Page 1 of 35 Articles in PresS. Physiol Genomics (October 2, 2007). doi:10.1152/physiolgenomics.00108.2007
Gene-Expression Profiling in Lung Fibroblasts Reveals New Players in Alveolarization.
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Olivier Boucherat , Marie-Laure Franco-Montoya , Christelle Thibault , Roberto Incitti , Bernadette 1,2
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Chailley-Heu , Christophe Delacourt
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and Jacques R. Bourbon
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Inserm, Unité 841, IMRB, Département de Biologie et Thérapeutiques Cardiorespiratoires et Hépatiques, Equipe 06, Créteil, F-94000, France. 2
Université Paris 12, Faculté de Médecine, IFR10, Créteil, F-94000, France. 3
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), F-67404, Illkirch, France. 4
IFR10, Plate-forme de Génomique, Institut National de la Santé et de la Recherche Médicale, Hôpital Henri Mondor, Créteil, F-94000, France.
Corresponding author: Jacques Bourbon, Inserm U841, Faculté de Médecine, 8 rue du Général Sarrail, 94000 Créteil, France. Phone: +(33) 1 49 81 37 33; Fax: +(33) 1 48 98 37 33; E-mail:
[email protected]
Running head: New genes involved in lung alveolarization
1 Copyright © 2007 by the American Physiological Society.
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ABSTRACT Little is known about the molecular basis of lung alveolarization. We used a microarray profiling strategy to identify novel genes that may regulate secondary septation process. Rat lung fibroblasts were extemporaneously isolated on postnatal days 2, 7, and 21, i.e. before, during, and after septation, respectively. Total RNA was extracted and cRNAs were hybridized to Affymetrix rat genome 230 2.0 microarrays. Expression levels of a selection of genes were confirmed by real-time PCR. In addition to genes already known to be up-regulated during alveolarization including drebrin, midkine, Fgfr3, and Fgfr4, the study allowed us to identify two remarkable groups of genes with opposite profiles, i.e. gathering genes either transiently upor down-regulated on day 7. The former group includes the transcription factors RXR , Hoxa2, a4, a5, and genes involved in Wnt signaling (Wnt5a, Fzd1, and Ndp); the latter group includes the extracellular matrix components Comp and Opn, and the signal molecule Slfn4. Profiling in whole lung from fetal life to adulthood confirmed that changes were specific of alveolarization. Two treatments that arrest septation, hyperoxia and dexamethasone, inhibited the expression of genes that are up-regulated during alveolarization, and conversely enhanced that of genes weakly expressed during alveolarization and up-regulated thereafter. The possible roles of these genes in secondary septation are discussed. Gene expression profiling analysis on freshly isolated cells represents a powerful approach to provide new information about differential regulation of genes during alveolarization and pathways potentially involved in the pathogenesis of bronchopulmonary dysplasia.
Key words: bronchopulmonary dysplasia, hyperoxia, Hox, Wnt, Opn
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INTRODUCTION New Bronchopulmonary Dysplasia (BPD) is chronic lung disease of preterm infants, most often consecutive to respiratory distress. BPD is featured by disruption of the normal process of alveolarization (or alveologenesis), leading to enlarged and simplified airspaces (26). Although BPD represents major health problem, mechanisms that inhibit distal lung growth are poorly understood (6). Establishing new basis for safe and effective intervention to prevent or reverse arrested alveolarization represents a pressing need. This can be achieved only by a better understanding of the mechanisms that regulate alveolarization. Compared with earlier periods of lung development, particularly branching morphogenesis, the secondary septation process through which alveolar sacs are subdivided into definitive alveoli has been the object of a fewer numbers of studies. Myofibroblasts express smooth muscle actin ( SMA) and synthesize elastin that forms deposits in the thickness of the walls of terminal air-sacs. At the level of elastic fibers, crests surge that progressively elongate to give rise to secondary septa (10). This