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First published online 8 June 2005
doi: 10.1242/dev.01895

Development 132, 3175-3184 (2005)
Published by The Company of Biologists 2005
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Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans

Naoharu Watanabe1, Yasuko Nagamatsu1, Keiko Gengyo-Ando2, Shohei Mitani2 and Yasumi Ohshima1,*,{dagger}

1 Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan
2 Department of Physiology, Tokyo Women's Medical University School of Medicine, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan



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  		Fig. 1. DIC microphotographs of an L4 stage animal of N2 (A), sma-5(n678) (B) and sma-5; Ex[Y75H1, kin-8::gfp] (C). (D-F) Higher magnification views of a part of the worm shown in A-C, respectively. These pictures were taken using an AxioCam CCD camera mounted on Zeiss Axiophot 2.

 


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  		Fig. 2. Growth curves of N2, sma-5(n678), sma-5 (tm448), i24-1 (sma-5 (n678);Ex[W06B3.2 genomic gene, kin-8::gfp]) and i52-1 (sma-5(tm448); Ex[sma-5 genomic gene, kin-8::gfp]. Error bars indicate standard deviations. Number of worms examined for each time point was 8-39.

 


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  		Fig. 3. Cloning of the sma-5 gene, mRNA structure and RNAi experiments. (A) Physical map of the region around sma-5 gene. The YAC DNA clone, cosmid DNA clones and PCR fragments used for identification of the sma-5 gene are shown. Thick lines represent regions carrying functional sma-5 gene. (B) Location of deletions in W06B3.2 and structure of mRNAs. White and grey rectangles indicate coding and non-coding exons, respectively. (C) RNAi experiments. Average volumes of progeny born after injection of dsRNA of W06B3.2 into N2 (RNAi-1, 2, 3), control progeny born from a parent injected with water or sma-5(n678) mutant worms are shown together with standard deviations. Two-day-old adult animals were used for measurement.

 


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  		Fig. 4. Alignment of amino acid sequences of W06B3.2a/c (upper line) and human MAPK7/ERK5 (lower line). A vertical line indicates identical amino acid, a colon indicates strong similarity and a dot indicates weak similarity between amino acids. A broken line indicates absence of corresponding amino acids.

 


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  		Fig. 5. Expression patterns of sma-5::gfp reporter genes. (A-C) A dorsal fluorescent image (A), a DIC image (B) and the merge of A and B (C) of an entire L4 animal transgenic for pPDW06-1a carrying a promoter for W06B3.2c. (D) A merge of dorsal fluorescent and DIC images of a young adult expressing a SMA-5/GFP fusion protein in intestine from pPDW06-9. (E) A confocal microscopic, dorsal/ventral image of a part of the body showing H-shaped excretory cell and the anterior end of intestine in an L4 worm expressing pPDW06-1a. (F) A similar confocal microscopic image, but color-coded depending on the depth, showing intestine (green), excretory cell (blue or yellow), and nuclei and faint cytoplasm of hypodermis (blue). (G,H) Lateral confocal microscopic images of an L2 worm transgenic for pPDW06-c carrying a promoter for W06B3.2a that expresses GFP in hypodermis (G) and pharynx (H).

 


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  		Fig. 6. Three-dimensional reconstructed images of hypodermis (A), intestine (B) and muscles (C) in the sma-5(n678) background. Scale bar: 200 µm.

 


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  		Fig. 7. Effect of expression of extrachromosomal sma-5 genomic gene (second group of columns) or sma-5 cDNA under the indicated promoter (lower groups of columns) in the sma-5 (n678) mutant on body and organ volume in 2-day-old adults. Body volumes relative to those of the wild-type body volume (white bar), and intestinal volumes (gray bar) or hypodermal volumes (black bar) relative to those of the dss-1::gfp or col-19p::gfp transgenic lines in the wild-type background are shown by bars. The top group of columns show the results of the sma-5 (n678) mutant. T-shaped bars represent s.e.m., which was calculated by the formula (fDx2/y2+x2fDy2/y4)1/2/n1/2 where x and fDx are mean and s.d. of the volume of the indicated strain, and y and fDy are those of the wild-type volume (Bevington and Robinson, 2003Go). The smaller of the numbers of worms examined for the indicated and the wild-type worms was taken as n. An asterisk indicates significant difference in t-tests (P≤0.05) from the value for the sma-5 mutant. One-hundred percent (wild-type volume) corresponds to 4.39±0.41 nl, 0.88±0.068 nl and 1.01±0.20 nl for body, intestinal and hypodermal volumes, respectively. Hypodermal volume of a worm expressing sma-5 cDNA under dss-1 promoter was not obtained because a corresponding transgenic line was not obtained.

 




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