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First published online October 22, 2003
doi: 10.1242/10.1242/dev.00799

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Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

¹ Comparative and Developmental Genetics Section, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
² Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK
³ Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, Sydney 2010, Australia
⁴ Department of Molecular and Cell Biology, University of California Berkeley, 555 Life Sciences Addition #3200, Berkeley, CA 94720-3200, USA

View larger version (69K): [in a new window]   Fig. 1. sap mutants develop lesions in skeletal muscle where fibres detach from myosepta and retract. Fibres in wild type (WT) span the entire somite between myosepta (arrowheads in A; lateral views, 48 hours post-fertilisation), whereas lesions within sap somites are evident as cell-free spaces (arrowheads in B). Toluidine blue histology reveals detached, retracted fibres in association with lesions in sap (arrowhead in D; parasagittal sections, 72 hours post-fertilisation) but not in WT (C). Reconstruction of somites in 3D using confocal microscopy of fluorescence from the Tg(acta:GFP) transgene reveals extensive fibre loss in sap (arrowhead in F) but not WT (E). Anti-MyHC (green) reveals that whereas differentiation is normal in both WT (G) and sap (H), lesions in sap mutant somites lack contractile apparatus (arrowhead). Examination of head musculature using fluorescence from the Tg(acta:GFP) transgene shows that these muscles are unaffected in sap (J) compared with WT (I).

View larger version (103K): [in a new window]   Fig. 2. Dystrophin is associated with the wild type (WT) embryonic muscle attachments. dystrophin mRNA (dmd) localises intracellularly to WT somite boundaries both before (19 hours post-fertilisation, A) and after (27 hours post-fertilisation, B) muscle fibre differentiation (arrowheads, lateral views). At 19 hours post-fertilisation, a crescent of mRNA is present at one side of undifferentiated cells that abut somite boundaries. Dystrophin protein (dys) is localised embryonically to fibre ends at somite boundaries, and at NMJs but not at the sarcolemma (C, horizontal section, 72 hours post-fertilisation). Dystrophin (green) in fibre ends sandwiches the ECM of the vertical myoseptum at somite boundaries, which contain tenascin-C (tn-c, red; arrowhead in D, horizontal section, 72 hours post-fertilisation). Dystrophin localises to fibre ends and NMJs but not to the sarcolemma embryonically (arrowheads in E, transverse section, 72 hours post-fertilisation). Dystrophin is detectable at muscle attachments (arrowheads in E-G), but triple labelling using anti-dystrophin (green), Alexa594--Bungarotoxin to label NMJs (rbtx, red), and DAPI (blue) reveals that dystrophin within the myotome is at NMJs, co-localising with rbtx to produce an overlapping yellow signal (arrows in F,G; horizontal sections 72 hours post-fertilisation).

View larger version (108K): [in a new window]   Fig. 3. sapta222a is a mutation of dmd that causes fibre detachment and is phenocopied by knocking down dmd function. Dystrophin C-terminal immunoreactivity is localised to somite boundaries in wild type (WT) (A, 27 hours post-fertilisation, lateral views) but lacking in both sap (B) and dystrophin-morphant embryos (C). Evans blue dye (EBD, red), does not appear in WT somites (D), but labels fibres in both sap (E) and dystrophin-morphant embryos (F). Labelled fibres are visible that have both detached and retracted (arrowheads) or still span a somite and show a retraction zone (between arrows in F, lateral views, 72 hours post-fertilisation). By 72 hours post-fertilisation, dystrophin is present in the neural tube and notochord in both WT (G) and sap (J, asterisks) but lacking from muscle attachments in sap (arrowheads in G,J, transverse sections). ß-dystroglycan is also localised to muscle attachments (arrowheads) and other sites (asterisks) in WT (H), but unlike dystrophin is preserved at muscle attachments in sap (K, 72 hours post-fertilisation, transverse sections). Utrophin is not detectable at muscle attachments in either WT (I) or sap (L, arrowheads). Anti-utrophin immunoreactivity in the epidermis provides an internal positive control (asterisks).

