Gene expression profiling identifies regulatory pathways involved in the late stage of rat fetal lung development

Abstract

Fetal lung development is a complex biological process that involves temporal and spatial regulations of many genes. To understand the molecular mechanisms of this process, we investigated gene expression profiles of fetal lungs on gestational days 18, 19, 20, and 21, as well as newborn and adult rat lungs. For this analysis, we used an in-house rat DNA microarray containing 6,000 known genes and 4,000 expressed sequence tags (ESTs). Of these, 1,512 genes passed the statistical significance analysis of microarray (SAM) test; an at least twofold change was shown for 583 genes (402 known genes and 181 ESTs) between at least two time points. K-means cluster analysis revealed seven major expression patterns. In one of the clusters, gene expression increased from day 18 to day 20 and then decreased. In this cluster, which contained 10 known genes and 5 ESTs, 8 genes are associated with development. These genes can be integrated into regulatory pathways, including growth factors, plasma membrane receptors, adhesion molecules, intracellular signaling molecules, and transcription factors. Real-time PCR analysis of these 10 genes showed an 88% consistency with the microarray data. The mRNA of LIM homeodomain protein 3a (Lhx3), a transcription factor, was enriched in fetal type II cells. In contrast, pleiotrophin, a growth factor, had a much higher expression in fetal lung tissues than in fetal type II cells. Immunohistochemistry revealed that Lhx3 was localized in fetal lung epithelial cells and pleiotrophin in the mesenchymal cells adjacent to the developing epithelium and blood vessel. Using GenMAPP, we identified four regulatory pathways: transforming growth factor-beta signaling, inflammatory response, cell cycle, and G protein signaling. We also identified two metabolic pathways: glycolysis-gluconeogenesis and proteasome degradation. Our results may provide new insights into the complex regulatory pathways that control fetal lung development.

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

“…Our previous real-time PCR and Western blot results have shown that PTN mRNA and protein expression levels were high at the late stage of fetal lung development (19). The current detailed immunohistochemical studies of fetal lungs at different stages confirmed that PTN was mainly expressed in mesenchymal cells surrounding the epithelium at E18 to E20.…”

“…PTN is expressed in fetal lungs (10) and lung cancer cell lines (18). Our DNA microarray analysis of fetal lungs reveals that ptn is highly expressed during the late stage of fetal lung development (19). This is confirmed by real-time PCR and Western blot.…”

“…The primers were designed using Primer Express software (Applied Biosystems, Foster City, CA): PTN, 5Ј-ATACCAGC-AGCAACGTCGAAA (forward) and 5Ј-GCACACACTCCAT-TGCCATT (reward); Dlk1, 5Ј-GTGAAGAACCATGGCAGT-GTGT (forward) and 5Ј-GACAGTCCTTTCCAGAGAA-TCCA (reward); 18 S rRNA 5Ј-TCCCAGTAAGTGCGGGT-CATA (forward) and 5Ј-CGAGGGCCTCACTAAACCATC (reward). Real-time PCR was performed on an ABI 7500 system using SYBR Green I detection as previously described (19). To eliminate the effects of primer dimers, we included a data acquisition step at a temperature of 2ϳ5°C lower than the annealing temperature of the products.…”

“…have previously shown that PTN mRNA and protein are highly expressed in the late stage of fetal lung development by analyzing fetal lungs at different gestational days as well as the lungs of newborn and adult rats using DNA microarray analysis, realtime PCR, and Western blots (19). We further investigated temporal and spatial expression of PTN during rat lung development by ABC immunostaining.…”

Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development

“…Mouse Lhx3a and Lhx3b mRNAs have distinct temporal expression profiles during development and are differentially expressed in cell lines that model the specialized pituitary cell types (Zhadanov et al, 1995a;Sloop et al, 1999;Sloop et al, 2001a;West et al, 2004). During rodent embryogenesis, Lhx3 mRNAs can be detected in the ventral spinal cord, the pons, the medulla oblongata, the pineal gland, the lungs, and in the developing and established anterior/intermediate pituitary gland (Seidah et al, 1994;Bach et al, 1995;Zhadanov et al, 1995a;Meier et al, 1999;Weng et al, 2006). Similar expression patterns are seen during human development (Sloop et al, 1999;Schmitt et al, 2000;Sobrier et al, 2004).…”