greatly increases gas-exchange surface area, and allows the animal to adapt successfully to extrauterine life. This process takes place postnatally for the major part in humans, and is totally postnatal in rodents. To date, only a few key molecules involved in the process have been identified, including extracellular matrix components, matrix-remodeling proteases, growth factors, and receptors (2, 5, 30, 42, 43, 47, 60). Four gene-expression profiling studies have been performed recently to identify new factors involved in lung development (4, 12, 14, 35). Although these studies identified a useful list of differentially regulated genes, only two studies (12, 14) addressed specifically the alveolar stage of development. Moreover, they dealt with limitations imposed by whole-organ approach, i.e. restriction in the extent of gene expression changes, alterations induced by changes in the proportion of various cell populations within the lung, and impossibility to associate changes with responsible cell type(s). Because the formation of alveoli occurs in a strictly defined period, we hypothesized that genes that regulate alveolar development must be differentially expressed during the period of active alveolar septation as compared to the preceding and following periods. In the rat, the formation of secondary alveolar septa takes place between the 4th and 14th postnatal days. Taking into account the above-emphasized pivotal role of interstitial cells, we searched for genes differentially expressed in rat lung fibroblasts isolated on postnatal days 2, 7, and 21. This allowed us to identify numerous genes that undergo significant changes in expression during alveolarization. A particular attention was paid to genes either up-regulated between postnatal days 2 and 7 and downregulated between days 7 and 21, or conversely, down-regulated between days 2 and 7 and up-regulated
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between days 7 and 21. Moreover we investigated the expression of some of these genes in two classical models of arrested alveolar development. These models, namely the chronic exposure of neonatal rats to 95% oxygen or to the synthetic glucocorticoid dexamethasone (Dex), are widely used to explore the mechanisms of BPD (9, 40). Data presented herein extend knowledge about normal alveolar formation, and consequently, they should help understanding the molecular impact of pathological disturbances on the developing lung.
MATERIAL AND METHODS Animals and lung tissue Dated pregnant Sprague-Dawley rats were purchased from Charles River (Saint Germain sur l’Arbresle, France). The day of mating was designated day 0 of gestation. Term is 22 days. Lung tissues were collected between fetal day 18 and postnatal day 21. Lung tissues from adult rats (8 wk of age) were also collected. Fetuses were retrieved by cesarean section from pentobarbital-anesthetized pregnant females. Lungs from fetuses and pups were either immediately frozen in liquid nitrogen then kept at -80°C until RNA extraction, or used for cell isolation. Procedures involving animals were in accordance with the rules of the Guide for Care and Use of Laboratory Animals, and were performed under the authority of the French Ministry of Agriculture.
Lung fibroblast isolation Rat lung fibroblasts were extemporaneously isolated on postnatal days 2 (saccular stage), 7 (alveolar stage, progressing secondary septation) and 21 (terminated secondary septation) as described previously (11). In brief, cells were dispersed by trypsin and collagenase, then plated in plastic Petri dishes in MEM-10% FBS, and allowed to adhere for 45 min. Non-adherent cells were removed, and the adherent fibroblasts were rinsed and immediately scrapped and pelleted. This method has been reported to result in fibroblast preparations with >95% purity (8, 11). Freshly isolated cells were stored at -80°C until RNA extraction for gene expression studies, without any culture step.
Generation of cRNA “target” and chip hybridization Total RNA was extracted from isolated fibroblasts using the guanidinium isothiocynate method (TRIzol reagent, Invitrogen, Cergy-Pontoise, France), followed by purification using Rneasy columns (Qiagen, Courtaboeuf, France). Three biological replicates from 3 different litters were prepared for each time point, i.e.
