) The average number of muscle arms extended by the indicated genotypes. The control transgenes are the DNAs common to all injection mixtures without the experimental unc-40-related transgene (see Materials and methods). Aside from trIs34, the transgenic arrays are maintained extra-chromosomally, independent lines of which are indicated with a number. Additional annotation is the same as for Fig. 1G. (G) The number of lateral muscle membrane extensions for the indicated genotypes, illustrating that unc-40 mutations suppress misdirected muscle arm extension to misguided lateral axons resulting from an unc-5 or unc-6 mutant background. Asterisks indicate significantly more defects than the controls (P<0.001) as indicated by the lines above the bars. The error bars in F and G represent s.e.m. (H) UNC-40::GFP expression directed by the unc-40 promoter. Localization to the muscle plasma membrane is indicated. (I) Muscle-specific expression of functional UNC-40::YFP, driven by the him-4 promoter. Enrichment at the muscle arm terminus is indicated. Scale bars: 50 μm in A-E′; 10 μm in H,I.
- Fig. 3.
UNC-73 is necessary for muscle arm extension and co-localizes with UNC-40 at muscle arm termini. (A,B) The ventral muscle arm extension defects of C. elegans unc-73 mutants. (C) Muscle-specific expression of UNC-73B::YFP rescues the muscle arm extension defects of unc-73 mutants. The annotation for A-C is as described in Fig. 1C-F′, except that only the ventral quadrants are shown. VL11 is indicated with either a white arrow or an asterisk. (D) Summary of muscle arm extension in the background of unc-73-related mutants. The annotation for D is the same as that for Fig. 2F, except that M-UNC-73 and N-UNC-73 represent muscle-expressed and neuronally expressed UNC-73 using the him-4 and unc-119 promoters, respectively. ND, not determined. (E-E″) UNC-73B::CFP specifically expressed in muscles in the background of the trIs34 integrated transgene that expresses UNC-40::YFP specifically in muscles. The localization of fusion proteins to the muscle arm termini is indicated with a yellow arrowhead. Scale bars: 50 μm in A-C; 10 μm in E-E″.
- Fig. 4.
Dense body components are necessary for muscle arm extension. (A-D) The ventral muscle arms of C. elegans mutants with disrupted dense body components. The annotation is the same as that for Fig. 1C-F′. (E) A summary of the number of muscle arms for the indicated genotypes. The annotation is the same as that for Fig. 1G. For heterozygous control counts, see Table S1 in the supplementary material. ND, not determined. (F,G) Functional UNC-95::GFP (Broday et al., 2004) (F) and UNC-97::GFP (Hobert et al., 1999) (G) fusion proteins can be seen in the muscle arms (red arrows). Scale bars: 25μ m in A-D; 50 μm in F,G.
- Fig. 5.
Members of the WAVE complex are required for muscle arm extension. (A-E) The genotype or RNAi treatment is indicated; the annotation is the same as Fig. 1C-F′. For E, rrf-3(pk1426) C. elegans were treated with wve-1(RNAi). For rrf-3(pk1426) control animals, see Fig. S2 in the supplementary material. (F) Summary of the muscle arm numbers in WAVE-related backgrounds. The annotation is the same as for Fig. 1G. ND, not determined. (G-G″) GEX-2::CFP specifically expressed in muscles in the background of the trIs34 integrated transgene that expresses UNC-40::YFP specifically in muscles. The localization of fusion proteins to the muscle arm termini is indicated with a yellow arrowhead. The muscle cell body is indicated with a white arrow. Scale bars: 50 μm in A-E; 5 μm in G-G″.
- Fig. 6.
Members of the WAVE complex direct HSN axon outgrowth in an UNC-40 pathway. (A-F) Examples of C. elegans HSNl axons, visualized with the zdIs13[tph-1p::GFP] array in the indicated genetic or RNAi background. Ventral is down and anterior is to the left. The red arrowhead indicates the direction of HSNl axon outgrowth. Scale bars: 10μ m. (G) The percentage of HSNl (dark gray bars) and HSNr (light gray bars) axons that fail to extend ventrally within two cell diameters of the cell body. Asterisks indicate significantly more defects than the controls indicated by the lines above the bars (red, P<0.05; black, P<0.1). Error bars indicate s.e.m. For further details, see Fig. S3 in the supplementary material.
