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Am J Physiol Lung Cell Mol Physiol 291: L1027-L1037, 2006. First published June 23, 2006; doi:10.1152/ajplung.00435.2005
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Gene expression profiling identifies regulatory pathways involved in the late stage of rat fetal lung development

Tingting Weng, Zhongming Chen, Nili Jin, Li Gao, and Lin Liu

Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma

Submitted 11 October 2005 ; accepted in final form 11 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

DNA microarray; cell differentiation

FETAL DEVELOPMENT OF THE PULMONARY system is a complex and important morphogenetic process. The lung is among the last of the fetal organs to mature functionally. Development of this complex air-exchange interface can be divided into five stages (6). In the embryonic stage (days 0–13), the lung originates as an outpouch from the ventral wall in the primitive esophagus and grows caudally into the splanchnic mesoderm to form the right and left buds. The terminal buds then dichotomously divide until the lung forms a glandlike structure. During the pseudoglandular stage (days 13–18), the bronchial buds repeatedly branch into the mesenchymal tissue to form the conductive airways of the lung. At this stage, there is little lumen in the epithelial tubes. In the canalicular stage (days 18–20), the lung grows considerably, with multiple generations of bronchioles. Capillaries are closely arranged with the terminal and respiratory bronchioles. The columnar epithelial cells undergo morphological changes to become cuboidal epithelial cells. In the saccular stage (day 20 to full term), the terminals of bronchioles become saccular, with the formation of alveolar ducts and air sacs. Capillaries bulge into terminal sacs to establish blood-air barriers. In the alveolar stage (from full term to adult), the alveolar-capillary barrier becomes progressively thinner, with an increasing capacity for gas exchange. The number and size of capillaries and alveoli increase markedly.

Coordinated regulation of signaling molecules and their pathways is required for fetal lung development (7, 41, 63, 80). These molecules include, for example, retinoic acid, fibroblast growth factors, transforming growth factors (TGF), and sonic hedgehog. Complex interactions occur between epithelial, mesenchymal, and extracellular matrix components. Through temporal and spatial regulations of these molecules and their interactions, the stem cells proliferate and differentiate into ≥40 distinct cell lineages, resulting in formation of the whole lung tissue.

DNA microarray has been used for gene expression profiling of pulmonary diseases, including lung cancers (3), emphysema (16), and hyperoxia or ventilator-induced lung injury (35, 53). This approach has been used previously to study mouse fetal lung development (2, 38). However, DNA microarray analysis of rat fetal lung development has not been reported. Gene expression in response to hypoxia differs among species (19). Our main objective in this study was to examine gene expression profiles during the late stage of rat fetal lung development. We focused on the late stage of fetal lung development because our research interests involve alveolar epithelial cell proliferation and differentiation. We used a DNA microarray representing 10,000 known rat genes and expressed sequence tags (ESTs) to profile gene expression of fetal lungs on gestational days 18, 19, 20, and 21 and newborn and adult rat lungs. Clustering analysis identified an important gene cluster involving lung development. Real-time PCR and Western blot confirmed the changes in gene expression of selected genes. Several pathways were also identified using GenMAPP and literature review.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Microarray slide preparation. The DNA microarray slides were prepared in-house using the Pan-Rat 50-mer 10,000K oligonucleotide set (MWG Biotech, High Point, NC), which includes 6,221 known rat genes, 3,594 rat ESTs, and 169 Arabidopsis-negative controls. The oligonucleotides were diluted to 25 µM with 3x saline-sodium citrate (SSC) and printed on epoxy-coated slides (CEL Associates, Pearland, TX) by a microarrayer (OmniGrid 100, GeneMachine, San Carlos, CA). The samples were spotted on each slide in triplicate using 16 ChipMaker Micro Spotting Pins (Telechem International, Sunnyvale, CA). This resulted in three identical 18 x 18 mm blocks on each slide, which allowed us to hybridize six samples on a single slide (Fig. 1A). The diameter of each spot was 120 µm, and the distance between two spots was 180 µm. The printed slides were incubated in 65% humidity for 48 h, dried, and kept at room temperature. Slide quality was assessed using SYBR Green II staining. No autofluorescence was found when the blank slides were scanned.