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a set of fibroblasts was isolated from pups from each of the 3 litters at each time point. Integrity and purity of RNA were checked by spectrophotometry and capillary electrophoresis, using the Bioanalyser 2100 and RNA 6000 LabChip kit from Agilent Technologies (Palo Alto, CA). cDNAs were synthesized using Superscript Choice system (Invitrogen). Biotin-labeled-cRNAs were then synthesized with the Affymetrix IVT labeling kit (Affymetrix, Santa Clara, CA, USA). After purification, 10 µg of fragmented cRNA were hybridized to the Affymetrix GeneChip Rat Genome 230 2.0 Array (31,042 probe sets including >28,000 rat genes), and the chips were automatically washed and stained with streptavidine-phycoerythrin using a fluidics station. Finally, arrays were scanned at 570 nm with a resolution of 1.56 µm/pixel, using the Gene Chip Scanner 3000 from Affymetrix. Expression values were generated using Microarray Suite v5.0 (MAS5, Affymetrix). Each sample and hybridization experiment underwent a quality control evaluation, including percentage of probe sets reliably detecting between 50% and 60% Present call, and 3’-5’ ratio of GAPDH gene 95% or with 21% (room air) FiO2, from day 0 to day 6. O2 concentrations were monitored regularly. Because adult rats have limited resistance to high O2, the dams were exchanged daily between O2-exposed and room air-exposed litters. Pups from two different litters were mixed so that there were littermates in both conditions. Chambers were opened for 20 min every day to switch dams between air and O2 environments and to clean cages. Following the 6-d 95%- O2 exposure period, some of the pups were allowed to restore in room air for 4 days. - Dexamethasone treatment. Two different litters were subdivided into three groups that received an intraperitoneal injection of either 0.1 or 0.5 µg/g/day water-soluble Dex (Sigma, L’Isle d’Abeau, France), or the vehicle alone (saline, control group). Six animals were used for each experimental condition. Pups were sacrificed on the day that followed the last injection of Dex or saline. - Sacrifice. On day 6 or 10, rat pups were killed by an intraperitoneal overdose of sodium pentobarbital (70 mg/kg, Ceva, Libourne, France), and were exsanguinated by aortic transsection. Lungs were either fixed at 20cm-H O constant pressure for morphologic analysis, or immediately dropped in liquid nitrogen and kept 2
frozen at -80°C until further RNA or protein extraction.
Real-time quantitative polymerase chain reaction (qPCR) RNAs from each extraction sample were reverse-transcribed into cDNAs using 2 µg of total RNA, Superscript II reverse transcriptase, and random hexamer primers (Invitrogen) according to the supplier’s protocol. Realtime PCR was performed on ABI Prism 7000 device (Applied Biosystems, Courtaboeuf, France) using the
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following protocol: initial denaturation (10 min at 95°C), then two-step amplification program (15 s at 95°C followed by 1 min at 60°C) repeated 40 times. Melt curve analysis was used to check that a single specific amplified product was generated. Reaction mixtures consisted of 25 ng cDNA, SYBR Green 2X PCR Master Mix (Applied Biosystems), and forward and reverse primers (Table 2) in a reaction volume of 25 µl. Primers were designed using Primer Express software (Applied Biosystems). Real-time quantification was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green dye to double-stranded DNA at the end of each amplification cycle. Relative expression was determined by using the
Ct (threshold cycle)
method of normalized samples ( Ct) in relation to the expression of a calibrator sample, according to the manufacturer’s protocol. Each PCR run included a no-template control and a sample without reverse transcriptase. All measurements were performed in triplicates.
Immunoblot analysis Rat lung tissue was homogenized in RIPA buffer containing protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein content was assayed using Bradford assay. Eighty micrograms of total proteins were electrophoresed on 12% SDS-polyacrylamide gels then transferred onto polyvinylidene-fluoride membrane (Millipore, Saint-Quentin en Yvelines, France). To document equivalent protein loading, membranes were stained with Ponceau S dye (Sigma, L’Isle d’Abeau, France) and photographed prior to antibody incubations. After blocking with 5% nonfat dry milk in Tris-buffer saline containing 0.05% Tween-20 (TTBS) at room temperature for 2h, membranes were exposed overnight at 4°C to either one of the following antibodies: mouse anti-osteopontin (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-schlafen4 (Santa Cruz Biotechnology), goat anti-norrin (R&D Systems), or rabbit anti-RXR (AbCys, Paris, France), all diluted in 2% nonfat dry milk in TTBS. After five rinses in TTBS, the membranes were incubated for 1h with the appropriate secondary IgG peroxidase-conjugated antibody. Membranes were then incubated for 1 min in chemiluminescent detection reagent (ECL, GE Healthcare Life Sciences, Velizy, France) before exposure to KODAK BioMax MS film for 2 min.
Immunohistochemistry Isolated cells adherent to plastic were methanol-fixed at -20°C and immunostained with goat anti-vimentin (Santa Cruz Biotechnology), or mouse monoclonal anti-pan-cytokeratin (Sigma), or goat anti-CD31 (Santa Cruz Biotechnology), or mouse monoclonal anti-CD68/ED1 (Abcam, Paris, France) antibodies. Secondary antibodies were donkey anti-goat Alexa fluor 594 and donkey anti-mouse Alexa fluor 488 (Invitrogen).
