rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion
Annette Rau1,*,
Detlev Buttgereit1,*,
Anne Holz1,*,
Richard Fetter2,
Stephen K. Doberstein2,
,
Achim Paululat1,
Nicole Staudt1,
,
Jim Skeath4,
Alan M. Michelson3 and
Renate Renkawitz-Pohl1,
1 Developmental Biology, Philipps-Universität Marburg, 35032 Marburg, Germany
2 Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
3 Brigham & Womens Hospital/Howard Hughes Medical Institute, 20 Shattuck Street, Room 1021, Boston, MA 02115, USA
4 Department of Genetics, Box 8232, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA
* These authors contributed equally to this work
Present address: MPI für Biophysikalische Chemie, Abt. Molekulare Entwicklungsbiologie, Am Fassberg, 37077 Göttingen, Germany
Present address: Exelixis, 170 Harbor Way, P.O. Box 511, South San Francisco, CA94083-0511, USA
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Fig. 1. Myoblast fusion is severely disturbed in rols mutants. The muscle phenotype of rols mutants (C-H) is compared with the wild-type (A,B) pattern of the body wall muscles. The mesoderm is visualized with antibodies against ß3-tubulin. (C) Df(3L)BK9 embryo, stage 16, lateral view; the number of myotubes as well as their orientation is altered significantly. (D) Same embryo as in C but at a higher magnification; the disarranged myotube pattern as well as single, unfused myoblasts (arrow) are shown. (E) Df(3L)BK9 embryo, stage 16, dorsal view; the dorsal vessel is nearly undisturbed; most unfused myoblasts are arranged nearby formed myotubes. (F) Same embryo as in E, but at higher magnification; the alignment of unfused myoblasts (arrow) at individual muscles is evident. (G) rolsAD328, EMS-induced allele, late stage 16, lateral view; the pattern of the somatic muscles is also severely disturbed, however, owing to the later embryo stage, the number of unfused myoblasts is reduced in comparison with C. (H) rolsP1729ber, a P-element induced allele, stage 15, dorsolateral view. As in the other alleles, muscle number is reduced, and many unfused myoblasts are visible; also the pharynx (arrow) shows fusion defects. Again, the dorsal vessel is only weakly affected.
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Fig. 2. Muscle founders/precursors are determined correctly in rols mutants. All nuclei of muscle founder cells are visualized at late stage 13 in the enhancer trap line rP298 by the anti-ß-galactosidase antibody. The expression of rP298 was monitored in embryos homozygous for the deficiency Df(3L)BK9 (B) and revealed essentially a wild-type pattern (A). However, at stage 15/16, the number of rP298 positive nuclei in rols mutant embryos (D) is significantly reduced compared with wild type (C). Eve expression in wild-type embryos marks the nuclei of dorsal muscle 1 (DA1, marked by a white circle in one segment) and pericardial cells (pc) (E). In rolsP1729 mutants, Eve staining in DA1 is visible only in two to five nuclei (F, marked by a white circle in one segment). This is shown for a younger embryo homozygous for the deficiency (H) where two to three nuclei of dorsal muscle 1 are stained compared with a wild-type embryo with five to ten Eve-positive nuclei (G).
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Fig. 3. Ultrastructural analysis of rols mutants show that Rols is required for alignment of muscle precursor cells with FCMs. Electron micrograph of an early stage 13 rolsAD328 mutant embryo (A) reveals closely associated cells (arrow) but in contrast to the wild type, no fusion complexes are evident. At stage 14 (B) myoblasts in rols mutant embryos have lost contact with muscle precursor cells compared with wild-type where prefusion complexes (Doberstein et al., 1997) are evident. Arrowheads indicate myoblasts, whereas the double arrowhead shows a trinucleated precursor cell. Regions of the endoderm (en) and the visceral mesoderm (vm) are marked with arrows.
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Fig. 4. (A) Genomic organization of the rols locus. The scheme summarizes the data of our genomic walk, the data from the Drosophila Genome Project and the sequence analysis of rols cDNAs, as well as the determination of the P-element insertion sites. The rols gene extends about 60 kb, as indicated by the scale and is flanked towards the centromer by an open reading frame coding for Semaphorin 5c (CG5661; S, gray box). The exons common to rols7 and rols6 are shown as black boxes, specific exons for rols7 are shown as black and white hatched boxes, while the specific exons for rols6 are shown as white boxes. The transcription initiation site of rols6 is localized within the large second intron of rols7. The translation initiation codons for Rols7 and Rols6 are unique, they are localized in the second exon unique to each message. The P-element in the mutant rolsP1027 is localized 70 bp upstream of the transcription initiation site of rols6, while the P-element P1729 of the rolsP1729 mutant is localized within the common part of the second intron. (B) Stage-specific mRNA levels detected by RT-PCR. Poly (A)+ RNA was prepared from embryos collected for the times indicated, and RNA from 0-4 and 4-8 hours was purified twice in order to remove genomic DNA completely. For details of the RT-PCR see Material and Methods. Whereas the ß1-tubulin mRNA (arrow) is present during all stages, rols6 and rols7 are first detected at 4-8 hours, corresponding to embryonic stages 8-11; rols7 reaches maximum levels between 8-12 hours (stages 12-14), while rols6 shows the highest levels at 12-16 hours (stages 15-16), whereas rols7 already declines. During 16 hours until hatching, rols6 is present at relative high levels compared with rols7.
