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doi: 10.1242/10.1242/dev.00541

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Regulation of myosin expression during myotome formation

Department of Medicine, Stanford University, School of Medicine, Stanford, CA 94305-5151, USA

View larger version (33K): [in a new window]   Fig. 1. RT-PCR analysis of somites from HH 10, 12, 15, 17 and 19 embryos, as well as ED6 wing bud, using primers specific for sMyHC1, 2, 3, efast MyHC and nfast MyHC. Total RNA was isolated from the three rostral-most somites of embryos at each stage and subjected to 36 cycles of RT-PCR. The first transcripts to appear are efast MyHC and sMyHC3, followed by sMyHC1 and finally sMyHC2. Transcripts of nfast MyHC were not detected in the somites at these stages, but were easily detected with 36 cycles in the wing bud at ED6.

View larger version (32K): [in a new window]   Fig. 2. Developmental expression of MyHC transcripts in the myotome detected by in situ hybridization. Staged embryos were hybridized with probes specific for sMyHC1, 2, 3 or efast MyHC. The number of somites expressing MyHC was plotted against the total number of somites that had formed in that embryo. efast MyHC (black, open square) was the first myosin expressed, followed a few hours later by nearly simultaneous expression of sMyHC1 (blue circle) and sMyHC3 (red square). There was a significant delay before the expression of sMyHC2 (green triangle).

View larger version (119K): [in a new window]   Fig. 3. Intracellular expression patterns of slow and fast MyHC mRNA within myotomal fibers. HH 15 (A,B) and HH 19 (C,D) embryos were hybridized with probes for efast MyHC and sMyHC3. (A) At HH 15, efast MyHC mRNA is found throughout the cytoplasm of myotomal fibers. (B) sMyHC3 mRNA is localized to the myotomal nuclear domain. Notice also that fewer somites express slow MyHC then efast MyHC at this stage. (C) At HH 19, efast MyHC expression has expanded along the dorsoventral axis of each somite, but retains its cytoplasmic localization. (D) sMyHC3 expression has expanded in the dorsoventral direction and some signal is now apparent in the cytoplasm. (E) In sagittal sections through immature somites, in situ hybridization indicates a restriction of the sMyHC3 mRNA transcripts to the central domain of myotomal fibers where the nuclei are located. (F) In more mature somites, sagittal sections show sMyHC3 mRNA transcripts distributed beyond the nuclear domain.

View larger version (132K): [in a new window]   Fig. 4. Myotomal fibers express both fast and slow MyHC proteins. A cross section (A,B) through a rostral somite of a HH 20 embryo and a longitudinal section (C-E) through a rostral myotome of a HH 24 embryo, double-stained with F59 for fast and S58 for slow MyHC protein. (A,C) Fast MyHC (red) is present in fibers of the myotome. (B,E) Slow MyHC (green) appeared in essentially all the fibers as well. (D) Nearly total overlap of the two fluorescent markers (yellow) demonstrates colocalization of fast and slow MyHC proteins in all fibers. The outline of the embryo was visualized by counterstaining nuclei with DAPI (blue). A and B, and C-E depict the same field viewed in different fluorescent channels.

View larger version (95K): [in a new window]   Fig. 5. Implantation of sonic-hedgehog-soaked beads results in an increase in slow and fast MyHC expression, and a thickening of the myotome. Embryos were implanted with beads soaked in either BSA (A,C) or N-Shh (B,D,E,G) and assayed by whole-mount in situ hybridization with probes specific for either sMyHC3 (A-D) or efast MyHC (E-G). The effect on both myosin isoforms was the same. (A,C) Implantation of a BSA-soaked bead does not cause an alteration to sMyHC3 expression when viewed dorsally (A) or laterally (C). (B,D) Beads soaked in Shh protein result in a marked increase in MyHC expression and thickening of the myotome. (E) When viewed in cross section, it is apparent that Shh-soaked beads cause a thickening of the adjacent myotome, whereas the contralateral myotome is unaffected. (F,G) At higher magnification of the areas shown in E, it is apparent that myotomal fibers adjacent to the Shh-soaked bead are hypertrophied.

