Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.
NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.
Varju
P
P
Department of Neural Cell Biology, Institute of Experimental Medicine, Budapest, Hungary.
Schlett
K
K
Eisel
U
U
Madarász
E
E
D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D017931:DNA Primers; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D016548:Prosencephalon / Q000166:cytology; D012333:RNA, Messenger / Q000032:analysis; D017470:Receptors, Glutamate / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D000970:Antineoplastic Agents; D017931:DNA Primers; D018690:Excitatory Amino Acid Agonists; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; D012333:RNA, Messenger; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D016159:Tumor Suppressor Protein p53; C108929:glutamate receptor delta 1; D014212:Tretinoin; D016202:N-Methylaspartate; D002118:Calcium