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Fig. 5. Deduced amino acid sequence of Rols7 and Rols6. From the analysis of the cDNA sequences, the possible open reading frames were deduced. According to conceptual translation, rols6 encodes a protein of 1670 amino acids, 79 of which at the very N terminus are unique to Rols6. rols7 encodes a protein of 1900 amino acids, 309 of which are unique to Rols7. The amino acid sequence of Rols7 is shown in total. The beginning of the common part of Rols7 and Rols6 is indicated by a black triangle, which corresponds to the first common intron-exon boundary of the mRNAs. Structural motifs were determined using the SMART-tool at the EMBL-server at Heidelberg (http:/smart.embl-heidelberg.de) (Schultz et al., 1998; Schultz et al., 2000). This analysis predicts four structural motifs within the common part of both proteins which are underlined and nominated in the sequence: a RING-finger motif in the N-terminal part, a predicted nucleotide binding motif, nine ankyrin motifs and a TPR-repeat (broken underline) close to the C terminus.
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Fig. 6. Differential distribution of rols-transcripts during Drosophila embryogenesis. (A,B) Expression patterns from the enhancer-trap lines rolsP1027 and rolsP1729. (A) rolsP1027 embryo, stage 12, lateral view, focus on the interior. ß-galactosidase expression is detectable in both midgut primordia. (B) rolsP1729, embryo stage 16, lateral view, focus on the epidermis. The nuclei correspond to the positions of the epidermal muscle attachment sites (apodemes). (C) rols7 is first detected in the mesoderm at the extended germ band stage. (D) During germband retraction, the number of rols7 expressing cells increases but remains restricted mainly to the somatic mesoderm; weak, transient expression is detected in the visceral mesoderm (arrow). (E) After stage 12, the mRNA remains restricted to a subset of myoblasts derived from the somatic mesoderm. (F) Higher magnification of muscle precursors of the embryo depicted in E reveals expression surrounding one of the nuclei in a precursor. (G) Anti Mef2 stains all nuclei of the somatic mesoderm. (H) rP298 stains a subset of myoblasts (the muscle founder/precursor cells in every segment), while the unfused fusion-competent cells are not stained. (I,J) rols6 is mainly expressed during the invagination of the anterior and posterior gut primordium, and later on in the developing pharynx, the malpighian tubules (arrow) and in some ectodermal cells. (K) rols7 is expressed in a small group of mesodermal cells of the clypeolabrum. (L) rols6 is expressed in the ectoderm beneath the rols7-positive cells. (M) The rP298 enhancer trap pattern coincides with the rols7 expression pattern in the clypeolabrum. (N) sns is transcribed in a group of mesodermal cells of the clypeolabrum flanking the rols7-positive cells on the dorsal side.
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Fig. 7. rols mutant cells are able to fuse with wild-type myoblasts as fusion-competent cells and exhibit fusion defects as muscle precursor cells. Descendants of the transplanted cells are visualized by histochemical ß-galactosidase staining only in syncytial tissues that co-express GAL4 and UAS-lacZ. Clones in the ventral and lateral larval muscles after transplantation of homozygous rols null mutant cells are shown. (A) Ventrolateral larval muscle clone spanning two segments derived from a transplantation of three to five homozygous mutant cells. (B) Most descendants of rols mutant cells behave normally in wild-type background. This ventral muscle clone shows two additional structures, one spindle-like (arrow) and one more compact (arrowhead) with two to three nuclei each. (C) Ventral bilateral clone in two larval muscles, a normal syncytium and a small elongated striated syncytium with four nuclei, probably representing a muscle precursor. (D) Part of a ventral muscle clone with a very shortened mini-muscle attached correctly to the epidermis.
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Fig. 8. Rolling pebbles signaling between precursor cells and FCMs is necessary to stabilize cell contacts and to initiate progression in fusion such as prefusion complex formation. In wild-type myoblasts fusion proceeds after adhesion of FCMs to precursor cells by forming the prefusion complex of paired vesicles followed by plaque formation of the opposing membranes and subsequent membrane breakdown. In rols mutants the initial adhesion of precursor cells with FCMs is observed but cells scatter instead of proceeding to prefusion complex formation, showing its first function before sns and blow but after the initial Duf function.
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© The Company of Biologists Ltd 2001