Figure 1
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  		Fig. 1. DNA microarray slides and experiment design. A: slides printed in-house with 3 identical blocks, each containing 10,000 rat genes, which allowed us to hybridize 6 samples on a single slide. B: loop design for microarray hybridization study. Each RNA sample was divided into 2 parts: one was labeled with Alexa 546 and the other with Alexa 647. Two cDNAs with different dyes were paired and hybridized to 1 block. Arrows correspond to hybridizations between 2 RNA samples: blunt end labeled green, and arrow end labeled red. Number 4 on the arrow indicates 4 biological replicate hybridizations. Opposite directions of the 2 arrows represent dye flipping. D18, D19, D20, and D21, fetal lungs at gestational days 18, 19, 20, and 21; NB, newborn lung; AD, adult lung.

 
Isolation of rat fetal lungs. We chose six lung tissues, with a focus on the late stage of fetal lung development, for our DNA microarray experiments: four fetal lungs on gestational days 18, 19, 20, and 21 (D18, D19, D20, and D21) and newborn (NB) and adult (AD) lungs. The Oklahoma State University Animal Care and Use Committee approved all the animal protocols used in the study. For day 18–day 21 fetal lungs, timed-pregnant Sprague-Dawley rats were killed by CO2 anesthesia. After the whole uterine horns were exposed and excised, the fetuses were removed and the fetal lungs were harvested. The isolated lungs were placed in ice-cold DMEM (Invitrogen, Carlsbad, CA). Newborn and adult rat lungs were collected from day 0 (neonatal) and 2-mo-old rats. Lung epithelial type II cells were isolated from fetal or newborn lungs according to the method of Fraslon-Vanhulle et al. (15). The purity and viability of the cells exceeded 90%, as assessed by alkaline phosphatase staining and trypan blue exclusion. Adult lungs and alveolar type I and type II cells were isolated as previously described (9). Total RNAs were extracted from the lung tissues or cells with TriReagent (Molecular Research Center, Cincinnati, OH).

DNA microarray hybridization. A loop design was used for our microarray hybridization to compare changes in gene expression between two adjacent time points (Fig. 1B). Because fetal lung development is a sequential event, a loop design has some advantages over a reference design. In a reference design, indirect comparison of adjacent time points through a common reference results in accumulated errors. In a loop design, direct comparison between two adjacent time points significantly increases the efficiency and accuracy of the experiments. Sixteen hybridizations were performed for each sample (4 biological replications, dye flip and hybridizations with 2 adjacent time points). Total RNAs were divided into two aliquots; each (10 µg) was reverse transcribed to cDNA using the modified oligo(dT) dye-specific primers from the 3DNA Array 50 kit (Genisphere, Hatfield, PA). The cDNA was purified with Microcom YM-30 columns (Millipore, Billerica, MA) and mixed with 2x hybridization buffer (50% formamide, 6x SSC, and 0.2% SDS). The cDNA concentrations were adjusted to 300 ng/µl. Five microliters of cDNAs from two adjacent time points were mixed and hybridized to one of three blocks on a microarray slide for ≥24 h at 42°C. After the unbound cDNAs were washed away with 2x SSC buffer (Sigma-Aldrich, St. Louis, MO), the slides were further incubated with 3 DNA capture reagents labeled with green (Alexa 546) or red (Alexa 647) dye for 2–3 h. Then the slides were washed again and scanned (ScanArray Express, Perkin Elmer, Boston, MA). The laser power and photomultiplier tube were adjusted so that ~5% of the spots were saturated.

Microarray data analysis. The scanned images were first inspected visually to uncover systematic biases or macroartifacts. Because of weak or bad hybridizations, 8 of 48 hybridizations were discarded. The signal intensity for each spot was obtained from the scanned slides using GenePix 5.0. The ratios between adjacent time points were normalized by LOWESS normalization using RealSpot software, which was developed in our laboratory (10). A quality index (QI) for each spot, based on signal intensity and signal-to-background ratio, was exported from RealSpot. The mean QI values were calculated by Excel. Any spots with mean QI < 1 were filtered. One-class significance analysis of microarray (SAM) statistical test was applied to the remaining genes using a cutoff q value of <0.01 (http://www.stat.stanford.edu/~=tibs/SAM/). The genes that passed the SAM test were further filtered using a fold change cutoff value of <2 and a coefficient of variation cutoff value of >0.5. The final genes were clustered by K-means clustering using Cluster and TreeView (http://rana.lbl.gov/index.htm?stanford/). For each cluster, the expression patterns were analyzed and the major functional categories were assigned using Gene Ontology (http://www.geneontology.org/). Biological pathways were identified using GenMAPP (http://www.genmapp.org/), a recently developed tool for visualizing expression data in the context of biological pathways. The log2(ratios) of day 18 to adult (D18/AD) and newborn to adult (NB/AD) were used for comparison of prenatal, postnatal, and adult lungs.

