Heathcote RD et al. (2000), Dystroglycan overexpression in vivo alters acet...

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Dev Biol 2000 Nov 15;2272:595-605. doi: 10.1006/dbio.2000.9906.

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Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction.

Heathcote RD , Ekman JM , Campbell KP , Godfrey EW .

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Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.

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Species referenced: Xenopus laevis
Genes referenced: dag1 dmd dmd.2 musk

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	FIG. 1. Dystroglycan distribution. (A) A frozen sagittal section of a normal embryo at 1.6 days (st 31). Dystroglycan is marked with the FP-B antibody (green) and AChRs are marked with rhodamine labeled bungarotoxin (red). Stacks of confocal images for each color have been projected and red is layered on top of the green. The broad green region is the intermyotomal septum, where synapses normally form. Less intense staining outlines the other surfaces of muscle fibers. AChR aggregates are predominantly located within the broad region of dystroglycan immunoreactivity. (B) A frozen horizontal section of an embryo at 1.8 days (st 33/34) which had one cell injected with dystroglycan RNA at the two-cell stage. A stack of images has been projected. Dystroglycan expression in several segments show much brighter staining on the right side, indicating that dystroglycan protein was successfully overexpressed. Note that on both sides, the outer edge of the spinal cord is stained. (C) Plot profile of equal rectangular areas from each side of the spinal cord of the animal shown in B. The plot is from section 6 of seven in the confocal stack. The peaks correspond to the regions of high intensity at the intermyotomal septum (synaptic regions). The intensities of both the peaks and the background were approximately doubled on the right (injected) side. For all seven sections, the three control peak areas were 54% of the injected side (P , 0.0001; paired t test) and the three control baseline areas were 48% of the injected side (P , 0.0001; paired t test). Even though the differences were consistent and significant, there was variability in the intensity on the injected side (between the second and the third peaks). Calibration: A, 20 mm; B, 100 mm.
	FIG. 2. Synaptic AChR distribution following overexpression. (A) Receptor aggregates in muscles overexpressing GFP. Image from a whole-mount embryo at 1.6 days (st 31) that was injected with GFP RNA at the one-cell stage. Projected stack of confocal images of a synaptic region stained with rhodamine–a-bungarotoxin. The AChR aggregate area is 1.24% of the entire image stack. (B) Dystroglycan overexpression. Image from a sibling injected with GFP and dystroglycan RNAs. The AChR aggregate area is reduced to 0.68%, approximately half that of the GFP control in A. Both images are from myotome 5, have extrasynaptic AChR aggregates, and appear similar except for the AChR aggregate area. Calibration: 20 mm.
	FIG. 3. Dystroglycan overexpression decreased AChR aggregation. (A) Raw AChR aggregation data. Each set of experiments came from siblings: some injected with GFP RNA alone (control, GFP) and others injected with a mixture of GFP and dystroglycan RNAs (experimental, DG). Confocal image stacks from six synaptic regions in each set were analyzed. The AChR aggregate area for each synaptic region sampled in two different sets of experiments is shown, as well as the average for each treatment. Variation occurred within and between experiments. In experiment a, the dystroglycan average was 62% of control (P 5 0.009; df 5 10; t test) and in experiment b, it was 45% of control (P 5 0.003; df 5 10; t test). Figure 2 images come from experiment a. 17 of 24 experiments showed a decrease in AChR aggregation of DG versus GFP. (B) The AChR aggregate area for all synaptic regions receiving each treatment showed a significant (P 5 0.005; df 5 250; t test) dystroglycan-associated decrease to 77.8% of control.
	FIG. 4. Rostral–caudal gradient of AChR expression. Top: Images from a whole-mount embryo (1.6 day, st 31) injected with GFP RNA (control). Projected confocal stacks from somites 5 (A), 6 (B), and 7 (C). The greatest AChR aggregation area was at somite 5 (1.82%); it decreased to 1.59% at somite 6 and 1.12% at somite 7. Bottom: Similar series from a sibling embryo injected with GFP and dystroglycan RNAs (experimental). Images are from somites 5 (D), 6 (E), and 7 (F). The respective AChR aggregate areas were 1.14, 0.89, and 0.42%. The inset in E plots the quantitative amounts for each image. Both control and experimental animals showed a rostral–caudal gradient of AChR aggregation. Calibration: 20 mm.
	FIG. 5. Dystroglycan inhibits AChR aggregation along a rostral– caudal gradient. Average AChR aggregation volume (with SEM) for all synaptic regions at each myotome border is shown. All three myotomes were analyzed in control (GFP; n 5 54) and experimental (DG; n 5 86) animals. A gradient is evident with the greatest and least AChR aggregation in the most rostral (5) and caudal (7) myotomes, respectively. Repeated-measures ANOVA showed significant overall differences between AChR aggregation areas across the three myotomes in both GFP and DG animals (P , 0.001 for each). Of all combinations within each group, only the synaptic regions between myotomes 5 and 6 in GFP-treated animals were not significantly different (P 5 0.08; compared using an unpaired t test). Regression analysis of the data for each experimental group showed similar slopes (20.12 for GFP and 20.09 for DG). The roughly parallel lines suggest that the experimental treatment inhibited AChR aggregation by a constant amount at each segment. The experimental (DG) value at myotome 5 is similar to the control (GFP) value at myotome 7, indicating that development in experimental animals lagged two segments behind controls.
	FIG. 6. Dystroglycan overexpression affects embryonic survival. (A) RNA injection decreased the survival of cleaving embryos to tail bud stage. Data are from more than 15,000 embryos produced by 16 females. Repeated-measures ANOVA shows that survival was significantly different across the three groups (P , 0.001). The Newman–Keuls posttest showed that the survival of animals injected with GFP RNA or a mixture of dystroglycan and GFP RNAs was significantly decreased from that of uninjected controls (P , 0.001) but not from each other. (B) Survival from cleavage to tail bud was examined for animals injected with different concentrations of dystroglycan (n 5 492 (Control), 661 (GFP; 0.5 ng), 690 ([DG]; 2.3 ng), 596 (2[DG]; 4.6 ng), 666 (4[DG]; 9.2 ng), 473 (4[AG]; 9.2 ng), 157 (6[AG]; 13.8 ng). Animals receiving the greatest amount of dystroglycan RNA (4[DG] or 9.2 ng) showed decreased survival but this was not evident in animals injected with equal or greater amounts of agrin RNA (4[AG] or 9.2 ng and 6[AG] or 13.8 ng) in other experiments. (C) To determine when additional dystroglycan disrupts embryogenesis, survival was analyzed at several stages. Since the effects of GFP and [DG] were similar to those of 2[DG] RNA, only the latter is shown. The largest quantities of DG RNA decreased survival the greatest during gastrulation.
	FIG. 7. Developmental defects following overexpression of dystroglycan. Embryos injected with dystroglycan showed decreased survival between cleavage and neurulation. Many of the animals showed defects in gastrulation, including multiple sites of gastrulation, incomplete gastrulation, and exogastrulation. A number of defective embryos injected with dystroglycan RNA are shown next to a single normal sibling (top) that gastrulated and underwent successful neurulation. Calibration: 1 mm.