Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus.
The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.
Ghorbel
Mohamed T
MT
The Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, United Kingdom.
Sharman
Greig
G
Hindmarch
Charles
C
Becker
Kevin G
KG
Barrett
Tanya
T
Murphy
David
D
D000818:Animals; D003681:Dehydration / Q000235:genetics*; D005786:Gene Expression Regulation; D015723:Gene Library; D017403:In Situ Hybridization; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D010286:Paraventricular Hypothalamic Nucleus / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013495:Supraoptic Nucleus / Q000166:cytology / Q000378:metabolism*
D012333:RNA, Messenger