Real-time PCR. The gene verification was carried out by real-time PCR on the same RNA samples used for the DNA microarray hybridization. Total RNAs were treated with RNase-free DNase (Ambion, Austin, TX) to remove DNA contamination and reverse-transcribed into cDNA using random hexamers and Maloney's murine leukemia virus reverse transcriptase (Invitrogen). Real-time PCR primers were designed using Primer Express software (Applied Biosystems, Foster City, CA). The length of each primer was 20–25 bp, with a melting temperature of 58–60°C (Table 1). Real-time PCR was performed on an ABI 7500 system using SYBR Green I detection (Qiagen, Valencia, CA), as previously described (79). To eliminate the effects of primer dimers, we included a data acquisition step at 2–5°C lower than the annealing temperature of the products. After the amplification, a dissociation curve was generated to check the specificity of the amplification. A standard curve was constructed on the basis of serial dilutions of a standard (107 to 10 copies) and corresponding threshold cycles. Standards were obtained by normal PCR and purified from agarose gel. The copy number was calculated from the standard curve and normalized to 18S rRNA.


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  		Table 1. Real-time PCR primers

 
Immunohistochemistry. The isolated fetal lungs were briefly washed with deionized water and immediately fixed with 4% formaldehyde in PBS. After 24 h of incubation, the lungs were rinsed with PBS, dehydrated in graded alcohol and xylene, and embedded in paraffin (60°C). Paraffin-embedded lungs were sectioned (4 µm) and placed on polylysine-coated glass slides. The lung sections were boiled for 30 min in 20 mM citrate buffer (pH 6.0) for antigen retrieval. The sections were permeabilized with 0.3% Triton X-100 in PBS for 15 min and blocked with 10% fetal bovine serum in PBS for 30 min and then incubated in goat anti-pleiotrophin (Ptn) or rabbit anti-LIM homeodomain protein 3a (Lhx3) antibodies (1:100 dilution; Abcam, Cambridge, MA) overnight at 4°C. The slides were washed and then incubated with biotinylated anti-goat or anti-rabbit antibody for 30 min and for an additional 30 min with ABC reagent (Vector Laboratories, Burlingame, CA). The slides were developed with 3,3'-diaminobenzidine until the desired staining appeared. Finally, the slides were mounted with antifade medium (5% n-propyl gallate and 80% glycerol in PBS) and examined using a Nikon Eclipse E600 microscope.

Western blot. The lung tissues were homogenized in lysis buffer (10 mM Tris·HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) on ice. Protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA). Total proteins were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. The blot was stained by Ponceau S to check transfer efficiency. The membrane was blocked with 5% dry milk in Tris-buffered saline for 1 h, washed with Tris-buffered saline containing 0.05% Tween 20, and incubated with goat anti-Ptn antibody (1:1,000 dilution) or mouse anti-beta-actin antibody (1:1,000 dilution; Bio-Rad) overnight at 4°C. The membrane was washed again and incubated with horseradish peroxidase-conjugated anti-goat or anti-mouse IgG (1:2,000 dilution) for 1 h. Finally, the membrane was developed with enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) and exposed to X-ray film.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
DNA microarray data analysis. To identify the genes that changed during the late stage of fetal lung development, we performed DNA microarray (10,000 genes) analysis. Six samples (D18, D19, D20, D21, NB, and AD) were arranged for hybridization using a loop design (Fig. 1B). There were a total of 48 hybridizations (4 biological replications and dye-flip): 8 were excluded because of poor images and 40 were used for further data analysis [D18/AD (n = 6), D19/D18 (n = 7), D20/D19 (n = 6), D21/D20 (n = 8), NB/D21 (n = 5), and AD/NB (n = 8)]. The data were subjected to LOWESS normalization. Bad or weak spots were removed by filtration using a cutoff QI < 1. Of these 10,000 genes, 1,512 (928 known genes and 584 ESTs) passed the one-class statistical SAM test using a false discovery rate (q value) of <0.01. Additional criteria were used to identify 583 genes (402 known genes and 181 ESTs): a fold change of ≥2 and a coefficient of variation of <0.5. The gene names, gene identifications, average log2[(ratio)s], Gene Ontology (GO) identifications, and GO terms are listed in supplemental Table 1 (see online version of this article). The microarray dataset is available at GEO database (http://www.ncbi.nlm.nih.gov/geo; GSE2160 [NCBI GEO] ).

