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First published online 22 February 2006
doi: 10.1242/dev.02300

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FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Department of Medical Genetics and Microbiology, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 1A, Canada.

View larger version (83K): [in a new window]   Fig. 1. Disruption of sem-5 results in ectopic membrane protrusions (EMEs). (A) An illustration of the BWMs (yellow and brown) and DA and DB commissural motor neurons (blue) of the left side of C. elegans. Both the outer row (brown) and the inner row (yellow) of BWMs express Mb::YFP from the trIs10 integrated array, whereas only select BWMs of the outer row (brown) express Mb::YFP from trIs30. The boxed area indicates the area of the worm seen in D-I. (B) A cross-section of A, with the dorsal, ventral and lateral hypodermal ridges (HR) indicated. (C) Disruption of sem-5 by RNAi or hypomorphic loss-of-function mutations results in EMEs as visualized with trIs10, as does sem-5(RNAi) in the background of an different muscle reporter strain RP168 [trEx(C26G2.1p::dsred2; B0285.6::mb::yfp)] (Dixon and Roy, 2005), denoted as trEx. Colored asterisks indicate significant differences (P<0.001) versus the relevant controls: black, versus trIs10;Ø(RNAi); red, versus trIs10; blue, versus trEx;Ø(RNAi). n>80 for each genotype. Error bars represent the standard error of the mean (s.e.m.). (D,E) The lateral BWM membrane of trIs10 worms fed upon Ø(RNAi) is not disrupted (blue arrow in D) and commissural motor axon guidance is normal (red arrow in E). The unc-129nsp::cfp promoter also drives expression of CFP in the hypodermal seam cells (orange arrows in E,I). (F,G) trIs10; unc-5(e53) worms extend muscle arms (yellow arrow in F) towards misguided motor axons (red arrow in G). (H,I) trIs10; sem-5(RNAi) worms show numerous EMEs (yellow arrow in H) but commissural axon guidance is normal (red arrow in I). Scale bar: 50 µm.

View larger version (68K): [in a new window]   Fig. 2. sem-5 expression in BWMs is necessary to prevent EMEs. (A) In a trIs10 I; sid-1(qt9) V; Ex(myo-3p::gfp(fRNA); myo-3p::gfp(rRNA); myo-3p::NLS::DsRed) background, BWM cells that are positive (NLS::DsRed2, white arrows) for the extra-chromosomal array directing the production of dsRNA targeting gfp do not produce Mb::YFP. Blue arrows indicate DsRed2-positive nuclei of ventral cord motoneurons. The expression of DsRed2 in these cells is from the trIs10 transgene, not the extra-chromosomal array. (B) A negative control expressing dsRNA targeting unc-115. White arrows indicate BWM cells that express NLS::dsRed2 and therefore are positive for the extra-chromosomal array. YFP expression in the BWMs is retained. (C) Expression of dsRNA targeting sem-5 in BWM results in numerous EMEs (white arrows). (D) Quantification of the EME phenotype in four lines (1-4) of unc-115(dsRNA) worms, four lines (1-4) of sem-5(dsRNA) worms and two lines (1 and 2) of egl-15(dsRNA) worms. Except for the trIs10 control, all strains are in the trIs10; sid-1(qt9)-null background. The full genotype of each strain can be found in Table S3 in the supplementary material. Red asterisks indicate statistical significance at the P<0.001 level versus the sid-1(qt9); trIs10 negative control. n>30 for all genotypes and error bars represent the s.e.m. The black asterisk indicates that one of the control lines displayed significantly more EMEs than the other three control lines. Scale bars: 50 µm.

View larger version (106K): [in a new window]   Fig. 3. An FGF pathway regulates muscle membrane extension. (A-D) Disruption of egl-15, let-756 and let-60 results in numerous EMEs. (E-H) The EMEs of sem-5(RNAi) worms (see Fig. 1H) are suppressed by clr-1(e1745) loss of function and let-60(n1046) gain of function, neither of which displays significant EMEs on their own. (A-H) Red arrows indicate individual EMEs, green arrows indicate intact lateral BWM membranes. (I-M) A dorsal right-hand side view of adult worms, showing BWMs numbers 9, 11, 13 (green arrowhead) and 15 [right to left, according to our numbering scheme (Dixon and Roy, 2005)], extending muscle arms (red arrowheads) to the dorsal nerve cord (blue arrowheads). (J) clr-1(e1745); trIs30 worms extend significantly fewer muscle arms to the nerve cord than do controls. (K) sem-5(RNAi); trIs30 worms extend muscle arms normally and display EMEs (yellow arrowheads). (L,M) The MAD phenotype of clr-1(e1745) worms is partially rescued in the background of sem-5(RNAi) (L) and fully rescued in the background of egl-15(n1783) (M). Scale bar: in A, 50 µm for A-H; in I-M, 50 µm.

