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First published online 1 June 2005
doi: 10.1242/dev.01883

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Muscle arm development in Caenorhabditis elegans

Department of Medical Genetics and Microbiology, Collaborative Program in Developmental Biology, University of Toronto, Toronto, ON, M5S 1A8, Canada

View larger version (92K): [in a new window]   Fig. 1. The muscle arms of C. elegans. (A) An illustration of the BWMs (red) of the left side of C. elegans. Each BWM quadrant is organized into two rows. (B) A cross-section of A adapted, with permission, from White et al. (White et al., 1986). Each muscle of the two dorsal BWM quadrants and each muscle of the two ventral quadrants (red) extends arms to the nearest nerve cord (dark green) in either the DHR or the VHR, respectively. Only the muscle arms of the ventral left quadrant are indicated by green arrows. The LHR is indicated. (C) A transmission electron micrograph by John White (with permission) of the boxed region in B. BWMs are shaded pink. (D) The dorsal right (top) and dorsal left (bottom) BWMs that express MB::YFP from the trIs30 integrated transgene in a young adult. The Cpppaaa BWM is indicated (yellow arrow), along with four muscle arms (green arrows). (E) The ventral left (top) and ventral right (bottom) BWMs. The red asterisk indicates the position of the vulva. (F,G) Tracings of the dorsal and ventral BWMs, respectively. The identities of the distal muscles that express Mb::YFP at high levels are bright yellow, and those that express Mb::YFP at lower levels are light yellow. The head BWMs are shaded in dark grey, while the neck BWMs are shaded in light grey. The black `X' in (G) represents the vulval muscles, which do not express Mb::YFP from trIs30 transgene. Anterior is to the left in all panels except in B and C. Scale bars: 50 µm. DHR, dorsal hypodermal ridge; LHR, lateral hypodermal ridge; VHR, ventral hypodermal ridge.

View larger version (135K): [in a new window]   Fig. 2. Larval muscle arm outgrowth and migration. (A) Dorsal view of eight BWMs of an early RP127 trIs25 L1. Muscles 9-15 of the dorsal right quadrant (blue arrows) each BWM extends a single arm (red arrow) to the dorsal cord (yellow arrow). (B) Dorsal view of the same eight BWMs as in A, but in a different animal of the early L2 stage. The four muscles of the dorsal right quadrant (blue arrows) extend 3-4 arms to the dorsal cord. In both A and B, anterior is to the left. (C) Four dorsal left BWMs of an adult RP242 Ex[him-4p::MB::YFP; hmr-1b:: DsRed2; unc-129nsp:: DsRed2]; dpy-5(e61) animal, which serves as the control for muscle arm counts in a lin-6 mutant background. BWM identity numbers are shown, along with the number of muscle arms in brackets. (D) The same four BWMs as in C, but in an RP226 Ex[him-4p::MB::YFP; hmr-1b:: DsRed2; unc-129nsp:: DsRed2]; lin-6(e1466) dpy-5(e61) animal. (E-P) RP235 animals mosaic for [myo-3p::Mb::YFP; unc-25p::DsRed; unc-129nsp::CFP]. Muscle arms are indicated with a red arrowhead. (E-H) The same ventral left muscle 13 from four different animals. (I-L) The same dorsal right muscle 13 from four different animals. (M-P) The same ventral right muscle 13 from four different animals. Scale bars: 20 µm.

View larger version (102K): [in a new window]   Fig. 3. Mutant muscle arm phenotypes. In all panels, BWMs of the dorsal right quadrant are shown. Dorsal is up, and anterior is to the right. All animals shown contain the trIs30 transgene. (A) An RP247 trIs30 animal with muscles 9-19 indicated with the number of muscle arms shown in brackets. The dorsal cord (not shown) is indicated with the blue arrow. In all subsequent panels, muscle number 11 is indicated with a green arrow for reference, select muscle arm termini defects are indicated with a red arrow, some flowing lateral muscle ends are indicated with a red arrowhead. (B) Note the arborized muscle arms (red arrow) and flowing lateral muscle ends (red arrowheads). (C) Note the fewer muscle arms, the errant membrane projections (yellow arrowhead), and flowing lateral muscle ends (red arrowhead). A commissural neuron whose fluorescence is bleeding through to the YFP channel is indicated with a blue arrow in E-H. Scale bars: 50 µm.

