Synapse formation and agrin expression in stratospheroid cultures from embryonic chick retina.
Stratospheroids are three-dimensional cellular spheres which develop in vitro through the proliferation and differentiation of retinal neuroepithelial precursor cells. We investigated synapse formation in stratospheroids by analyzing the development of aggregates of synapse-associated molecules and of electron microscopically identifiable synaptic specializations. Our results show that the first aggregates of the GABA(A) receptor, the glycine receptor, and gephyrin appear in the inner plexiform layer after 8 days in culture simultaneously with the development of the first active zones and postsynaptic densities. In contrast, presynaptic molecules including synaptophysin could be detected in the inner plexiform layer before synaptogenesis, suggesting functions for these molecules in addition to neurotransmitter exocytosis at mature synapses. Similar to the retina in vivo, synapses were not found in the nuclear layers of stratospheroids. We also analyzed the isoform pattern, expression, and distribution of the extracellular matrix molecule agrin, a key regulator during formation, maintenance, and regeneration of the neuromuscular junction. In stratospheroids, several agrin isoforms were expressed as highly glycosylated proteins with an apparent molecular weight of approximately 400 kDa, similar to the molecular weight of agrin in the retina in vivo. The expression specifically of the neuronal isoforms of agrin was concurrent with the onset of synaptogenesis. Moreover, the neuronal agrin isoforms were exclusively found in the synapse-containing inner plexiform layer, whereas other agrin isoforms were associated also with the inner limiting membrane and with Müller glial cells. These results show that synapse formation is very similar in stratospheroids and in the retina in vivo, and they suggest an important role for agrin during CNS development.
Hering
H
H
Department of Neuroanatomy, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, Frankfurt, D-60528, Germany.
Kröger
S
S
D018171:Agrin / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D005109:Extracellular Matrix / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009457:Neuroglia / Q000378:metabolism*; D009924:Organ Culture Techniques; D020033:Protein Isoforms / Q000378:metabolism; D018079:Receptors, GABA / Q000378:metabolism; D012160:Retina / Q000196:embryology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013569:Synapses / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism
D018171:Agrin; D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D018079:Receptors, GABA; D016708:Synaptophysin; D014746:Vimentin; C073441:gephyrin