Cluster analysis and major functional categories. We used K-means cluster analysis to cluster the 583 above-mentioned genes into 5–20 nodes. We found that the best approach was to group the genes into seven clusters on the basis of their expression patterns (Fig. 2A). Of the 583 genes, 272 were annotated by GO (http://www.geneontology.org). The percentage of major functions for each cluster was calculated and is shown in Fig. 2B.


Figure 2
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  		Fig. 2. Tree view and functional categories for clustered genes. A: K-means clustering analysis on 583 genes, which significantly changed between ≥2 time points (P < 0.01, fold change ≥2, coefficient of variation ≤ 0.5) using cluster and tree view. Genes were clustered into 7 clusters. Each row corresponds to 1 gene, and each column corresponds to 1 log2(ratio). Numbers in parentheses adjacent to ratios represent number of hybridizations used for data analysis. Red, upregulation; green, downregulation; black, no change. Brightness of the color represents the value of the ratio. B: expression patterns and functional analysis of each cluster. The 7 clusters identified from K-means clustering analysis were plotted as log2(ratio) vs. time. Distribution of major functional categories for biological process terms are shown in pie charts. The log2(ratio) is each time point vs. adult. Values are means ± SD.

 
GenMAPP. To identify signaling pathways, metabolic pathways, and other functional groups that may be involved in fetal lung development, we used GenMAPP to visualize the changes in gene expression. The log2(D18/AD) and log2(NB/AD) were chosen to represent prenatal, postnatal, and adult lungs. The data sets were imported into GenMAPP. Four major regulatory pathways (TGF-beta signaling, inflammatory response, cell cycle, and G protein signaling) and two metabolic pathways (glycolysis-glycogenesis and proteasome degradation) were identified. These pathways are shown in supplemental Fig. 1 (see online version of this article).

Real-time PCR validation. We next focused on cluster 5, the "differentiation cluster," for further studies. Gene expression in this cluster increased in the canalicular stage (D18–D20) and then decreased (D20 to AD). This group consists of 5 ESTs and 10 known genes. Among the 10 known genes, 8 were directly or indirectly involved in development or cell differentiation. Table 2 shows the major functions and the cellular locations of these 10 known genes.


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  		Table 2. Cluster 5 genes and their functions

 
We attempted to verify the microarray data from the 10 known genes in cluster 5 using absolute quantitative real-time PCR. The copy number was obtained from the standard curve constructed from the purified PCR products and normalized to 18S rRNA. The expression levels of these genes are shown in Fig. 3A. Delta-like homolog Drosophila 1 (Dlk1) had the highest expression, followed by glycogenin, Ptn, and alcohol dehydrogenase-3 (Adh3). In contrast, expression of Lhx3 was the lowest. The log2(ratio) between adjacent time points was calculated and compared with the microarray data. As shown in Fig. 3B, 88% of the log2(ratios) from real-time PCR were consistent with the microarray data, even though absolute changes varied. In general, fold change of real-time PCR was greater than that of the microarray, probably because of the difference between the two methods (solid-state vs. solution hybridization).


Figure 3
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  		Fig. 3. mRNA expression of cluster 5 genes during fetal lung development: comparison of microarray with real-time PCR. Total RNA was extracted from fetal lungs on gestational days 18–21 and from newborn and adult lungs and reversed transcribed to cDNA. mRNA levels were determined by real-time PCR, and data were normalized to 18S rRNA. A: relative mRNA expression levels of cluster 5 genes. B: comparison of microarray and real-time PCR: D18/D19 (1), D19/D20 (2), D20/D21 (3), D21/NB (4), NB/AD (5), and AD/D18 (6). Values are means ± SE; n = 8 (4 animal groups and each assay performed in duplicate) for microarray and real-time PCR.

 
mRNA expression of Ptn and Lhx3 in fetal lung cells. The mRNA levels of Ptn and Lhx3 in the isolated lung cells were determined using real-time PCR. Ptn was highly expressed in fetal lung tissues (D18, D19, D20, D21, and NB), but there was essentially no expression in adult lung tissues (AD; Fig. 4A). Ptn mRNA expression in isolated alveolar epithelial type II cells from fetal lungs on days 18 and 19 and newborn and adult lungs was negligible. Also the mRNA of Ptn was not found in alveolar epithelial type I cells or macrophages isolated from adult lungs. On the other hand, mRNA expression of Lhx3 was high in type II cells isolated from fetal lungs on days 20 and 21 and newborn lungs (Fig. 4B) but low in type II cells isolated from fetal lungs on days 18 and 19 and adult lungs and low in type I cells isolated from adult lungs and in macrophages. Relatively low expression of Lhx3 was observed in fetal and adult lung tissues, indicating its specific expression in fetal type II cells.


