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. 2000 Jun 15;14(12):1498-511.

Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion

B A Bour  1 , M Chakravarti, J M West, S M Abmayr
Affiliations

Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion

B A Bour et al. Genes Dev. .

Abstract

The Drosophila sticks-and-stones (sns) locus was identified on the basis of its mutant phenotype, the complete absence of body wall muscles and corresponding presence of unfused myoblasts. The genetic location of the mutation responsible for this apparent defect in myoblast fusion was determined by recombination and deficiency mapping, and the corresponding wild-type gene was isolated in a molecular walk. Identification of the SNS coding sequence revealed a putative member of the immunoglobulin superfamily (IgSF) of cell adhesion molecules. As anticipated from this homology, SNS is enriched at the membrane and clusters at discrete sites, coincident with the occurrence of myoblast fusion. Both the sns transcript and the encoded protein are expressed in precursors of the somatic and visceral musculature of the embryo. Within the presumptive somatic musculature, SNS expression is restricted to the putative fusion-competent cells and is not detected in unfused founder cells. Thus, SNS represents the first known marker for this subgroup of myoblasts, and provides an opportunity to identify pathways specifying this cell type as well as transcriptional regulators of fusion-specific genes. To these ends, we demonstrate that the presence of SNS-expressing cells is absolutely dependent on Notch, and that expression of SNS does not require the myogenic regulatory protein MEF2.

