Identification of cell-specific messenger ribonucleic acids in oxytocinergic and vasopressinergic magnocellular neurons in rat supraoptic nucleus by single-cell differential hybridization.
Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system synthesize high levels of the peptides oxytocin (OT) and vasopressin (VP) in separate cells. We used RT-PCR amplification of the RNA from single-cells dissected from supraoptic nuclei of lactating rats to produce cDNAs from identified OT or VP MCNs, which were used to construct OT- and VP-MCN-specific cDNA libraries. These cDNA libraries were then screened using labeled probes from the OT- and VP-cells' amplified cDNAs. Differentially hybridized colonies were isolated and characterized by slot blot hybridization, Southern blot hybridization, DNA sequencing, and in situ hybridization histochemistry. Using this approach, several novel cell-specific mRNAs were identified in the MCNs. One cell-specific clone, phosphofructokinase-C, was isolated from the OT-cell library, and five cell-specific clones were isolated from the VP-cell library. These were identified as paternally expressed gene (Peg)5/neuronatin, metallothionein III, Peg3, synaptotagmin V, and a 3'-phosphoadenosine 5'-phosphosulfate synthase 2-related mRNA. None of these genes would have been predicted to be differentially expressed in OT and VP MCNs, based on our current knowledge; and hence, this single cell differential gene expression approach has begun to further define the MCN phenotypes by identifying selectively expressed molecules in them.
Yamashita
Mitsuo
M
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Glasgow
Eric
E
Zhang
Bing-Jun
BJ
Kusano
Kiyoshi
K
Gainer
Harold
H
D000818:Animals; D015139:Blotting, Southern; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D018392:Genomic Imprinting; D007031:Hypothalamus / Q000196:embryology; D017403:In Situ Hybridization; D007774:Lactation; D009474:Neurons / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010121:Oxytocin / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013495:Supraoptic Nucleus / Q000737:chemistry*; D014667:Vasopressins / Q000235:genetics* / Q000502:physiology
D018076:DNA, Complementary; D012333:RNA, Messenger; D014667:Vasopressins; D010121:Oxytocin