Figure 4
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  		Fig. 4. Real-time PCR analysis of pleiotrophin (Ptn) and LIM homeodomain protein 3a (Lhx3) in fetal lung cells. Total RNA was extracted from lung tissues and reverse transcribed to cDNA. mRNA abundance of Ptn (A) and Lhx3 (B) was determined by real-time PCR in adult alveolar type I cells (TI-AD), adult alveolar type II cells (TII-AD), fetal epithelial type II cells on gestational days 18–21 (TII-D18 to TII-D21), newborn alveolar type II cells (TII-NB), adult microphages (M-AD), fetal lung tissue on gestational days 18–21, and newborn and adult lung tissue. Values are means ± SE; n = 8 (4 biological preparations, each assay performed in duplicate). *P < 0.05 vs. AD or TII-AD.

 
Ptn and Lhx3 protein expression. We used Western blot to quantify Ptn protein expression in fetal lung tissues. Ptn protein was significantly upregulated in fetal lungs on days 20 and 21 and newborn lungs compared with fetal lungs on days 18 and 19. However, very little Ptn protein was found in adult lungs (Fig. 5A).


Figure 5
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  		Fig. 5. Protein expression and cellular location of Lhx3 and Ptn. A: Western blot analysis of Ptn in fetal lungs on gestational days 18–21 and in newborn and adult lungs. beta-Actin was used as loading control. B: D20 fetal lung tissue sections stained using anti-Lhx3 (a and c) and anti-Ptn (b and d) antibodies. Panels c and d are enlarged fields enclosed in boxes of panels a and b, respectively. Lhx3 was located in the nucleus of columnar epithelial cells (arrow). Endothelial cells (*) and mesenchymal cells (arrow) around the epithelium and blood vessels stained positive for Ptn. Results are representative of lung tissue sections from 4 animals; all show similar staining.

 
To determine the cellular localization of Ptn and Lhx3 proteins in fetal lungs, we performed immunohistochemistry using the ABC reagent. Lhx3 was primarily expressed in the columnar cells of the developing airways. The positive signal was located in the nucleus and showed a purplelike color on counterstaining with hematoxylin (Fig. 5B). Ptn was located in the mesenchyme adjacent to the developing epithelium and endothelium (Fig. 5B). Some staining was noted on the endothelial cells. No positive staining was found in the negative control without primary antibodies (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The gene expression profiles of fetal lung development in mice have been reported elsewhere (2, 38). However, the present study is the first to investigate rat fetal lung development using DNA microarray and is different from the previous studies in several ways. 1) We used a loop design to directly compare two adjacent time points. Therefore, the number of accumulated errors was decreased. 2) We performed 16 hybridizations for each sample and, thus, had a relatively large number of biological and technical replications. As a result, the accuracy of the experiments was increased. Indeed, real-time PCR verified 88% of the selected genes. 3) We focused on the late stage of fetal lung development (D18 to NB) to identify the genes involved in cell proliferation and differentiation. This also enabled us to perform a detailed study during this stage with a fetal lung sample from each day. 4) Using cluster analysis combined with GO, we were able to identify a novel differentiation cluster composed of 10 known genes and 5 ESTs involved in cell proliferation, cell differentiation, and development.

Some genes identified in our study have expression patterns similar to those in mice, such as Dlk1, glyceraldehyde-3-phosphate dehydrogenase, and a number of genes in the tyrosine kinase family. However, a significant number of the genes identified in our study are different from those identified in the mouse studies (2, 38), perhaps because of species difference. Comparative studies revealed different gene profiles of the lungs in response to hypoxia between the rat and the mouse (19). Another possible reason is the difference in DNA microarray platforms and data analysis.