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Simultaneous nuclear labeling was obtained with Vectashield mounting medium with dapi (Vector Laboratories, Burlingame, CA). Immunostaining of postnatal day-10 lung tissue was performed using 5µm paraffin sections that were deparaffinized and rehydrated. For epitope retrieval, the sections were boiled in 10mM sodium citrate for 20 min and cooled for 20 min. Endogenous peroxidase activity was quenched for 15 min in a bath of 3% H2O2 in H2O. All the sections were incubated with 2.5% normal horse serum for 30 min, then with goat anti-norrin (R&D Systems), rabbit anti-frizzled1 (MBL International, Woburn, MA), goat antiWnt5a, goat anti-Hoxa5 (Santa Cruz Biotechnology), or rabbit anti-RXR (AbCys) antibodies overnight at 4°C in a humidified chamber. After PBS washing, the sections were incubated for 15 min with horse biotinylated secondary antibody. After an extensive wash with PBS, all the sections were exposed to a streptavidin/peroxidase preformed complex for 15 min. Horse serum, biotinylated secondary antibody, and streptavidine/peroxidase were from Vectastain Universal Quick Kit (Vector Laboratories). Finally, sections were covered with diaminobenzidine tetrahydrochloride (Vector Laboratories) for 2 min, counterstained with methyl green, dehydrated, and observed with light microscope. As negative controls, primary antibodies were omitted.
Statistical analysis of whole lung studies Data are presented as mean ± se. Multiple group comparisons were made by non-parametric Kruskal-Wallis analysis, and two-group comparisons were made by non-parametric Mann and Whitney U test. P=0.05 was considered as the limit of statistical significance.
RESULTS Microarray analysis identifies gene-expression patterns associated with alveolarization. In an attempt to identify developmentally regulated genes whose expression changes are associated with lung alveolarization, microarray transcription profiles were obtained from lung fibroblasts isolated at postnatal days 2, 7 and 21. Fibroblast-enrichment of cell preparations was checked by immunolabeling for vimentin (fibroblast marker), cytokeratins (epithelial markers), CD31 (endothelial marker), and CD68/ED1 (macrophage marker). As shown in Figure 1A, almost all cells were vimentin-positive, indicating their interstitial cell character. Only a few cells stained for cytokeratin (Figure 1B). Rare cells displayed very faint CD31 labeling (not shown). No CD68/ED1 labeling was detected. The proportion of genes designated as “Present” in isolated fibroblasts by
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the Affymetrix software was 57.0 ± 1.27, 57.7 ± 0.92, and 53.9 ± 1.62% at postnatal days 2, 7, and 21, respectively, and did not fluctuate significantly (Table 1). Based on the hypothesis that genes that regulate alveolar development must be differentially expressed during the period of active septum formation as compared to preceding and following periods, we focused our analysis on genes simultaneously increased between days 2 and 7, and decreased between days 7 and 21 (genes designated up-regulated during septation, Figure 2), or conversely, simultaneously decreased between days 2 and 7, and increased between days 7 and 21 (genes designated down-regulated during septation, Figure 3).
Validation of microarray data To validate microarray data using independent methods, we selected 13 genes and analyzed their expression by qPCR, using the same samples as in the microarray experiment. Among up-regulated genes, we selected midkine (MK) because of its previously known lung-expression pattern (38), and homeobox (Hox) genes a2, a4 and a5 as well as the retinoic acid X receptor
(RXR ) for their important role in embryogenesis and their
potential contribution to the transcriptional regulation of genes involved in secondary septation. Frizzled-1 (Fzd1), wingless-type MMTV integration site 5a (Wnt5a) and Norrie disease protein or norrin (Ndp) were also retained for validation given their prominent roles in developmental events during embryogenesis (31). Among down-regulated genes, we selected extracellular matrix (ECM) components or cell-matrix adhesion molecules, including tenascin-X (TnX), cartilage-oligomeric matrix protein (Comp), osteopontin (Opn), and osteoactivin (Gpnmb). Lastly, a member of the schlafen (Slfn) protein family implicated in the regulation of cell growth, Slfn4 (7), was selected because its expression in the lung had not been reported previously. For each selected gene, the fold-change determined by qPCR was similar to or higher than that determined by the microarray analysis (Table 3), indicating high degree of concordance between results from both methods. This suggests a high degree of confidence in our overall microarray data.