View larger version (110K): [in a new window]   Fig. 4. Actin distribution in muscle arms. All muscles shown express GFP::ACT-1 from the extra-chromosomal array trEx[pPRRF207(him-4p::gfp::act-1) in the background of the dpy-5(e61) mutation, which enables better visualization of the muscle arms. (A-C) GFP::ACT-1 in a wild-type background. (A) Magnification of the muscle arm shown in B. Note the long actin cables in A and B (green arrows). (C) A lighter exposure of the same muscle depicted in B. Note the incorporation of GFP::ACT-1 in the thin filaments (blue arrow). Orange arrows point to arborized arm termini, yellow arrows point to disorganized sarcomeres, red arrows point to actin bundles within arms, green arrowheads point to discrete muscle lateral ends, and red arrowheads point to enriched actin in flowing lateral ends of muscle. Scale bars: 25 µm.

View larger version (101K): [in a new window]   Fig. 5. unc-54(RNAi) interacts with act-1,2,3(st15) and unc-60(su158) to generate highly arborized muscle arm termini. All animals shown contain the trIs30 transgene and express Mb::YFP in select BWMs, and DsRed2 in commissural motoneurons. Muscles 9 and 11 of the dorsal right quadrant are shown in the first column, the corresponding motoneurons in the middle column, and the merged image, with YFP false coloured green, is in the third column. Red arrows indicate arborized muscle arm termini, while green arrows indicate normal muscle arm termini, irrespective of the defects of the arm stalk. Red arrowheads indicate membrane extension defects, yellow arrowheads indicate errant flowing lateral muscle ends, while white arrowheads indicate discrete lateral muscle ends. In the second column, blue arrows indicate a normal dorsal cord, while an orange arrow indicates a defasciculated dorsal nerve cord. Note that the defasciculated nerve cords correlate with muscle arm termini arborization. Also note that in unc-60(su158); lev-11(RNAi) (G) and in unc-60(su158); unc-54(RNAi) (I) animals, the nerve cords are defasciculated, where none of the single mutants or RNAi-treatments result in defasciculated cords (C-E). Scale bars: 25 µm.

View larger version (16K): [in a new window]   Fig. 6. A comparison of average muscle arm widths. (A) The muscle arm widths are expressed as an average percentage of muscle cell length (see Materials and methods). (B) The average total arm width per BWM for select genotypes. A single asterisk at the end of the bars indicates a significant difference with trIs30 (P<0.001) (grey bar). A double asterisk at the end of the bars indicates a significant difference with the total arm width of trIs30 controls (white bar) (P<0.001). All animals were raised at 20°C. Errors bars represent the standard error of the mean.

View larger version (79K): [in a new window]   Fig. 7. Muscles mosaic for MHC-A::GFP. (A-C) The same muscles of the same RP339 trIs30; myo-3(st386); stEx30[myo-3p::myo-3::gfp; pRF4(rol-6(su1006))] animal (A) Muscles photographed with a YFP filter. Two muscles are indicated (white and yellow arrow) and both express Mb::YFP. (B) The same muscles in A photographed with a CFP filter to exclude the YFP signal, but to capture the GFP signal. One muscle expresses MHC-A::GFP (white arrow), while the other does not (yellow arrow). (C). Merge of A and B. YFP is false coloured green, and the GFP is false coloured red.

View larger version (15K): [in a new window]   Fig. 8. Temperature shift experiments with the temperature-sensitive unc-54(e1157) allele. Shown is the average number of muscle arms extended by muscles 9 through 19 for the indicated genotypes. Counts for genotypes marked with an asterisk represent the average for muscles 9 to 15. For each muscle n=15, except for trIs30 (20°C) and trIs30 (20°C) hatchlings, for which n=30. The allele name is followed by either the temperature at which the animals were constitutively raised or a description of the time point (TP) (in hours) at which the animals were shifted to a different temperature. The coloured bars on the left represent the temperature at which animals were incubated during the indicated developmental times. Green, incubated at 15°C; red, incubated at 25°C; E, embryogenesis; L, larval development; A, adulthood. The error bars represents the standard error of the mean. Averages less than two arms per BWM are all significantly different than averages greater than two (P<0.001).

View larger version (15K): [in a new window]   Fig. 9. A two-phase model of muscle arm development. (A) Three myoblasts (red), which juxtapose a motoneuron (green) during mid-embryogenesis. The nucleus of the centre myoblast is shown. (B) Myoblasts either migrate away or are displaced from the motoneuron, leaving a muscle arm membrane attachment behind. This is the first phase of muscle arm development. (C) The activity of unc-54/MHC-B and lev-11/tropomyosin are required to modify the morphology of the embryonic muscle arm. (D) The myoblasts complete differentiation, including the obliquely arranged sarcomeres (dark red lines represent anisotropic bands). (E) After embryogenesis and early in larval development, post-embryonic neurons develop (additional green lines). These additional axons may induce larval muscle arm extension through the secretion of a chemotropic cue. This is the second phase of muscle arm development and is dependant on actin, unc-54/MHC-B, lev-11/tropomyosin and the F-actin depolymerization activity of unc-60B/ADF/cofilin.