During fetal lung development, cell proliferation and cell population undergo dynamic changes. The rate of cell proliferation increases at the pseudoglandular stage and declines at the canalicular and saccular stages. However, the percentage of dividing interstitial cells decreases from gestational day 17 to day 20 and then markedly increases on day 20. An opposite trend is seen for epithelial cells (1). This raises the possibility that some of the changes in gene expression may reflect the difference in cell population in the developing lung. Although we cannot rule out the possibility, the changes in gene expression in cluster 5 seem not to correspond to cell population, at least in the fetal stages (1). The change in Ptn mRNA between prenatal and adult stages is tremendous (>100-fold) and is probably not due to fewer mesenchymal cells in the adult lung.

Two genes, Ptn and Lhx3, were further characterized at mRNA and protein levels. Ptn and Lhx3 were located in fetal lung fibroblasts and epithelial cells, respectively. Ptn, an 18-kDa heparin-binding cytokine (13), is highly expressed in the late stage of embryogenesis (17, 33, 77) and postnatally in the nervous system (66). It shares 50% sequence homology with midkine, which may play a role in lung morphogenesis. In contrast to Ptn, midkine is primarily localized in epithelial cells of mouse lung from gestational day 18 to birth. Its expression is high from gestational days 13–16 but low from day 18 to birth and is undetectable in adult lungs (55). Midkine expression is stimulated by retinoic acid but inhibited by glucocorticoid (26).

Ptn acts through two cell surface receptors, anaplastic lymphoma kinase (ALK) and the protein tyrosine phosphatase receptor RPTPZ1. ALK is the receptor tyrosine kinase that is expressed in the developing nervous systems and some tumor cells (25, 44). On binding with Ptn, ALK activates the Ras-MAPK and the phosphatidylinositol 3-kinase-Akt signaling pathways (54, 70), leading to the stimulation of cell proliferation and the inhibition of apoptosis.

Ptn also regulates beta-catenin phosphorylation through RPTPZ1. In the absence of Ptn, beta-catenin is associated with E-cadherin. The binding of Ptn to RPTPZ1 results in the dimerization and inactivation of the receptor and, thus, an increase of the tyrosine phosphorylation of beta-catenin (42). Phosphorylated beta-catenin rapidly dissociates from E-cadherin. Recently, Fyn, a member of the Src family (51), and beta-adducin (52) have been found to be downstream targets of the Ptn/RPTPZ1 signaling pathway. Because it is the central component of the Wnt signaling pathway (82), beta-catenin may be a link between Ptn and Wnt signaling pathways. However, the association between these two pathways and the possible role of the Ptn/RPTPZ1 pathway in fetal lung development remain to be determined.

Lhx3, one of the LIM homeodomain transcription factors, is essential for pituitary development and function (11). Mutations in the human Lhx3 gene result in severe endocrine disease and cause a deficiency in all anterior pituitary hormones except adrenocorticotropin (47, 68). Lhx3 is thought to be one of the genes that play an important role in the development of Rathke's pouch, beginning at around embryonic day 8.5 in the mouse (64). In situ hybridization reveals the highest expression of Lhx3 in the developing lung and pituitary (61). However, its function in lung development has not been studied.

Ephrin (Eph) A3 is a GPI-anchored membrane glycoprotein and a ligand for Eph receptors. The binding of clustered Eph to Eph receptors activates the downstream cascade of the Eph signaling pathway and regulates vascular aggregation and homeostasis. Recently, EphA3 has been identified as a downstream protein of the Wnt-Frizzled signaling pathway (32). Wnt modulates lung morphogenesis through the canonical beta-catenin/lymphocyte enhanger factor-T cell factor pathway (45). In the presence of Wnt, beta-catenin is stabilized and accumulates in the nucleus to activate transcription of its downstream genes, such as N-myc, bone morphogenetic protein 4 (Bmp4), and fibroblast growth factor (FGF) (65).

Dlk1 belongs to the family of epithelial growth factor-like homeotic proteins that are involved in regulating growth and differentiation through cell-cell interaction. Dlk1 inhibits the differentiation of preadipocytes to mature adipocytes (67). Dlk1 acts through the Notch pathway (24). The binding of their ligands, Delta-like or Jagged, with Notch receptors results in a proteolytic cleavage of the Notch intracellular domain. The latter translocates into the nucleus and associates with a transcription factor, RBP-Jk. As a result, it activates expression of the downstream target genes, including hairy and enhancer of split (Hes) and Hes-related repressor protein transcriptional repressors. In situ hybridization of fetal lung mouse tissue shows that Dlk1 expression is high on gestational day 12.5 but downregulated on day 16.5 (85), similar to our microarray results.

