Cary RB and Klymkowsky MW (1994), Desmin organization during the differentiation ...

XB-ART-21425

Differentiation 1994 Apr 01;561-2:31-8. doi: 10.1046/j.1432-0436.1994.56120031.x.

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Desmin organization during the differentiation of the dorsal myotome in Xenopus laevis.

Cary RB , Klymkowsky MW .

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The reorganization of desmin-type intermediate filaments during muscle differentiation has been studied primarily in cultured cell systems. Here we describe the process of desmin reorganization during the differentiation of the dorsal myotomal muscle of the clawed frog Xenopus laevis. This muscle differs from those described previously primarily in that the desmin system forms de novo, i.e., without the presence of a pre-existing vimentin filament system. The most striking observation is that prior to myotomal segmentation and rotation desmin is concentrated at the medial and lateral tips of the myocytes. It remains concentrated in these regions following somite rotation and is located primarily to the intersomite junctions as late as the stage 33-35 tadpole. As the muscle matures (stage 30 and later) desmin becomes increasingly associated with the sarcolemma and with the Z-discs. The concentration of desmin at the nascent intersomite junction suggests that desmin is involved in coupling somites to one another in the early Xenopus embryo.

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Species referenced: Xenopus laevis
Genes referenced: pprc1 vim was

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	Fig. 1. Changing desmin organization during myotomal maturation. Xenopus embryos at various stages of development were fixed. bleached and stained with the mouse monoclonal anti-desmin antibody DE-B-5. Embryos were then either stained with a peroxidase conjugated secondary antibody for whole-mount examination (a. c, e, g) or were stained with a fluorescein-conjugated secondary antibody for section-based examination (b. d, f. h). a At stage 22 desmin can be detected throughout the dorsal myotomc, i.c. in both prc- @ R ) and post-rotation (R) myotomc ( A and P. mark anterior and posterior ends of thc embryo). Immature myofibrils are first visible at this stage (see Fig. 2b). b Prior to the segmentation and rotation of the myotome, desmin is concentrated at the ends of the myocytes (urrows). NT. neural tube; M. myotome. c By stage 30/31 myotomal rotation is complete, although somites are still forming, by a different mechanism. in the tail bud. Desmin remains concentrated at the ends of the cell (+ S+. marks a single somite). Faint staining can also be seen associated with the lateral membranes. Myofibrils are present at this stage but are not yet mature (see Fig.' 2c). d Section analysis of the same stage provides a clearer image of the desmin concentrated at the intersomite junction ( I S 4 and the desmin associated with the lateral myocyte membrane (arrows). An enlargement of the ISJ is presented in the insert. e By stage 36/37 desmin staining is present throughout the muscle cell, although focal concentrations a t the 1SJ are still apparent (arrowhead). f Immunofluorescence microscopy of sections from this stage clearly reveal the association between desmin and the Z-discs (small arrows) and with the sarcolemma (large arrows). At the sarcolemma, desmin is often punctate (hlack arrow). g At stage 48, desmin is present throughout the muscle cell with little if any preferential accumulation a t the ends of the cell. h Immunofluorescence microscopy at this stage indicates that desmin is concentrated at the sarcolemma and is associated with Z-lines (arrows). Bar a, 0.2 mm; bur c, 0.2 mm for c-g; barb, 50 pm for b-h
	Fig. 2. Electron microscopy of early stages. a At stage 22, the space between adjacent somites is wide (c J - , ISJ) and free of collagen fibrils. There is also a wide gap between the lateral membranes of the myocytes (double-headed arrow). b At this stage, laterally aligned fibers, presumably thin (actin) filaments can been seen (double arrows); occasionally Z-discs can be seen (black arrows). c By stage 30/31, Z-discs are prominent (double arrows) but irregular; only a few myofibrils are present in each cell. It is often possible to see the developing t-tubule system associated with the Z-discs. The ISJ has narrowed (c ISJ-+) and contains numerous extracellular fibrils. Close junctions are frequent along both lateral and terminal membranes (solid arrows). The extracellular fibers can be seen more clearly at higher magnification (d) (small arrows). Filaments of 6-8 nm, presumably actin filaments, can be seen approaching the junctional membrane (larger arrow) and electron dense material has begun to accumulate at the terminal membrane (double arrow). Bars, 10 pm (a); 1 pm (b, c); 500 nm (d)
	Fig. 3. Electron microscopy of later stages. a At stage 37/38 the lateral spaces between adjacent myocytes is greatly reduced (arrows). Close junctions between the terminal membranes are now absent and myofibrils occupy a substantial portion of the cellular volume. The myofibrils clearly anchor to the myocyte membrane at the ISJ (arrowheads). b In the region of the ISJ the membrane of the muscle cell has become highly involuted; the cytoplasmic Face is characterized by electro? dense material and the thin filaments of the forming myofibrils appear to attach to the ISJ membrane (arrows). The ISJ space (asrerix) is filled with fibers. c By stage 47/48 the volume of the myocyte occupied by myofibrils has increased substantially. The lateral spaces between myocytes (arrowheads) has largely disappeared. Z-lines are straight and well aligned within each individual cell (arrows). d The electron dense material associated with the cytoplasmic face of the ISJ membrane is now largely concentrated at the tips and lateral surfaces of the folds. Junctional structures connecting the myofibrils to the dense extracellular network of fibrils, can be seen (arrows). Bars, 2 pm (a & c); 500 nm (b); 1 pm (d)
	Fig. 4. Confocal image of desmin aggregates. Here two myocytes of a stage 48 embryo, stained in whole-mount with anti-desmin and antiMlg-FI, have been visualized using confocal microscopy. This revealed the presence of large irregular or ‘astral ’ aggregates of desmin ( A ) . Desmin was also clearly associated with Z-discs (white on block arrows) and with the subsarcolemmal membrane (ivhire arrows)