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Abstract
The protein components of mature skeletal muscle have largely been characterized, but the mechanics and sequence of their assembly during normal development remain an active field of study. Chaperone proteins specific to sarcomeric myosins have been shown to be necessary in zebrafish and invertebrates for proper muscle assembly and function. The Xenopus tropicalis mutation dicky ticker results in disrupted skeletal muscle myofibrillogenesis, paralysis, and lack of heartbeat, and maps to a missense mutation in the muscle-specific chaperone unc45b. Unc45b is known to be required for folding the head domains of myosin heavy chains, and mutant embryos fail to incorporate muscle myosin into sarcomeres. Mutants also show delayed polymerization of alpha-actinin-rich Z-bodies into the Z-disks that flank the myosin-containing A-band. The dicky ticker phenotype confirms that a requirement for myosin-specific chaperones is conserved in tetrapod sarcomerogenesis, and also suggests a novel role for myosin chaperone function in Z-body maturation.
Figure 2. Unc45b expression in Xenopus tropicalis. (A) WISH showing unc45b expression in developing heart (hrt) and somites (som) at stage 28. (B) ventral view of heart expression at stage 28. (C) unc45b expression at stage 40 throughout the somites and (D) in jaw (jw), heart (hrt) and body wall muscles (bw).
Figure 3. Myofibrillogenesis is disrupted and sarcomere formation is delayed in dit. Confocal analysis of wild type (left panels) and dit embryos (right panels) for sarcomeric myosin heavy chain (MyHC) and Z-disc (α-actinin) immunoreactivity. (A, C) Anteriorsomites of stage 28 wild type embryos show sarcomeric myosin (MyHC) organized into A-bands separated by regularly spaced Z-discs (α-actinin). (B) Anterior dit somites show disorganized MyHC staining, and (D) α-actinin staining is punctate, with occasional ordered Z-bodies consistent with the appearance of incipient sarcomeres (arrowheads). (E) More posteriorly at st. 28, α-actinin staining shows wild type somites 15-16 (arrow indicates intersomitic boundary) are beginning to organize Z-discs (arrowheads), while (F) dit somites at this level show only background staining (arrow indicates intersomitic boundary). Scale bars 10 μm.
Figure 4. MyHC immunoreactivity is lost and Z-disc formation recovers in dit. Confocal analysis of wild type (left panels) and dit embryos (right panels) stained with MyHC A4.1025, α-actinin and titin T12 antibodies. (A, C, E) A-bands and Z-discs are well-organized in wild type somites at stage 43. (B) Sarcomeric myosin staining in dit is virtually absent at these stages, but Z-disc-containing sarcomeres are observed (D, F). Phalloidin stained thin filaments are present but disorganized in dit somites.
Figure 5. Morpholinos against unc45b phenocopy dit. Unc45b knockdown closely resembles the dit phenotype and results in depletion of MyHC staining and disorganized sarcomeres. Confocal analysis of control MO (A, C), ATG MO-injected (B, D), and splice MO-injected embryos (E, F) for sarcomeric myosin heavy chain (MyHC) and Z-discs (α-actinin), co-stained with phalloidin.
Figure 6. unc45b expression is upregulated in dit. WISH showing expression of unc45b at stage 43 in (A) wild type and (B) dit, and at stage 40 in (C) control MO and (D) unc45b splice-MO injected embryos. Both splice-MO injected and dit embryos show increased unc45b expression relative to controls.
Additional file 3:
Muscle structure is disrupted in dit tails. (A) Birefringence of polarized light in stage 43 wild type tail. (B) Birefringence is greatly reduced in dit tails of the same stage. (C) Phalloidin staining of stage 43 wild type tailmuscle showing orderly myofibril structure. (D) Phalloidin staining in dit embryo tails shows disorganized myofibrils.
Additional file 4:
Western blot analysis of wild type and dit embryos with MyHC F59 antibody. Presence of MyHC as detected by the F59 antibody is reduced in dit compared to wild type embryos at both stages 40 and 43.
unc45b (unc-45 homolog B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
unc45b (unc-45 homolog B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anteriorleft, dorsal up.
Figure 1. dicky ticker maps to a lesion in the myosin co-chaperone unc45b. (A) Pools of gynogenetically derived wild type and dit embryos were assayed for linkage to polymorphisms near the 10 X. tropicalis chromosomes. Clear linkage (*) was observed at Linkage Group 6 (Chromosome 2) (B) Linkage analysis using SSLP and SNP markers with 562 mutant embryos placed the dit locus in a 230 kb interval of Scaffold_72 containing four gene models including unc45b. (C) cDNA sequence of dit unc45b revealed a thymidine to cytidine transition, substituting Arg for Cys779 in the UCS domain. (D) Cys779 is phylogenetically conserved in unc45b orthologs from Drosophila (Dm) to human (Hs).
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