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First published online March 6, 2009
doi: 10.1242/10.1242/dev.026302

Development 136, 1159-1168 (2009)
Published by The Company of Biologists 2009
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The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts

Claude Shelton1,*, Kiranmai S. Kocherlakota1,2,*, Shufei Zhuang1 and Susan M. Abmayr1,3,{dagger}

1 Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
2 The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, PA 16802, USA.
3 Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 3038, Kansas City, KS 66160, USA.


Figure 1
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  		Fig. 1. Fusion of the Eve-expressing DA1 founder cell in various mutant embryos. (A-O) Abdominal segments 3-5 are shown in embryos stained with anti-Eve to mark the nuclei of the DA1 muscle and two pericardial cells per hemisegment. (A-C) Wild-type, (D-F) mbcD11.2/mbcD11.2, (G-I) snsXB3/snsXB3, (J-L) Df(1)w67k30/Y and (M-O) lmd1/lmd1. The founder cell for DA1 remains mononucleate in embryos lacking mbc, kirre and rst [Df(1)w67k30], or lmd at developmental stages when significant fusion is observed in wild-type embryos (arrows). In embryos mutant for sns, by contrast, the Eve-expressing DA1 founder cell undergoes limited fusion to generate bi- or tri-nucleate syncitia (arrowheads). (P) The average number of DA1 nuclei per hemisegment was quantitated in late stage 15 embryos of each mutant genotype. (Q) The fusion profile of precursor formation in wild-type and snsXB3 embryos shown as the average number of DA1 nuclei per hemisegment. (R) The percentage of embryos observed with any hemisegments showing DA1 precursor formation. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Hbs, but not Rst, substitutes for Sns in directing precursor formation. (A-H) Three representative abdominal segments of late stage 15 embryos. (A-F) Eve marks DA1 and pericardial cell nuclei. A similar amount of fusion is observed in snss660 mutants (A) and rstirreC1; snss660 double mutants (B). (C,D) Pericardin (Prc) staining identifies the Eve-expressing pericardial cells. Multinucleate DA1 precursors are present in snsXB3 mutants (C, arrowheads) but not in snsXB3, hbs2593 recombinants (D, arrows). (E,F) Eve and Kr expression identify nuclei in DA1, whereas Kr expression identifies nuclei in DO1. Fusion to form the DA1 and DO1 precursor is observed in snsXB3 mutant embryos (E, arrowheads) but not snsXB3, hbs459 recombinants (F, arrows). (G,H) Nau expression marks nuclei of the VA1 muscle. Again, fusion is observed in snsXB3 mutants (G, arrowheads) but not in snsXB3, hbs2593 recombinants (H, arrow). (I) The average number of nuclei per hemisegment is shown for DA1 (Eve), DO1 (Kr) and VA1 (Nau) in the indicated genotypes. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3. Hbs and Sns have comparable aggregation behavior with Kirre-expressing cells and undergo similar biochemical modifications. (A) Kinetics of trans-heterotypic aggregation of S2 cells expressing Kirre co-cultured with S2 cells expressing Sns (diamonds, bold line) and S2 cells expressing Hbs (squares, dashed line). (B,C) Sns-HA or Hbs-HA expressed pan-mesodermally with mef2Gal4 and immunoprecipitated with anti-HA resin. (B) Immunoblots of untreated (Mock) or PNGaseF-treated Sns-HA and Hbs-HA reveals modification by N-linked glycans. (C) Immunoblots of Sns-HA, Hbs-HA or control (mef2Gal4/+) samples probed with anti-HA or anti-phosphotyrosine antibodies. (D) Alignment of transmembrane and cytodomain sequences from Sns and Hbs. The entire Hbs cytodomain is shown. Only the membrane proximal half of the Sns cytodomain is shown. The caret positions correspond to (1) AA1113, (2) AA1164, (3) AA1232 and (4) AA1278 of SNS, and served as deletion endpoints in a structure/function analysis of Sns. Conserved sequences from Hbs, important PxxP motifs and tyrosine residues crucial for Sns function are highlighted.

 

Figure 4
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  		Fig. 4. Comparison of the ability of various Sns-Hbs chimeric proteins to rescue the sns mutant myoblast fusion defect. (A-U) Stage 16 embryos stained with anti-myosin heavy chain antibody. In all orientations, anterior is to the left and dorsal is up. (A,D,G,J,M,P) Dorsolateral view; (B,E,H,K,N,Q) lateral view; (C,F,I,L,O,R) ventrolateral view. Expression of the indicated transgenes (for sequences, see Fig. S2 in the supplementary material) was directed in snsZf1.4/snszf1.4 mutant embryos with snsGal4 at 25°C. (S-U) Ventrolateral views of snsXB3 mutant (S), and rescued snszf1.4 mutant embryos using snsGal4 to drive expression of UAS-hbs (T) or UAS-sns (U). Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. Sns interacts with Hbs genetically and biochemically. (A) The average number of unfused myoblasts was determined by staining for β-galactosidase and quantitated in four contiguous abdominal segments of six embryos for control (snslacZ/+; mef2Gal4/+) and test (snslacZ/+; mef2Gal4/UAS-sns). Representative late stage 15 embryos fluorescently stained for myosin heavy chain (red) and β-galactosidase (green) are shown to the right. (B) Late stage 15 embryos of the indicated genotypes immunofluorescently stained for Sns (red) to highlight unfused myoblasts, and myosin heavy chain (green) to mark muscles. The bar chart shows the average number of unfused myoblasts for the indicated genotypes, quantitated in four contiguous abdominal segments per embryo. (C) In the left-hand panel, expression of pUAST-sns-Flag and/or pUAST-hbs was under the control of actinGal4 (pWAGal4) in transiently transfected S2 cells. In the right-hand panel, expression of UAS-sns-V5 and/or UAS-hbs was directed pan-mesodermally in embryos using mef2Gal4. Sns was immunoprecipitated from lysates using the indicated epitope tag, and the immunoblots probed with anti-flag, anti-V5 or anti-Hbs as indicated. (D) In the left-hand panel, S2 cells were transiently transfected with pUAST-sns-V5 and/or pUAST-sns-HA. Expression was directed by actinGal4 (pWAGal4). In the right-hand panel, expression of UAS-sns-V5 and/or UAS-sns-HA was directed pan-mesodermally in embryos using mef2Gal4. Sns was immunoprecipitated from lysates using the indicated epitope tag, and the resulting immunoblots probed with anti-HA or anti-V5 as indicated. Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. The Sns extracellular domain alone is not able to direct precursor formation. (A-E) Embryos were immunofluorescently stained with anti-Eve and anti-Kr antibodies. (A-D) Expression of the UAS-sns20-5HA transgene (A,B) or the UAS-hbs{Delta}ICD-HA transgene (C,D) were driven pan-mesodermally with the mef2Gal4 driver in an snsXB3, hbs2593 mutant background. Note the presence of single Eve-positive or Kr-positive nuclei per hemisegment (arrows). The asterisks in B and D indicate the presence of anti-Kr staining not associated with the mesoderm. (E) Mid-stage 15 snsXB3 mutant embryo; note the regular occurrence of multiple Eve-positive or Kr-positive nuclei per hemisegment (arrowheads). (F) The average number of nuclei per hemisegment for abdominal segments 2-7 was calculated for the DA1 muscle (Eve-positive, Kr-positive) and the DO1 (Kr-positive) muscle for embryos where SNS20-5HA or Hbs{Delta}ICD-HA expression was directed in snsXB3, hbs2593 mutants. Scale bar: 20 µm.

 

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© The Company of Biologists Ltd 2009