View larger version (24K): [in a new window]   Fig. 4. Temperature-shift time-course experiments with temperature-sensitive alleles of FGF pathway components. (A) Synchronized egl-15(n484); trIs10 L1 hatchlings that were raised at 20°C were either kept at 20°C or shifted to 25°C as hatchlings (0 hours), or 6, 12, 18, 24 or 30 hours after hatching. The number of EMEs was counted in these animals upon reaching young adulthood. The number of EMEs in egl-15(n484); trIs10 animals raised at 25°C is also shown. A blue asterisks indicates that the number of EMEs is significantly greater than those in animals raised at 20°C (P<0.001). A black asterisks indicates that the number of EMEs is significantly different compared to animals raised at both temperatures (P<0.001). A red asterisks indicates that the number of EMEs is significantly lower compared to animals raised at 25°C (P<0.001). The number of animals counted is shown at the base of each bar. Error bars represent the s.e.m. (B) Synchronized trIs10; sos-1(cs41) hatchling L1s that were raised at 20°C were either kept at 20°C or shifted to 25°C as hatchlings (0 hours), or 6, 12, 18, 24 or 30 hours after hatching. (C) Synchronized egl-15(n484); trIs10 hatchling L1s that were raised at 25°C were either kept at 25°C or shifted to 20°C as hatchlings (0 hours), or 6, 12, 18 or 24 hours after hatching. (D) Synchronized sos-1(cs41); trIs10 hatchling L1s that were raised at 25°C were either kept at 25°C or shifted to 20°C as hatchlings (0 hours), or 6, 12, 18 or 24 hours after hatching. For B-D, the notation on the chart is the same as that in A.

View larger version (78K): [in a new window]   Fig. 5. let-756 functions non-autonomously to regulate muscle membrane extension. (A-D) LET-756::YFP expression from RP195 Ex[let-756p::LET-756::YFP; let-756p::DsRed]; let-756(s2887) unc-32(e189) III animals. Scale bar: 5 µm. (A) The anterior dorsal BWMs show LET-756::YFP expression in the nucleus (black arrow) and at the sites of BWM-BWM contacts (yellow arrow). (B) Nuclear localization of LET-756::YFP (arrow) in a CAN neuron. (C) RFP channel of the same cell in B. (D) A DIC image of the same cell in B. The arrow indicates the same spot as in B. (E,F) An RP175 Ex[let-756p::YFP; let-756p::LET-756]; let-756(s2887) unc-32(e189) III animal. Expression of let-756 in two BWMs (arrow, F) is sufficient to rescue the lethality associated with a let-756(s2887) null mutation, and can locally rescue the scrawny phenotype (arrow, E). Scale bar: 50 µm. (G) let-756 expression from either the 3.0 kb let-756 promoter/enhancer sequence, a pan-neuronal promoter (F25B3.3p) or a hypodermal promoter (dpy-7p) in a let-756(s2887) null background confers significantly fewer EMEs (red asterisks) than let-756(s2887) null escapers without the rescuing arrays (P<0.001). Black asterisks indicate significantly more EMEs than in trIs10 or trIs10; unc-32(e189) controls (P<0.001). n values are indicated for each genotype within each bar and error bars represent the s.e.m.

View larger version (120K): [in a new window]   Fig. 6. Disruption of laminin and integrin genes results in EMEs that can be suppressed by clr-1 mutants. (A-H) All images correspond to the boxed area in Fig. 1A. Red arrows indicate individual EMEs and green arrows indicate intact lateral BWM membranes. EMEs are seen in trIs10 worms fed lam-1(RNAi) (A), lam-2(RNAi) (B), epi-1(RNAi) (C) or pat-2(RNAi) (D). (E-H) The EME phenotype is strongly suppressed for each genotype in the background of clr-1(e1745) at 20°C. (I-K) A dorsal right-hand side view of adult worms in the background of trIs30, showing BWMs numbers 9, 11, 13 (green arrowhead) and 15, right to left, according to our numbering scheme (Dixon and Roy, 2005), extending muscle arms (red arrowheads) to the dorsal nerve cord (blue arrowheads). A MAD phenotype is seen in lam-1(rh219); trIs30 (I), pat-2(RNAi); trIs30 (J) and unc-52(e998); trIs30 (K) worms. Scale bar: in A, 50 µm for A-H; 50 µm in I-K.