Differential gene expression at gestational days 14 and 16 in normal and nitrogen-induced hypoplastic murine fetal lungs with coexistent diaphragmatic hernia.

The purpose of this study was to identify differentially expressed genes in normal and nitrofen-induced hypoplastic lungs in fetal mice. Such genes may play a role in the regulation of lung development. CD-1 pregnant dams were gavaged with 25 mg of nitrofen on gestational day (Gd) 8 to induce pulmonary hypoplasia and diaphragmatic hernia (DH). Normal and nitrofen-treated fetuses were removed on Gd 14 and Gd 16. Lungs were examined in all nitrofen-exposed fetuses and only those that had developed severely hypoplastic lungs with coexistent diaphragmatic hernia were taken for molecular analyses. RNA was extracted from normal and nitrofen-treated lungs, reverse transcribed, and PCR-amplified using 48 combinations of anchor and arbitrary primers for each condition. The resulting cDNAs from normal and hypoplastic lungs were run on 6% polyacrylamide differential display gels. In Gd 14 lungs, we observed 10 differentially expressed cDNA bands, of which 6 were identified to be inhibited and 4 were reduced in the hypoplastic lungs compared to normal fetal lungs. From the Gd 16 lungs, a total of 29 differentially expressed cDNA bands were found, of which 11 were reduced, 4 were inhibited, 11 were enhanced, and 3 were induced in the hypoplastic compared to the normal lungs. All 39 differentially expressed cDNAs were cloned, sequenced, and identified through BLAST searches. Among the sequences that were identified, results were as follows: 1) Hypoplastic Gd 14 lungs had two unknown cDNA sequences with reduced/inhibited expressions, whereas one was a known sequence having 77% similarity with a promoter region regulating various cytokines such as IL-1, IL-2, and IL-11. The expression of this sequence was inhibited in the hypoplastic lungs. This sequence also had similarity to lipid-binding proteins. 2) On Gd 16, hypoplastic lungs had one cDNA sequence with reduced expression which had 82% similarity with thyroid hormone receptor gene exon 1 and two other cDNA sequences with enhanced expressions. One of these enhanced cDNA sequences in hypoplastic lungs had 98% similarity with the fibroblast growth factor receptor-3 gene, and the other was an unknown sequence. Northern blot hybridizations were performed to confirm the differential expression of the two sequences of interest, which were identified as thyroid hormone receptor and fibroblast growth factor (FGF) receptor-3. Overall, out of a total of 39 RT-PCR products (i.e., cDNAs), the abundance of which was altered by nitrofen, 6 were found to be homologous to sequences in Gen Bank through BLAST searches. These 6 sequences became the products of interest, and 3 of these 6 products were similar to previously identified genes. Our results may shed some light on regulatory aspects of lung development and open avenues for treatment of hypoplastic lungs and other respiratory problems in human neonates.