Anderson MJ et al. (1995), Erratic deposition of agrin during the formatio...

XB-ART-19552

Dev Biol 1995 Jul 01;1701:1-20. doi: 10.1006/dbio.1995.1191.

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Erratic deposition of agrin during the formation of Xenopus neuromuscular junctions in culture.

Anderson MJ , Shi ZQ , Grawel R , Zackson SL .

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In order to disclose the mechanism that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo, agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.

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Species referenced: Xenopus laevis
Genes referenced: avd sdc2

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	FIG. 1. Distribution of basal lamina components in frozen sections of mature Xenopus tadpole tails after indirect fluorescent staining with monoclonal antibodies and TRITC-labeled goat anti-mouse lgG. Note that stained basement membranes extended around the spinal cord (AC) and around all myotomal muscle fibers {D- F). Dense accumulations of 2AC2-stained HSPG (A), C3-stained agrin (B), and 9DC3 (C) are visible in the pial ECM that surrounds the central nervous system. In myotornal muscle 2AC2-stained HSPG (F), C3-stained agrin (D), and 9DC3 (E) were present in the basal laminae surrounding each myofiber. Agrin and HSPG were also concentrated at neuromuscular junctions (G) revealed by counterstaining with biotin-aBGT and FITC-labeled anti-biotin, here illustrated for the same field shown in F . Among these components, only HSPG is concentrated at myotendinous junctions, which are aligned with synapses at the myocommata separating individual myotomes (compare F, G). Similar myocommata are marked with arrows in D, E. Bar in C represents 30um in A- C; that in G corresponds to 20 um in D-G.
	FIG. 2. Extracellular matrix deposited on the substratum by epithelial cells present in Xenopus muscle cultures, as revealed by indirect staining for agrin with biotinylated antibody C3, FITC~ labeled avidin, and FITC-biotin-dextran (A), and for basal lamina HSPG with TRITC-labeled antibody 2AC2 (B). Note the elaborate web of ECM filaments containing nearly congruent distributions of both substances. A similar distribution was shared by 9DC3 (not shown). Bar in B is 20 um.
	FIG. 3. Association between epithelial agrin deposits (A) and Xenopus myotomal myocytes, shown in phase-contrast optics (B), after indirect staining with digoxygenin-C3 and TRITC-labeled mouse anti-digoxygenin (35/20) and goat anti-mouse IgG. Note that a dense carpet of agrin-containing ECM particles extends over the substratum around several neural tube-derived epithelial cells (see arrowheads in the phase-contrast image in B) to become closely associated with surrounding myocytes. The bar in B represents 30um.
	FIG. 4. Association of TRITC~stained agrin (A) with accumulations of FITC-stained AChR (B) on the upper surface a noninnervated myocyte (shown in phase-contrast optics in C) in a Xeno~ nervemuscle culture. Agrin was stained as in Fig. 3, while AChR were stained with biotin-aBGT, FITC-avidin, and FITC-biotin-dextran. Note the presence of AChR microaggregates colocalized with some (but not all) sites of agrin deposition. Agrin accumulations in A are on the curved upper cell surface that cannot be recorded entirely in a single focal plane. Bar inC represents 20 um.
	FIG. 5. Accumulation of agrin (A) at dense HSPG plaques (B) on the flat substratum-attached surface of a noninnervated myocyte in a Xenopus nerve-muscle culture. Agrin was decorated with biotin-C3, FITC-avidin, and FITC-biotin-dextran, while basal lamina HSPG was stained with TRITC-labeled antibody 2AC2. Note that agrin occasionally deposited at these sites of dense HSPG accumulation, when they were situated near the cell perimeter. Similar HSPG plaques in less accessible regions under myocytes were routinely stained by anti-HSPG antibodies, but almost never acquired detectable agrin deposits. The bar in B represents 20 um.
	FIG. 6. Accumulation of TRITC-stained agrin (A) and FITC-stained AChR (B) at a living Xenopus neuromuscular junction developing in culture. The staining procedure was similar to that described in Fig 4. The path of nerve-muscle contact is shown in the corresponding phase-contrast view in (C). Examples of such extensive codistribution between agrin and AChR were relatively uncommon during development in cell culture. Note also the nearby agrin-producing epithelial cell (see* in C). Bar inC represents 20um.
	FIG. 7. Absence of detectab)e agrin deposition at other synapses, again identified by counterstaining AChR as described for Fig 4. Agrin was stained as noted in Fig 3. Note that, while scattered agrincontaining ECM particles were visible over the culture substratum underneath the muscle cells (A), no agrin accumulation was detectable at the postsynaptic AChR clusters (shown in B) along the path of nerve-muscle contact (shown in phase-contrast in C). This culture had been fixed in formaldehyde after staining, and was mounted with agents that retard photobleaching (see Materials and Methods). The bar inC represents 20 um.
	FIG. 8. Variation in agrin deposition (A and B) at different neuromuscular junctions, despite their accessibility to staining with antibodies against basal lamina HSPG (C and D). Agrin was stained overnight with biotin-C3, followed by Cy3-streptavidin. During overnight agrin labeling HSPG was counterstained with FITC-labeled antibodies 2AA5, 2BA5, and 2BD5. Note the near congruence of agrin (A) and HSPG (C) accumulations along one of the two paths of synaptic nerve-muscle contact on a muscle cell (see arrowheads in phase-contrast image in E) and the virtual absence of detectable agrin (B) at another site of nerve-muscle contact (F) with similarly advanced postsynaptic differentiation. Since these sites were all accessible to ·staining with HSPG-reactive antibodies, agrin fails to accumulate at some synapses in culture. Bar in F represents 20um.
	FIG. 9. Survival of synaptic agrin accumulations after nerve degeneration in an older Xenapus nerve-muscle culture, as revealed by staining for agrin (A) and AChR (B) as in Fig 4. Note the persistence of synaptic agrin and AChR after the degeneration of the nerve fiber, which is no longer visible in phase contrast (C). Such examples show that agrin accumulations remain relatively stable and do not dissociate from synapses before the degeneration of the motor neurite. Note the agrin-generating epithelial cells (see * in C). Bar in C represents 20 Jl.ffi.
	FIG. 10. Variable deposition of salamander agrin from Ambystorna neural tube cells on Xenopus myocytes developing in culture. Ambystmna agrin (A and B) was revealed by staining with biotin-labeled 5Bl followed by Cy3-streptavi.din, while HSPG (C, D) was counterstained with FITC-conjugated antibodies 2AA5, 2BA5, and 2BD5. Note that some sites of synaptic nerve-muscle contact (see arrows in phase-contrast images in E and F) showed extensive HSPG (C) and agrin (A) accumulation, while other synaptic HSPG accumulations (D) were virtually devoid of detectable agrin (B). Note also the scattered accumulations of A mhystoma agrin away from the regions of nerve-muscle contact on the same cells. Bars in E and F represent 20 .um.
	FIG. 11. Deposition of agrin-containing ECM by salamander neuroendothelial cells in cultures of Xenopus myotomal and A mbystoma neural tube cells. The staining method for salamander agrin with antibody 5Bl (A) was similar to that noted in Fig. 5, revealing agrin associated with one of the islets of neuroendothelial cells (see phase-contrast image in B) that contaminate chimeric nerve-muscle cultures. Agrin secreted by such cells may bind to basal laminae on nearby myoeytes, including those at developing neuromuscular junctions. Bar in B is 20 um.