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Dev Biol
1999 Jan 01;2051:22-32. doi: 10.1006/dbio.1998.9104.
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Overexpression of agrin isoforms in Xenopus embryos alters the distribution of synaptic acetylcholine receptors during development of the neuromuscular junction.
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Synapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules is agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin's synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA into Xenopus laevis embryos at the one- or two-cell stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100-200%. Rat agrin protein was colocalized with AChR aggregates in innervated regions of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation.
FIG. 1. Innervation of myotomal muscles in the Xenopus embryo,
stage 31. (a) Diagram of stage 31 embryo showing myotomal
muscles (shaded). (b) Innervation of both ends of muscle cells by
motor axons is indicated by arrows. Bands of synapses from
adjacent myotomes are closer together than indicated and cannot
always be distinguished by light microscopy.
FIG. 2. GFP is a marker for successful RNA injection and indicates the extent of diffusion and translation of injected RNA and the
persistence of the protein product. One blastomere of a two-cell stage embryo was injected with GFP RNA. GFP fluorescence is seen in
most cells on the right (upper) half of this tadpole (stage 35/36, 2.1 days of development).
FIG. 3. Rat agrin is colocalized with AChR aggregates in confocal images of the innervated region of myotomal muscles. Embryos were
injected at the one-cell stage with rat agrin RNA (neuronal isoform 4,8) in the absence of GFP RNA. Whole mounts of fixed stage 31 (1.6
day) embryos were stained as described under Methods. (A) Rat agrin immunofluorescence (FITC-labeled antibody). (B) AChR aggregates
labeled with rhodamine–a-bungarotoxin. (C) Superimposed images from A and B showing colocalized rat agrin and AChR aggregates
(yellow). Arrows indicate synaptic regions adjacent to the myotomal septum. Bar, 20 mm.
FIG. 4. Injected agrin RNA persists throughout embryogenesis.
Rat agrin RNA was detected in RNA-injected embryos by RT-PCR.
(a) Rat agrin sequence. (b) Ornithine decarboxylase sequence from
Xenopus as an internal control. Lanes: 1, no RNA control; 2â5,
embryos injected with agrin RNA (10 ng) at the one-cell stage and
analyzed at stages 2, 9, 32, and 35/36 (2 hâ2.1 days); 6 and 7,
uninjected control embryos (stages 9 and 32).
FIG. 5. Overexpression of neuronal and muscle agrin isoforms
increases AChR aggregate area in innervated regions of myotomal
muscles. Xenopus embryos were injected at the one-cell stage with
RNAs encoding GFP (A), rat neuronal agrin (4,8) and GFP (B), or rat
muscle agrin (0,0) and GFP (C). AChRs were labeled with
rhodamineâa-bungarotoxin and images were obtained by confocal
microscopy at stage 31 (1.6 days). Overlaid series of four images,
taken at 1-mm intervals, are shown. Bands of AChR aggregates
(bright clusters) running horizontally across the images represent
innervated regions at the ends of two adjacent myotomes; muscle
cells run vertically, parallel to the anteriorâposterior axis of the
embryo. The images shown were taken from the same experiment;
AChR aggregate areas were 0.61% (A), 2.94% (B), and 2.16% (C) of
area imaged. Bar, 20 mm.
