Kay BK et al. (1988), Patterns of N-CAM expression during myogenesis ...

XB-ART-27443

Development 1988 Jul 01;1033:463-71.

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Patterns of N-CAM expression during myogenesis in Xenopus laevis.

Kay BK , Schwartz LM , Rutishauser U , Qiu TH , Peng HB .

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The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.

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Species referenced: Xenopus laevis
Genes referenced: ncam1

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	Fig. 1. During the formation of the tail, N-CAM is expressed solely on neural tissues in Xenopus embryos. A shows a phase-contrast image of a longitudinal cross section of a stage-22 embryo. B is the corresponding immunofluorescence image obtained with rabbit N-CAM antibodies and goat anti-rabbit Ig antibodies linked to FITC. The cell membranes of the neural tube are labelled, whereas the mononucleated muscle cells arranged in somites are not. C shows an N-CAM immunofluorescence staining of a stage-25 embryo. The level of background fluorescence is high in B and C due to the autofluorescence of yolk. D shows N-CAM immunofluorescence staining of a whole-mount strip of a stage-42 tadpole tail. The pigmented cells of the neuroepithelium do not stain, nor do the myotomal muscles separating each set of axons. N, row of nuclei. Bar, 120^m in A-C and 100Um in D.
	Fig. 2. N-CAM expression is neural-specific in tadpole heads. A is the fluorescent image of a stage-41 tadpole stained with rhodamine-labelled phalloidin. In this ventral view, the mylohyoid muscle (M) and ocular muscle (O) groups stain strongly. B displays the N-CAM antibody immunofluorescence staining of a comparably staged tadpole whole-mount preparation. The ventral side of the head is shown, with bright staining of the brain, and the optic and olfactory nerves. Bar, 500um.
	Fig. 3. N-CAM is expressed on the hindlimb muscles of tadpoles and on adult leg muscles after denervation. A shows a cross section of a hindlimb of a stage-55 tadpole in phase contrast, and B shows the immunofluorescence staining of the same section obtained with the monoclonal antibody 4D. Note the strong staining of the muscle fibres with N-CAM antibodies. C is the phase-contrast image of a hindlimb muscle of a comparably staged tadpole, and D is the staining pattern obtained with polyclonal anti-frog N-CAM antibodies. E-F display the N-CAM immunofluorescence staining of gastrocnemius muscles from the same adult frog with polyclonal anti-frog N-CAM antibodies; E is from the intact limb and F is the contralateral leg 4 days after denervation. The bright spots in E are autofluorescing red blood cells. Bar, 135um.
	Fig. 4. Western blots of Xenopus embryonic tissues with N-CAM antibodies. Various samples were homogenized in extraction buffer, electrophoresed in a 7 % SDSpolyacrylamide gel, blotted and reacted with the rabbit anti-frog N-CAM antibodies. Lane A contains chick embryonic brain (E15), B developing limbs of a stage-55 Xenopus tadpole, and C brain of a stage-55 tadpole. Sizes of the major reactive polypeptide chains are Mr x 10-3.
	Fig. 5. Lack of N-CAM expression in cultured Xenopus myotomal muscle cells. Tail muscle cells from stage-22 embryos were cultured and reacted in the living state with N-CAM antibodies and FITCconjugated secondary antibodies (A), or examined by phasecontrast microscopy (B). Cells were also double stained with N-CAM antibodies (C) and R-BTX (D); the spontaneously formed acetylcholine receptor clusters (marked with arrowheads in D) do not react with N-CAM antibodies. Neurites and growth cones (arrowhead) from neural tube explants, on the other hand, react quite well with N-CAM antibodies (E). Muscle cells from stage-50 tadpoles were also examined for N-CAM expression after dissociation from the tail musculature. After dissociation, these cells were immediately fixed and processed for binding of N-CAM antibodies (F) or R-BTX (G). Note the lack of N-CAM at the postsynaptic acetylcholine receptor clusters, marked by arrowheads in G, as well as along the extrajunctional sarcolemma. Bar, 30um.
	Fig. 6. N-CAM expression on cultured muscle cells from developing limb musculature of Xenopus. Left column: immunofluorescence staining for N-CAM; middle column: R-BTX staining to show the presence of acetylcholine receptors: right column: phase-contrast image (C,I) or DAPI staining (F). A-C shows a mononucleated myoblast and several fibroblasts (asterisks). D-F shows a developing myotube with four nuclei, a myoblast and a fibroblast (arrowhead). G-I shows a myoblast stained with preimmune serum (G) or R-BTX (H), and the corresponding phasecontrast image (I). Bar, 40um.