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First published online 19 November 2003
doi: 10.1242/dev.00863

Development 130, 6453-6464 (2003)
Published by The Company of Biologists 2003
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The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling

Jun Liang1, Robyn Lints2,3, Marisa L. Foehr4, Rafal Tokarz1, Ling Yu1, Scott W. Emmons2, Jun Liu4 and Cathy Savage-Dunn1,*

1 Department of Biology, Queens College, and PhD Program in Biochemistry, The Graduate School and University Center, The City University of New York, Flushing, NY 11367, USA
2 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
3 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA



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  		Fig. 3. Alternative splicing of sma-9 transcripts. sma-9 cDNA clones reveal a complex alternative splicing at both 5' and 3' ends. At least three N terminus and seven C terminus forms were found. Predicted exons 4, 15 and 20 are labeled. Approximate locations of the termination codons in qc3 and wk55 are shown above the predicted exon structure. Gray boxes represent unique C-terminal sequences; an alternative exon 15 with two extra bases is indicated in yellow. cDNA clones representing each variant are: class A - pCS234 from RT-PCR; class B - yk1285a11; class C - pCS272 from RT-PCR; class Ia - yk128a8, yk1136g02 and yk1109f01; class Ib - yk43h3; class Ic - G-dvp11458.x (Walhout et al., 2000Go); class IIa - yk6d10, yk864c1 and yk1134e06; class IIb - yk1237d01, which is SL2- spliced, yk856b10, yk1057a6, yk1216e10, yk1103h10 and yk127d10, which is also missing the intron between exon 21 and 22 (not shown); class IIc - yk1264e07; and class III - yk328c9. Only the trans-spliced cDNA clones yk1285a11 and yk1237d01 appear to be full length.

 


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  		Fig. 4. sma-9 is widely expressed. sma-9 upstream sequences (K) were fused with a GFP reporter gene. (A-C) GFP expression in wild-type transgenic animals. The transgenic animals display a strong fluorescence in the VNC, pharynx and intestine, from stages L1 (not shown) to adult (A,B). L2 stage (C) animals show expression in the seam cells that disappears after L3 (Nomarski image in D). Arrows in C and D indicate seam cell nuclei. (E,G,I) Immunostaining by anti-SMA-9 antibodies against the unique 70 aa C terminus present in class II isoforms. (F,H,J) DAPI staining of the same animals. (E,F) N2; (G,H) sma-9(wk55); (I,J) enlarged view of the anterior region of a wild-type late L1 stage animal. In all animals, anterior is to the left, arrowheads show pharyngeal nuclei, black arrows show intestinal nuclei and white arrows show hypodermal nuclei. (K) Different sma-9 upstream regions used to construct GFP reporters.

 


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  		Fig. 1. sma-9 mutants display Sma and Mab phenotypes similar to those of DBL-1 pathway mutants. (A,B) Male tail phenotype. Wild-type male tail (A) has nine bilateral pairs of sensory rays; sma-9(wk55) mutant (B) displays a ray 8-9 fusion. (C-F) Body size phenotype. sma-9(lf) mutant (D) is Sma compared with N2 (C). Cosmid T05A10 rescued sma-9(wk55) body size (E). (F) dsRNAi of sma-9 3' exons in him-5. (G-J) Genetic interactions. lon-1;sma-9 double mutant (G) is neither Lon nor Sma. dbl-1 overexpression in wild-type background is Lon (J). However, the animal displays a Sma phenotype in sma-9(wk55) background (H). (I) lon-1 single mutant. All animals in C-J are young adults photographed at the same magnification, with anterior oriented to the left.

 


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  		Fig. 2. sma-9 encodes a zinc finger transcription factor. (A) The longest conceptual SMA-9 sequence. Mutation sites in wk55 and qc3 alleles are boxed. Seven C2H2 zinc finger motifs, in three clusters, are shaded. NLSs are underlined. S/TPXK/R or SPKK motifs are double underlined. ARDs are bold and underlined. The asterisk indicates the site at which the alternative C terminus begins. The alternative C terminus unique in C. elegans is italic and underlined. The predicted N-terminal initiation sequence (predicted exon 1) is italic and bold. (B) Comparison of the SMA-9, Shn, MBP1 and {alpha}A-CBP1 zinc finger regions. The Cys and His residues of zinc finger motifs are boxed. The identical residues are shaded. (C) Comparison of the SMA-9, Shn and MBP1 zinc finger domains. The SMA-9 first pair of zinc fingers is highly conserved between invertebrates and vertebrates. The SMA-9 triplet appears to have been either eliminated during vertebrate evolution or acquired in worm-fly lineage. The SMA-9 second pair is unique in C. elegans. The Shn first pair is conserved in vertebrate homologs but not in C. elegans.

 


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  		Fig. 5. sma-9 functions in early stages to regulate body size. (A) Growth curve of sma-9 (cross), sma-4 (triangle), dbl-1(square) and N2 (diamond). In L1 stage larvae (12 hours), all animals are the same length. In L1 through L3 stages (48 hours), sma-9, sma-4 and dbl-1 show reduced a growth rate compared with N2. After L3, sma-9 mutants grow rapidly, whereas dbl-1 and sma-4 mutants continue to display a reduced growth rate. Data for N2 and dbl-1 is from Savage-Dunn et al. (Savage-Dunn et al., 2000Go). (B) At L3 stage, sma-9 worm length, pharynx length and seam cell length is indistinguishable from that of dbl-1 and sma-4. Data for N2 and dbl-1 is from Wang et al. (Wang et al., 2002Go). (C) Model of sma-9 function in the DBL-1 pathway. In body size development, sma-9 functions in early larval stages and may be replaced by other transcriptional cofactors in late larval stages. In male tail development, sma-9 prevents fusions in rays 8-9 specifically, but regulates cat-2 activity, the rate-limiting step in dopamine expression, in all rays. Other cofactors may be involved in ray 4-5 and 6-7 fusions. For spicule development, both sma-9 and other cofactors are probably required.

 

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© The Company of Biologists Ltd 2003