Expression profile of selected genes from fetal life to adulthood In order to determine whether (i) changes in isolated fibroblasts were detectable also in whole lung homogenate, and (ii) observed changes were specific of alveolarization, we evaluated the mRNA level of selected genes in the developing rat lung from fetal day 18 (canalicular stage) to adulthood. Genes of the first group, i.e. those up-regulated in fibroblasts, showed quite similar expression patterns although with variable amplitude (Figure 4A and 4B). The expression of all three Hox gene and RXR changed little until postnatal
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day 3, increased then 2- to 4-fold on day 5, remained elevated during alveolar septation, and returned to prenatal levels on day 20 to further decrease to extremely low levels in adulthood (Figure 4A). The expression profiles of Wnt5a, Fzd1, and Ndp were similar to that of transcription factors with sustained elevated expression between days 5 and 14 about 2.5-, 3.5 and 6.5-fold the day 3 level, respectively, and marked down-regulation on day 20 (Figure 4B).. In the second group, i.e. genes down-regulated in fibroblasts (Figure 4C), Slfn4 and Gpnmb expression peaked up on fetal day 21 and postnatal day 1, respectively (10- to 12-fold increase as compared with fetal day 18), decreased 2- to 3-fold during alveolar septation, then re-increased 3and 1.5-fold, respectively, after its completion; Sfln4 level stayed then elevated whereas that of Gpnmb dropped down in the adult lung (Figure 4C) TnX expression stayed unchanged from fetal day 18 to postnatal day 3, increased slightly on day 5 then sharply on days 14-20 when it reached about 12 times the fetal and postnatal level, and returned to very low level in the adult lung. These three genes therefore share the feature of peaking during the second phase of alveolarization that follows the completion of septation.
Localization of up-regulated gene expression in postnatal rat lung Immunostaining for WNT5A, NDP, FZD1, HOXa5, and RXR proteins was performed on sections from day-10 rat lungs. For each gene, immunostaining was detected in the thickness of septa, including at the tip of growing secondary septa (Figure 5, A-J). In addition, a strong immunostaining for NDP and RXR was also observed in bronchial epithelial cells (Figure 5, C and I).
Effects of Dex on selected-gene expression in postnatal rat lung Consistent with previous studies (40, 51), Dex (0.1µg/g/d) given from postnatal days 1 to 5 induced marked impairment of alveolar septation on postnatal day 6, with enlarged distal airspaces (Figure 6B) compared with normal alveolar structure of control littermates (Figure 6A). As regards up-regulated genes during septation, Hoxa5, Wnt5a and Ndp mRNA levels showed a 40-50% decrease with this dosage, whereas no change was observed for Hoxa2, a4 and Fzd1 (Figure 6C). When Dex dosage was increased to 0.5µg/g/d, the expression of all selected genes except RXR was decreased 40% to 80%. As regards down-regulated genes, Dex 0.1 and 0.5 µg/g/d caused a similar 2-fold increase in Gpnmb and Comp mRNA abundance as compared with controls (Figure 6D). The effect was stronger for Slfn4 and Opn mRNA abundance with 4- and 11-fold increases, respectively. No change was observed for TnX expression level at any Dex dosage.
