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A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anteriorsomitic mesoderm, and its concentration then declines in anteriorsomites from the tailbud stage onwards.
Fig. 5. XMyoD expression in gastrulae. (A) st 11.5 wholemount, showing collar of stained nuclei around the blastopore; (B) higher power view of the area surrounding the yolk plug of a stll embryo, focused to show the gap in dorsal staining, which corresponds to the prospective notochord; (C, D) slightly oblique sections of stll wholemount, cut in approximately the planes indicated in (B). Some of the yolk plug nuclei are faintly stained in (D), a non-specific consequence of rather heavy staining, that we have seen also with other primary antibodies.
Fig. 6. XMyoD is present only in myotomal nuclei of neurulae and tailbud embryos. Transverse sections of (A) an early neurula (st12.5-13), and (B) an early tailbudembryo (st23) stained with D7F2. (C) Section through the posterior part of the segmented region of a tailbudembryo (st26) stained for XMyoD (blue-purple) and 12/101 antigen (red), showing that all XMyoD-expressing nuclei are in 12/101-positive cells. (D) Section through mid-neurula (stl5-16) stained with D7F2 for XMyoD and with Hoechst 33258 to show all nuclei. XMyoD non-expressing nuclei show the bright, light blue Hoechst fluorescence; XMyoD expressing nuclei show a darker blue due to Hoechst fluorescence shining through the dark BCIP/NBT blue purple reaction product. All clearly myotomal nuclei express XMyoD.
Fig. 7. Expression of XMyoD in neurulae and tailbud embryos. Whole-mount immunocytochemistry with D7F2. (A) Early neurula (st12.5-13); (B) mid-late neurula (st17); (C-E) late neurulae (stl9), (D) same embryo as in C also stained with 12/101 (red), (E) side view of newly formed somites; (F) early tailbud (st24); (G, H) later tailbud (st27), (H) close-up of developing tail where myotomes are forming, the major site of XMyoD expression at this stage. Note that XMyoD and 12/101 staining in D can be distinguished on the basis of subcellular location, even if the secondary antibodies used to detect 12/101 react with any unremoved D7F2. Some staining of the archenteron contents is visible in F, which we judge to be a result of non-specific sticking of the antibodies, because we have seen it also with several other monoclonal antibodies and when no primary antibody was used, but not when all antibodies were omitted. Colour reactions were allowed to proceed so as to give the highest signal over background, that is for longer for the early neurula and tailbud embryos, than for the more strongly expressing late neurulae. Anterior of all embryos to the left.