Adh3 catalyzes the synthesis of retinoic acid, a biologically active derivative of vitamin A. Retinoic acid is important for lung branching morphogenesis (36). It regulates expression of the genes encoding for surfactant proteins and the enzymes that produce surfactant lipids (78). Adh3 expression is turned on at the late stages of liver development (76), but its temporal expression during fetal lung development is unknown.

Cadherin-22 is responsible for Ca2+-dependent cell-cell adhesion and is one of the important membrane proteins for mesenchymal-epithelial interaction in brain development (4, 22). Cadherin-22 is temporally and spatially expressed in the developing brain; its mRNA expression reached the highest level during the early postnatal stage and then declined at day 10 (71). Whether NADH/NADH thyroid oxidase-2 and glycogenin regulate fetal lung development has not been studied, but the similarity between their expression patterns and those of the other development-associated genes in this cluster suggests that they might have roles in this process.

The power of DNA microarray data is to generate hypotheses for further functional studies. From our gene expression data and the gene functions in cluster 5, we proposed a working model for regulation of fetal lung development and alveolar epithelial type II cell differentiation. However, we should emphasize that these pathways in fetal lung cells are highly speculative at this stage, because we have not determined the expression of each component in respective cells (except Ptn and Lhx3) as well as their interactions. In fetal lung fibroblasts or other mesenchymal cells, Adh3 catalyzes the synthesis of retinoic acid, which translocates into the nucleus and stimulates expression of Ptn. After its secretion, Ptn binds to the RPTPZ1 on the surface of epithelial type II cells. This inhibits the phosphatase activity of RPTPZ1 and rapidly increases the tyrosine phosphorylation of beta-catenin, which decreases its affinity to cadherin. Next, beta-catenin is released, enters the nucleus, and increases the expression of Lhx3 and other target genes. The increased Lhx3 expression may exert a positive-feedback regulation on its expression. Other genes in this cluster, Dlk1 via the Notch pathway and EphA3 via the Wnt pathway, may coregulate fetal lung development and fetal epithelial type II cell proliferation and differentiation.

The 583 identified genes were grouped into seven clusters. Cluster 1 is composed of 101 genes (21 ESTs and 80 known genes). The expression level of these genes was low in the fetal lung at day 18 and high the adult lung. The major functional categories of this cluster are metabolism and signal transduction. Seven genes are associated with cell proliferation. Phospholipase A2 group 1b (Pla2g1b) promotes cell proliferation and migration via receptor-mediated effects (29). Zinc finger protein hf-1b is a ubiquitous transcription factor. However, when it combines with a muscle factor (hf-1b/mef-2), hf-1b confers cardiac muscle-specific gene expression (46). Hes3, a member of the basic helix-loop-helix transcription factor family, regulates mammalian neural development (34). FGF receptor 2 (FGFR2) is the most common receptor for FGF-1, FGF-7, and FGF-10. FGFR2–/– mice are viable until birth and show severe lung defects (12). Prostaglandin-endoperoxide synthase 2 (ptgs2) is a nuclear membrane protein that may negatively regulate cell proliferation (59). Merlin (Nf2) inhibits cell cycle progression by decreasing cyclin D1 expression (84). 11beta-Hydroxysteroid dehydrogenase type 1 (Hsd11b1) regulates lung maturation by synthesizing glucocorticoid locally (20). RTI40 (also called T1{alpha}), a lung type I cell-specific protein, can be used as a biochemical marker for acute lung injury (40). The developmental expression of hsd11b and RTI40 in the lung is consistent with our microarray data (21, 81).

The 34 genes (9 ESTs and 25 known genes) in cluster 2 were decreased in the canalicular stage and then gradually increased in the saccular stage and declined after birth. Eight genes in this cluster are related to ion transport and five to development. Interestingly, four of the five development-related genes are involved in muscle development, including skeletal troponin I (Tnni2), cardiac troponin I (Tnni3), {gamma}-enteric muscle actin (actg2), and cysteine-rich protein 3 (csrp3). Tnni2 and Tnni3 are different isoforms in the thin filament-linked Ca2+ regulatory system in vertebrate striated muscle (49). Actg2 is a major component of the organized contractile apparatus of smooth muscle cells (60). Csrp3 is a LIM domain protein that promotes myogenic differentiation and is a novel regulator of myogenesis (5, 48). {alpha}-Crystallin B (Cryab), a small heat shock protein involved in sensory organ development, has cytoprotective and oncogenic functions (50).