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Effects of 95% O2 and recovery in air on selected-gene expression in postnatal rat lung Consistent with previous observations (9), newborn rats exposed to hyperoxia for 6 days displayed large and simplified air spaces (Figure 7B) as compared with the alveolar structure of littermate controls exposed to air (Figure 7A). When O2-exposed rats were subsequently allowed to recover in room air for 4 days, normal lung structure caught-up (Figure 7D) as compared with 10-day controls in air (Figure 7C). The expression level of all up-regulated genes (Figure 7E) was decreased approximately 50% under O2 as compared with controls. Restoration to levels no longer significantly different from those in controls occurred for Hox genes, RXR and Wnt5a after return in air for 4 days. By contrast, Fzd1 and Ndp mRNAs remained significantly lower than normal level, although they increased significantly as compared with day 6 under hyperoxia, indicating partial recovery. Similar to the Dex model, down-regulated genes (Figure 7F) displayed an opposite pattern. Comp and TnX mRNA levels were increased 2-fold by hyperoxia, and Gpnmb, Slfn4 and Opn mRNA levels were increased 5, 6 and 32-fold, respectively. Following the 4-day recovery in air, mRNA levels were similar to those in air-exposed controls for all genes. To determine whether changes in mRNA level were reflected in lung protein contents, we performed western blots for RXR , NDP, OPN and SLFN4, which displayed the largest variations of expression. As shown in Figure 7G, protein changes were concordant with RNA changes, since RXR
and NDP amounts were
decreased in O2, and reciprocally, OPN and SFLN4 amounts were increased.
DISCUSSION Rationale, limitations, and validity of the approach Understanding the molecular mechanisms of alveolarization is an important issue for the prevention and care of alveolar growth arrest in BPD, as well as for maintenance and regeneration of the alveolar structure in th
emphysema. In the rat, alveolar formation is an entirely postnatal event that initiates on the 4 postnatal day and is completed by 3 weeks (10). Alveolar number increases linearly over this period (50), but alveolar density reaches maximal value on day 8, and does not increase further thereafter (44). This is consistent with the concept of a two-phase model of alveolarization consisting of saccular subdivision by secondary crests until day 8, and acinar extension with alveoli added distally in a second step (39). Consequently, the choice of postnatal days 2, 7, and 21 for microarray analysis should allow one to discriminate between genes involved in secondary septation and those that are involved later. This is of particular interest considering that impaired
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secondary septation is the major feature of BPD. Formation of secondary septa is dependent on harmonious interactions between several cell types. Because fibroblasts are key players in alveolar septation, we searched for genes differentially expressed in fibroblasts that were isolated at the 3 stages listed above. This, along with investigating the effects of treatments that impair septation, allowed us to identify genes the expression of which appears to have to be either enhanced or repressed for proper course of the process. Considering the cell complexity of the lung, cell type-specific gene profiling studies present several advantages for interpreting the results, including removing a lot of noise from the system. Although our approach did not allow detecting expression changes in epithelial and endothelial cells, which is clearly a limitation of the present study, this approach facilitated the interpretation of data. Importantly, subsequent study in whole lung confirmed the significance of changes evidenced in fibroblasts. Moreover, our microarray data pointed out stage-associated changes in expression of some genes previously recognized to be involved in alveolarization. These include for instance midkine and drebrin. Midkine expression was previously shown to be transiently increased in normal lungs between postnatal days 2 and 7, diminished by hyperoxia or glucocorticoids, and upregulated by retinoic acid (24, 41). Drebrin, an actin-binding protein, has been detected in cell processes of myofibroblasts during the maturation of alveolar septa (63). The developmental expression patterns previously reported in whole lung for these genes are very similar to those obtained herein from isolated fibroblasts. Our microarray analysis also identified genes the targeted inactivation of which altered secondary septation, including Hoxa5, Fgfr3, and Fgfr4 (34, 49, 60). Taken together, these findings indicate that the transcriptome status of fibroblasts was unlikely to have been altered extensively by cell isolation, which validates the approach. Nevertheless, the pulmonary fibroblast population is heterogeneous. Fibroblasts are present in vascular and airway adventitia, and in the alveolar area two sub-classes known as myofibroblasts and lipofibroblasts (or lipid-laden fibroblasts) are present (56). Consequently, whole-lung digests followed by adherence-based cell selection results in a somewhat heterogeneous population of fibroblastic cells. We therefore cannot exclude the possibility that a subset of regulated genes during alveolarization evidenced in the present study may finally prove to not be expressed in alveolar myofibroblasts.