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Figures

Figure 1
Figure 1
Genetic and molecular map of the chromosomal region that includes sns. (A) Genetic map of cytological region 44F. (Horizontal bars) Deficiencies, (vertical dashed lines) lethal complementation groups, (gray boxes) P-element insertions. The genetic positions of lethal complementation groups in the region, including the sns locus, are indicated. As shown, sns maps between the proximal breakpoints of Df(2R)sns-04913HOa and Df(2R)RyaR-16109HO. (B) Enlargement of the genetic map shown in A onto which the positions of cloned sequences have been mapped. Black horizontal bars represent deficiencies as in A. The gray bars indicate the positions of two P1 clones (provided by the Berkeley Drosophila Genome Project) relative to the deficiencies. The STS 728 is indicated by the dark black box within each clone. As indicated, DS04320 begins at STS 728 and extends proximally. The proximal sequence of this clone is deleted in IN(2LR)P14[L]TE45F[R], whereas the distal end is deleted in both Df(2R)sns-04913HOa and Df(2R)sns-04913HOb. DS01342, which includes a significant portion of the sns genomic region, extends distally, and is deleted in Df(2R)RyaR-16109HO as shown. (C) Molecular walk within the P1 clone DS01342. The gray bar at the top represents the P1 clone used for a chromosomal walk to clone sns. The position of STS 728 is indicated. In the restriction map of the region, (E) EcoRI, (B) BamHI, (P) PstI, and (H) HindIII. Subclones are as indicated below the restriction map. A 2.9-kb EcoRI fragment (indicated RI4) is highlighted. This fragment includes sns coding sequence, and revealed the splicing defect in snsXB3 (refer to text).
Figure 2
Figure 2
MHC, NAU, and MEF2 positive cells are present in sns mutant embryos but do not fuse to form muscle fibers. All embryos are oriented ventrolaterally with anterior to the left. Wild-type embryos are shown in A, C, and E. (B,D,F) Embryos that are genetically snsA3.24/Df(2R)BB1. Embryos homozygous for snsA3.24 exhibit the same mutant phenotype (data not shown) and Df(2R)BB1 completely removes the sns genomic region (Results, Materials and Methods). Therefore, by genetic criteria, snsA3.24 appears to represent the null phenotype for sns. (A,B) Stage 15 embryos immunostained with an MHC antibody to visualize the musculature. By comparison with wild-type in A, embryos mutant for sns exhibit unfused MHC expressing myoblasts in the place of mature muscle fibers. (C,D) Stage 13 embryos immunostained with antisera against NAU to visualize a subset of founder cells. In wild-type embryos (C), NAU-expressing cells are distributed in an array of muscle founders and precursors. (D) The pattern of NAU-expressing cells is not affected in sns mutant embryos, suggesting that founder cell specification does not require sns. (E,F) Stage 15 embryos immunostained with antisera against MEF2 to visualize the entire myoblast population. MEF2 expression can be seen in developing muscle fibers in wild-type embryos (E) and in the corresponding population of unfused myoblasts in sns mutant embryos (F).
Figure 3
Figure 3
The putative sns sequence is altered in mutant alleles of sns. (A) A BamHI site located within an intron of the sns gene is altered in snsXB3 by a G to A transition in the first G of the BamHI recognition sequence. As confirmed by RT–PCR of mRNA from snsXB3 mutant embryos, this change introduces a splice acceptor site that results in a spliced product of 248 bp rather than the wild-type product of 195 bp. Direct sequencing of the 248 bp product confirmed the altered sequence indicated in A. (B–D) Non-isotopic RNase cleavage revealed mutations in the putative sns gene in other EMS induced alleles of sns (data not shown). Direct sequencing of these alleles revealed the indicated alterations. (B) snsZF1.4 contains a C to T transition that introduces a stop codon at amino acid position 356. (C) snsXH2 contains a C to T transition that introduces a stop codon at amino acid 465. (D) rost202 a putative mutant allele of the previously described rost gene (Paululat et al. 1995) contains a C to T transition that introduces a stop codon at amino acid position 367 of SNS.
Figure 4
Figure 4
Amino acid sequence of the putative SNS protein and structurally-related sequences in multiple organisms. The putative SNS open reading frame is indicated in the top line (GenBank accession no. AF254867). Structural homologs include the hypothetical ORF of the Drosophila hibris gene (GenBank accession no. AF210316; Artero and Baylies 1999), human Nephrin (GenBank accession no. AF035385; Kestila et al. 1998; Lenkkeri et al. 1999), and a C. elegans ORF (GenBank accession no. CAB63432). Alignment was done using ClustalW and is presented using Boxshade. (Black boxes) Amino acid identity; (gray boxes) amino acid similarity; (horizontal black lines) immunoglobulin homology domains; (dashed horizontal line) the fibronectin type III domain; (thick black horizontal bar) the transmembrane domain. Asterisks highlight the residues altered in various mutant alleles of sns.
Figure 5
Figure 5
Embryonic expression of the sns transcript and encoded protein are restricted to myogenic cells. (A–C,I) Localization of the sns transcript in wild-type embryos by in situ hybridization using a digoxigenin labeled probe. (D–H) Localization of the sns-encoded protein, detected by antisera generated against the cytoplasmic domain of SNS. All embryos are oriented laterally with anterior to the left. (A,D) Early Stage 12, (B,E) early Stage 13, (C,F) late Stage 14. Expression of SNS essentially mimics that of the transcript. (A,D) Expression at early stages is observed in both the developing somatic and visceral musculature (arrows and arrowheads, respectively). Expression persists in the somatic mesoderm throughout the time that myoblast fusion occurs. (G–I) High magnification of embryos at comparable stages to those shown in D–F. (G) SNS protein outlines the membrane of the entire cell initially (arrowhead). (H) As development proceeds, SNS becomes increasingly localized to distinct points in the membrane (arrowheads). (I) By Stage 17, expression of sns transcript has declined significantly in the muscles, but is observed at a low level in muscle attachment sites (arrow).
Figure 6
Figure 6
SNS is not detected in unfused rP298–lacZ-expressing founder cells. Embryos were analyzed for coincident expression of SNS and the enhancer trap line rP298–lacZ by immunofluorescence and confocal microscopy. SNS expression is indicated in red (A,B,D,E,G,H), and rP298-driven expression of β-galactosidase is indicated in green (B,C,E,F,H,I). One micron confocal sections are shown. (A–C) A dorsolateral view of a Stage 13 embryo where anterior is to the left and dorsal to the top. (D–F) A lateral view of a Stage 12 embryo where anterior is to the top and dorsal to the right. (G–I) A dorsal view of a late Stage 12 embryo where anterior is to the left and the midline is to the top. Unfused β-galactosidase-expressing founder cells are indicated by arrowheads in B, E, and H. As evidenced by comparisons with the locations of the SNS-expressing cells, none of these founder cells co-express SNS.
Figure 7
Figure 7
SNS is not detected in the unfused EVE-expressing founder cell. Embryos were analyzed for coincident expression of SNS and EVE by immunofluorescence and confocal microscopy. SNS expression is indicated in red (B,C,E,F,H,I,K,L), and EVE expression is indicated in green (A,B,D,E,G,H,J,K). (A–C) Stage 12, (D–I) Stage 13, and (J–L) Stage 14. A projection of 3 × 1 micron confocal sections are shown in all panels. All views are lateral, with anterior to the left and dorsal to the top. Unfused EVE-expressing founder cells are indicated by arrowheads in B and H.
Figure 8
Figure 8
SNS expression is not detected in morphologically distinct founder cells in embryos blocked for myoblast fusion. snsXS5 and mbcD11.2 mutant embryos were analyzed for the presence of morphologically distinct founder cells (Rushton et al. 1995) that express either MHC or SNS. (A,B) Lateral view of a Stage 16 embryo homozygous for the non-null sns allele snsXS5, immunostained for SNS (A) or MHC (B). (C,D) Lateral view of a Stage 16 embryo homozygous for mbcD11.2, immunostained for SNS (C) or MHC (D). Arrows in B and D indicate the positions of elongated founder cells that are visualized by their expression of MHC. By contrast, no such cells are revealed by expression of SNS.
Figure 9
Figure 9
SNS expression in the putative fusion competent cells is dependent on Notch. Wild-type and NXK11 mutant embryos were analyzed for expression of SNS and the founder cell marker VG (Williams et al. 1991). SNS expression is indicated in red (A,B,D,E), and VG expression is indicated in green (B,C,E,F). Both embryos are at Stage 13, and all views are ventral with anterior to the left. (A–C) Confocal projections of 22 × 1 micron sections of a wild-type embryo. As expected, both SNS- and VG-expressing cells are seen. (D–F) Confocal projections of 16 × 1 micron sections through an NXK11 mutant embryo at the same stage. As expected, the Notch mutant embryo exhibits a dramatically expanded population of VG-expressing founder cells compared to wild-type (compare B and C with E and F). By contrast, SNS expression is dramatically decreased (compare A and B with D and E), presumably due to the absence of fusion competent cells.
Figure 10
Figure 10
SNS expression is detected in the absence of the myogenic transcriptional activator MEF2. Expression of the sns transcript was detected immunohistochemically, by in situ hybridization using a digoxigenin labeled probe. All views are lateral, with anterior to the left and dorsal to the top. (A) Wild-type Stage 14 embryo. (B) Stage 14 embryo homozygous for the mef2 mutation mef222–21 (Bour et al. 1995). As shown, levels of sns transcript appear to be relatively unaffected in mef2 mutant embryos, indicating that MEF2 is not essential for sns expression.
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