View larger version (54K): [in a new window]   Fig. 4. (A) Homozygosity for the nonsense mutation (AAATAA) in sapta222a dmd exon 4 segregated exclusively with the sap phenotype. (B) Rooted tree showing dystrophin and utrophin proteins in vertebrates. The tree is rooted using Drosophila melanogaster dystrophin. The numbers represent the percentage of 1000 bootstrap trials that support the branch. Protein accession numbers: XP_081212 NP_000100 O97592 NP_031894 CAA31746 NP_009055 CAA58496. The Fugu sequences are manually corrected GENSCAN predictions from genomic scaffolds (www.jgi.doe.gov/fugu, Scaffold 234). The zebrafish utrophin sequence is predicted from the zebrafish genome project. The tree has been made from partial sequences corresponding to the zebrafish protein published in this paper. (C) Partial alignment of zebrafish (Dr_dys), predicted Fugu rubripes (Fr_predict), human (Hs_dys) and chicken (Gg_dys) dystrophin proteins including the two N-terminal calponin homology domains (CH, underlined). The position of the stop codon in sapta222a dmd is marked by an asterisk. Exon boundaries 2 to 7 are marked by green arrowheads, except between exons 6 and 7 (red) against which MO1 was directed. Chick, Gallus gallus; Dog, Canis familiaris; DYS, dystrophin; Fugu, Fugu rubripes; Hum, Homo sapiens; Mus, Mus musculus; Rat, Rattus norvegicus; UTRO, utrophin; Zebra, Danio rerio.

View larger version (103K): [in a new window]   Fig. 5. In vivo observation of muscle attachment failure and molecular analysis of detached free ends. A single fibre (A,B short arrows) viewed in vivo in the process of detaching myosepta, under differential interference contrast (A, lateral view, 5 days post-fertilisation) and labelled with EBD (B). A gap is visible between the separating posterior end of the fibre (right, short arrow) and the myoseptum (arrowhead). A narrowed retraction zone has formed where the contractile apparatus has withdrawn from the centre of the fibre (between the short arrows). The anterior end of the fibre (left, short arrow) is partly obscured by a second dye-positive detached cell (long arrow). Confocal microscopy of GFP revealed this example of a free end of a single detached fibre (D, bracket, lateral view, 6 days post-fertilisation). Stacked confocal images allow tracing of individual fibres to their insertion points at the muscle attachments. Fibres within sap homozygotes (D) exhibit a club-like or faceted appearance at their newly detached membranes, not evident in wild-type embryos (C). ßDG protein at the muscle attachments is not retained in the fibre membranes once detachment occurs. Two neighbouring detached and retracted mutant cells visible under DIC (arrowheads in E, 72 hours post-fertilisation, lateral view) are still attached to their posterior (right) myoseptum (arrows in E,F). Labelling with anti-ßDG (F) shows that this integral membrane protein has been lost from their free (left) ends upon detachment, possibly maintaining its binding to -dystroglycan and laminin at the myoseptum. Phosphorylated focal adhesion kinase is enriched in terminal cytoplasm at muscle attachments (arrows in G,H, lateral confocal images, 72 hours post-fertilisation). Unlike ßDG, however, p(tyr397)FAK remains visibly localised to the free end of detached cells, indicating the retention of terminal cytoplasm by detached and retracted fibres (arrowheads in H).

View larger version (131K): [in a new window]   Fig. 6. Electron microscopy shows that wild-type (WT) embryonic myofibrils align to form a regular sarcomeric array that attaches obliquely to the myosepta (asterisks) (A). In sap homozygotes, fibres showing detached ends (arrows in B,C,G) and shortening of both the entire fibre and the sarcomeres, are visible. In these cells, the separation and regularity of sarcomeric banding is greatly reduced or collapsed compared with that in intact neighbouring cells, and absent in some places (B,C). Actin filaments (AF) run longitudinally from the terminal sarcomeres to the vertical myoseptum in both WT and intact sap mutant muscle fibres (D,E). Nuclear changes were also followed by electron microscopy in order to examine whether detachment precedes or follows cell death in sapje mutants. Nuclear condensations indicative of apoptosis were only present in detached mutant fibres (G), but were not observed in either intact mutant or WT (F) fibres, demonstrating that detachment is not a secondary process resulting from apoptosis of muscle fibres. Four days post-fertilisation, parasagittal sections. AS, absent sarcomeres; CS, collapsed sarcomeres; IS, intact sarcomeres; N, nucleus.