Genes up-regulated during septation This study identified numerous genes that were up-regulated in lung fibroblasts during secondary septation, the involvement of which in the process had not been suspected previously. The expression study in whole
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lung of a selection of these genes indicated that they returned to low level after completion of the septation process, i.e. between postnatal days 14 and 20. Their protein products were localized in the thickness of growing septa. Taken together, these findings strongly argue in favor of their involvement in the alveolarization process. Interestingly, we found that in addition to Hoxa5, the mRNAs from two other Hox genes, Hoxa2 and Hoxa4, were increased during alveolarization and deeply diminished in animal models of arrested septation. Hox genes encode homeodomain transcription factors that are important for specifying tissue and cell-type identities along the anterior-posterior axis of the embryo (33). They have also a variety of functions in the development of a number of organs. Hoxa5 inactivation leads to severe respiratory tract defects (3) and impairs alveolarization because of mispositioning of alveolar myofibroblasts (34). By contrast, previous investigations on both other Hox genes had not revealed involvement in lung alveolar development. Mice homozygous for a targeted mutation of the Hoxa-2 gene are born with a bilateral cleft of the secondary palate associated with multiple head and cranial anomalies, and they die within 24 hr of birth (17). Consequently, evaluating consequences of this deletion for secondary septation of the lung is not possible. With regard to Hoxa4, its expression was found in structures partially derived from the lateral plate or para-axial mesoderm, including the fetal lung, intestine, and kidney (16). Lack of lung phenotype during the perinatal period in Hoxa4-null mice may be due to functional redundancy between Hox genes. It should be emphasized that although the latter have been implicated in the regulation of a variety of pathways, few target genes have been demonstrated to be placed under their direct regulatory control (53). Hence, to fully evaluate the role of Hox proteins in alveolar development, future research requires identifying their target genes, in addition to determining their respective specific functions.
Among the transcription factors whose expression is increased during alveolarization, we also found a member of the retinoic acid (RA) pathway, namely RXR . There are two classes of RA-activated transcription factors, the RARs and RXRs, that form homo- and heterodimers and bind both all-trans-RA and 9-cis-RA, or only 9-cis-RA, respectively. Each class possesses three isoforms,
,
, and
(25). Exogenous and
endogenous retinoids have been reported to positively influence alveolarization (15, 37). Consistently, the crucial role of several RA receptors for alveolar development has been demonstrated. For instance, mice deleted for both RAR and RXR genes fail to form alveoli correctly, whereas mice deleted for the RAR gene form too many alveoli (38, 42). The expression of several RARs has been reported to be enhanced in mouse lung when alveolar septation starts (18). Thus far, however, RXR had not been involved. Its developmental
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increase in fibroblasts coincidental with septation suggests an important function in these cells during the process, possibly in partnership with other receptors. The orphan receptor NR4A1 and the thyroid hormone receptor (THR ), which also display maximal gene expression on day 7 (Figure 2) and have previously been reported to interact with RXR , are potential partners. Identifying the role and targets of RXR
therefore
appears as an important objective in the understanding of alveolarization mechanisms.
Three genes involved in Wnt signaling also displayed a marked increase during secondary septation, including Wnt5a, Ndp and Fzd1. Wnt factors and NDP are diffusible mediators that bind to frizzled transmembrane receptors (62). After binding to frizzled receptors, intracellular Wnt signaling occurs through canonical and noncanonical pathways to regulate a lot of processes during development (31). The importance of Wnt signaling during lung development has been demonstrated by several ways. A number of Wnt ligands and frizzled receptors are expressed dynamically during lung development (55). Moreover, lung deletion of
-
catenin, an essential downstream effector of the canonical Wnt signaling, disturbs lung proximal/distal cell fate and leads to death from respiratory failure at birth (45). Few data are available specifically on Fzd1 and Wnt5a in lung development. Fzd1 was shown to be expressed in the developing lung mesenchyme, consistently with present findings, and in presumptive bronchial smooth muscle as well as in vascular smooth muscle tissues such as those of the aorta and large pulmonary vessels (58). It may mediate Wnt signaling originating from other lung cell type(s). In Wnt5a
-/-
mice, lung saccular development was delayed with thickened septa and -/-
increased cell proliferation both in epithelium and mesenchyme (28). Wnt5a neonates die shortly after birth, however, which prevents from evaluating consequences for alveolarization. To date, no published data highlight the involvement of Ndp in lung development. Ndp-deficient mice, a Norrie-disease animal model, develop blindness because of distinct failure in retinal angiogenesis and complete lack of the deep capillary layers of the retina. Taking into account on the one hand, the deficit of capillary/alveolar apposition in Wnt5a
-/-
fetuses (28), and on the other hand, that WNT5a and NDP enhance angiogenesis through frizzled-4 interaction (36, 62), the hypothesis that both mediators play a role in lung microvascular development appears likely. Microvascular development is known to be required for normal alveolarization and to be controlled by VEGF produced by epithelial cells (43). Moreover, expression of each of these genes was deeply depressed in animal models of impaired alveolarization (Figures 6C and 7E). We hypothesize that WNT5a and NDP may mediate another angiogenic control mechanism through mesenchymal-endothelial cell interaction in the process of secondary septa formation.