- Fig. 7.
UNC-40-induced myopodia are suppressed by mutations in cytoskeletal regulators and in unc-95. (A-J) Transgenic arrays created with 25-fold more UNC-40::YFP transgene than that used for rescue experiments induce fine membrane protrusions called myopodia (red arrowheads). Examples of body wall muscles extending myopodia induced by overexpressed UNC-40::YFP. The UNC-40::YFP is used to visualize the body wall muscle plasma membrane (white). The alleles or RNAi treatments used are indicated in K. Scale bars: 10 μm. (K) Summary of the number of myopodia observed per body wall muscle of the indicated genotype. All alleles used in this analysis are null except for those of unc-73. Myopodia counts for gex-2 and gex-3 mutant C. elegans were performed in homozygous offspring of heterozygous parents because ok1603 and zu196 are maternal-effect lethal. Error bars indicate s.e.m.
Tables
- Table 1.
The 23 Madd mutants recovered in our screen for genes required for muscle arm extension
Complementation group Allele LG* Map position* Failed to complement† Homolog Mutation‡ 1 gex-2 tr116 IV <–16 ok1603 Sra1/p140/Cyfip1 R420Stop (c5009t) 2 madd-2 § tr64 V 3 tr96 4 tr101 5 tr103 6 tr113 7 tr129 8 unc-33 tr114 IV –5 < +1 e204 Crmp2/Dpysl2 R502H (g6504a) 9 unc-40 tr63 I –0.7 < 1.0 n324 Dcc/neogenin Intron 6 splice donor (g4869a) 10 tr115 > –1.7 n324 W1107Stop (g8867a) 11 tr121 –4.5 < 1.0 n324 Exon 8 splice donor/D426N (g5765a) 12 unc-51 tr126 V +19.4 > +25.2 e369 Ulk2 I59T (t1240c) 13 unc-54 tr112 I LGI e190 MHC-B/Myh7 – 14 tr124 LGI e190 – 15 unc-60B tr125 V LGV su158 Cofilin/ADF G44E (g2241a) 16 tr50 –19.1 < –17.6 su158 – 17 unc-73 tr117 I –4.5 < 1.0 e936 E1335K (g9486a) 18 unc-93 tr120sd III –7.4 < –2 e1500 UNC-93 G388R (g2476a) 19 unc-95 tr61 I LGI su33 UNC-95 Intron 1 splice acceptor (g1693a) 20 – tr98d I –6.2 < –4.5 – – – 21 – tr105 I +4 < +23.5 – – – 22 – tr119 I +1.9 < +4 – – – 23 – tr123 I –4.8 < –1.6 – – – -
↵* The linkage group (LG) (i.e. chromosome) and map position are shown. Only the linkage group is shown if mapping did not proceed beyond bulk segregant analysis
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↵† The allele used in the complementation test is shown
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↵‡ The mutant residue is shown followed by the mutant nucleotide in brackets, which is relative to the adenine of the predicted start codon in the genomic sequence (WormBase release 187). For tr117 and tr125, the mutant nucleotides are with respect to F55C7.7b and C38C3.5c.1, respectively. tr63 carries a mutation in the invariant first base of the splice donor of intron 6, which is likely to result in the translation of 11 additional codons within intron 6 before a stop codon is reached, truncating the protein between the second and third IG domains. Similarly, tr121 is mutant in the last nucleotide of exon 8 and may also disrupt splicing (Farrer et al., 2002). For those aberrantly spliced tr121 transcripts, a stop codon is present 99 codons into intron 8, resulting in a predicted truncated protein between the fourth IG domain and the first fibronectin type III domain. For those transcripts without altered splicing, a D426N mutation is created between the fourth IG domain and the first fibronectin type III domain
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↵§ Details of the six madd-2 alleles isolated in our screen will be presented elsewhere
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