Differential display of genes in normal and hypoplastic fetal murine lungs.
To evaluate lung development at the level of gene expression, a comparison was made between normal and hypoplastic murine fetal lungs by using the mRNA differential display technique. We focused on altered gene expressions at gestational day (Gd) 19 in normal and hypoplastic murine lungs. Hypoplastic fetal lungs were created by gavaging pregnant mice at Gd8 with 25 mg of nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) [1]. Normal as well as gavaged mice were euthanized by an overdose of halothane at Gd19, and fetuses were removed by laparotomy. Lungs were excised and total RNA was extracted from normal and hypoplastic fetal lungs. Differential display technique was carried out using the RNAimage kit (GenHunter Corp., TN). Each reverse transcription and polymerase chain reaction (RT-PCR) was performed using one specific anchor primer H-T11M (5'HindIII-T11A/C/G3') and one arbitrary primer. We have used a total of 3 different anchor primers and 24 arbitrary primers for each sample. There were 20 differentially expressed cDNA clones, either induced, inhibited, enhanced, or reduced in hypoplastic fetal lungs as compared to normal. Of these, one clone (NL2) with reduced expression in Gd19 hypoplastic lungs had 100% homology with mouse nucleosome assembly protein I gene. Another clone (NT5) with induced expression in hypoplastic lungs is an unknown gene. Further, analyses of Northern blots of lungs from various gestational ages showed that the expression of NT5 was induced in hypoplastic lungs at Gd18, whereas in normal lungs it was first expressed at the neonatal stage and was increasingly expressed into adulthood. There is a single hybridized band, approximately 400 bp long for NT5 message. Dexamethasone induced expression of NT5 in normal Gd14 pseudoglandular lungs cultured for 7 days; however, different growth factors did not. Northern blot hybridization of multiple adult mice tissues showed NT5 expression in the lung, intestine, and spleen. The thyromimetic action of nitrofen and the interactive functional pathways of dexamethasone with T3 are known. Therefore, we suggest that the isolation and characterization of NT5 may provide valuable information on the regulation of lung development.
Zhang
L
L
Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033-0850, USA.
Zgleszewski
S E
SE
Cilley
R E
RE
Chinoy
M R
MR
D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005315:Fetal Diseases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D065630:Hernias, Diaphragmatic, Congenital; D008168:Lung / Q000002:abnormalities* / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D010647:Phenyl Ethers / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase
D017931:DNA Primers; D010647:Phenyl Ethers; D012333:RNA, Messenger; D003907:Dexamethasone; D012194:RNA-Directed DNA Polymerase; C007350:nitrofen