View larger version (88K): [in a new window]   Fig. 6. Nerve fiber outgrowth and innervation of the myotome. Explants were taken from HH 14 (A-C) and HH 23 (D) embryos, and stained with antibodies that identify neurons (red) and desmin (C,D) (green). (A) After 4 hours of incubation, no nerve fiber outgrowth was detected in explants of a HH 14 embryo. (B) Nerve fibers can be seen to extend laterally from the neural tube of explants of HH 14 embryos after 18 hours of incubation. (C) When explants were stained for both desmin and neurofilament protein, it became apparent that nerve fibers were extending to innervate the developing myotomal regions of the somite. (D) In an explant taken of the wing-level somites of a HH 23 embryo and incubated for 18 hours, it can be seen that mature nerve fibers enter the myotomal regions of the somite at two discrete sites (arrows).

View larger version (57K): [in a new window]   Fig. 7. Surgical separation of somites from the neural tube prevents initiation of sMyHC2 but not sMyHC3 expression. Explants from HH 14 embryos were cultured for 24 hours either with or without axial structures (neural tube/notochord) and were then assayed by in situ hybridization. (A) In the absence of neural tube, sMyHC2 was not expressed. (B) When the contralateral somites of (A) were incubated with the associated neural tube (nt), sMyHC2 is expressed. (C) Separation of somites from the neural tube/notochord does not inhibit sMyHC3 expression. (D) Somites contralateral to (C) and incubated in the presence of neural tube/notochord also express sMyHC3.

View larger version (41K): [in a new window]   Fig. 8. Exposure of explant cultures to d-tubocurare prevents expression of sMyHC2 but not of other MyHC genes. Explants of somites with the neural tube taken from HH 14 embryos were cultured for 24 hours in the presence (A-D) or absence (E) of 16 µM d-tubocurare and assayed by in situ hybridization with probes specific for sMyHC1, 2 and 3, and efast MyHC. (A) efast MyHC, (B) sMyHC1 and (C) sMyHC3 are expressed in the presence of dtubocurare. (D) sMyHC2 was not expressed in explants cultured in the presence of dtubocurare but (E) was expressed when cultured in its absence.

View larger version (135K): [in a new window]   Fig. 9. Localization of MyHC message within the developing myotome. Embryos hybridized with isoform-specific MyHC probes were subsequently stained with a monoclonal anti-desmin antibody (green). (A) In somites 26 and 27 of an HH19 embryo, the first efast MyHC expression is detected by in situ hybridization in the ventrolateral regions of the myotome (arrowhead), and desmin-positive, MyHC-negative fibers are positioned primarily in the dorsal region. (B) In more-mature (rostral) somites of the same embryo, the expression of efast MyHC extends more dorsomedially. (C) In somites 31 and 32 of an HH21 embryo, the first fibers to express sMyHC3 are detected near the middle of the myotome (arrowhead), and desmin-positive, slow-MyHC-negative fibers are present both dorsal and ventral to this region. (D) After an initial extension to the ventrolateral margin of the myotome, sMyHC3 expression expands in the dorsomedial direction in more-mature (rostral) somites. The broken line outlines the extent of the desmin-positive fibers in the myotome. Not all fibers are within the focal plane of the photo.

View larger version (32K): [in a new window]   Fig. 10. Model of fiber formation in the myotome. The myotome as defined by desmin staining is represented as closed boxes. Fast MyHC transcripts appear first in the myotome and are distributed throughout the width of myotomal fibers, whereas transcripts for slow MyHCs appear slightly later and are initially confined to the central nuclear domain. Fast MyHC expression begins near the ventrolateral edge of the myotome and expands dorsomedially as development continues. Slow MyHCs are initially expressed more centrally in the myotome and expression initially expands ventrolaterally. Subsequently, slow MyHC expression expands dorsomedially within the myotome. In the most-mature somites, slow MyHC transcripts become distribute more evenly throughout the myotomal fibers, eventually losing their nuclear localization completely to mimic the distribution of fast MyHC transcripts.