Cluster 3 is the largest cluster, consisting of 152 genes (112 known genes and 40 ESTs) that exhibit downregulation of expression levels after birth. More than half of these genes are involved in metabolism; most of the metabolic proteins are ribosome proteins for protein biosynthesis. Additionally, five genes have roles in development or cell proliferation. High-mobility group 1 (hmg1) may contribute to the pathogenesis of acute lung injury. Antibodies against hmg1 reduce lipopolysaccharide-induced acute lung injury (74). The synaptonemal complex protein 1 (Sycp1) is a component of the synaptonemal complexes involved in chromosome pairing during meiosis (14). Intestine and stomach expression 1 (Mist1) is a basic helix-loop-helix transcription factor that regulates developmental patterns through the G protein-coupled signaling pathway (31). Myosin Ib (Myo1b) is a widely expressed protein involved in cell motility (51).

Expression of the 115 genes in cluster 4 (86 known genes and 29 ESTs) was higher in adult than in fetal and newborn lungs. These genes may be important for normal lung function during adulthood. There are two major functional categories in this cluster: signal transduction and transport. Four genes are involved in cell growth. Latent TGF-beta-binding protein-1- or -2-like proteins (Ltbp1 or Ltbp2) are extracellular matrix proteins that regulate TGF-beta activity (57). Activin A receptor type II-like-1 (Acvrl1), a member of the TGF-beta type I receptor family, is highly expressed in endothelial cells and functions in the normal development of the arterial and venous vascular beds (62, 75). Bmp6 regulates embryonic tissue development, cell proliferation, differentiation, morphogenesis, and apoptosis in multiple systems via the TGF-beta-signaling pathway (18).

The gene expression patterns in cluster 6 (40 known genes and 10 ESTs) and cluster 7 (50 known genes and 65 ESTs) are similar. About 40% of the genes in cluster 6 and 30% of the genes in cluster 7 are related to metabolism. A significant percentage (10%) of the genes in cluster 6 are involved in development. EphB1 is a developmentally regulated membrane protein involved in neurogenesis (43). Its expression is induced by retinoic acid (58). In rat brain, EphB1 mRNA level is high in the fetal stages but low in the adult (8). {alpha}-Tropomyosin (56), {alpha}1-actin (39), and myogenin (83) are involved in muscle development. Protein arginine n-methyltransferase, a nuclear enzyme, catalyzes S-adenosyl-L-methionine-dependent methylation of arginine residues during organogenesis (37).

A relatively large number of genes in cluster 7 participate in transport, signal transduction, and proliferation. The proliferation-related genes include TGF-{alpha}, high-mobility group protein 2 (hmg2), Ran, pituitary tumor-transforming 1 (Pttg1), and topoisomerase (DNA)-2{alpha} (Top2a). Except for TGF-{alpha}, all the genes are nuclear proteins and involved in regulation of the cell cycle, protein transcription, or cell maintenance (27, 69, 72, 73). TGF-{alpha} is a growth factor that regulates the cell cycle. Overexpression of TGF-{alpha} disrupts lung morphogenesis (30) and surfactant homeostasis (23). Expression of TGF-{alpha} is decreased during the late stage of fetal lung development (28).

Gene expression profiles of fetal lungs at the late developmental stages were examined in this study. The 583 identified genes were clustered according to their expression patterns. Cluster 5 contained 10 known genes and 5 ESTs that are involved in cell proliferation, cell differentiation, and development. In general, expression of the genes in this cluster was high in the late stage of fetal lung development but low in adult lungs. Most of these genes have not been studied in the lung. These genes might be incorporated into several pathways, including retinoic acid signaling, Ptn-beta-catenin signaling, and the Notch and Wnt pathways. Our findings may provide important clues for further understanding of molecular mechanisms in fetal lung development.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-052146 and R01 HL-071628 (L. Liu).


   ACKNOWLEDGMENTS
 
We thank Candice Marsh and Tisha Posey for editorial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Liu, Dept. of Physiological Sciences, Oklahoma State Univ., 264 McElroy Hall, Stillwater, OK 74078 (e-mail: lin.liu{at}okstate.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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