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Genes down-regulated during septation and/or enhanced after its completion We found Slfn4, a negative regulator of cell proliferation (7), to be down-regulated in the lung from days 1 through 8, i.e. a period characterized by extensive overall cell proliferation (19). This period was strikingly flanked by two expression peaks. Moreover Slfn4 expression was increased in animal models of arrested alveolarization. This may account at least in part for the inhibitory effect of hyperoxia and dexamethasone on lung cell proliferation (32, 59). Further work will be necessary to clarify Sfln4 function in vivo and its mechanism of action. Several genes that share similar expression profile during alveolarization encode proteins that are integral part of ECM. Evidence has accumulated over recent years for a crucial role of ECM components in alveolar development (6). However, none of those genes identified in the present study had previously been related to this process. Cartilage oligomeric matrix protein (Comp) is a member of the thrombospondin family also referred to as thrombospondin-5 (Tsp5). To date, there is limited evidence of Comp/Tsp5 expression outside the skeleton. Although several members of the thrombospondin family have the ability to inhibit angiogenesis (1), Comp does not share this feature in vitro (57). Its function during alveolarization can only be a matter of speculation. In addition to Comp, we found that Opn gene expression was diminished during the window of secondary septation and dramatically increased in BPD models. OPN is a secreted RDG-containing phosphoprotein with structural and functional characteristics of a matricellular protein that has been implicated in a number of physiological and pathological events, although this is often controversial. OPN exists both as an immobilized ECM molecule in mineralized tissues and as a cytokine-like soluble protein that mediates cellular functions involved in inflammation and ECM remodeling (13). During development, OPN is essential to mammary gland differentiation (46). Altered wound healing was observed in Opn-deficient mice (29), whereas recombinant OPN enhanced migration and adhesion of murine fibroblast cell line (54). Furthermore, Opn-null mice developed more severe acute lung injury than wild type mice, and their survival times were shorter than those of their matched wild-type controls (65). Lastly, Opn is highly expressed in pulmonary fibrosis, a feature encountered to variable extent in BPD (48). These data suggest a protective role of OPN in vivo, and physiological functions during matrix reorganization after injury. Further studies should aim at determining the significance of decreased expression before septation and the role of increased expression in arrested septation. We also included in genes of interest that of the ECM protein tenascin-X. Deficiency of the latter was
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described as the molecular basis of a recessive type of Ehlers-Danlos syndrome characterized by abnormalities in elastic fiber morpholology (66). Consistently, a recent study suggested that TnX is important in derma for the stability and maintenance of established elastin fibers, rather than for initial phase of elastogenesis (67). The striking up-regulation of TnX gene expression at the end of alveolarization when elastin deposition is completed is therefore consistent with the assumption that TnX may play a similar role in alveolar septa. Last, the up-regulation of these various genes between days 14 and 20 and their return to low expression level in the adult lung are suggestive of an involvement in the last part of alveolarization, when septa get thinner and microvascular maturation takes place. Further studies should determine whether their changes and those of genes with similar expression profile are associated with these processes.
Gene expression balance/imbalance and effects on septation Strikingly, the expression of genes up- and down-regulated during secondary septation was invariably decreased or enhanced, respectively, in models of arrested septation. We cannot rule out the possibility that these changes may be due at least in part to changes in the proportions of the various lung cell types induced by the treatments. Dex indeed reduces the volume of interstitial fibroblasts (40). However, whereas this may account for reduced expression of genes that are up-regulated in fibroblasts during septation, it could hardly explain the considerable increase of those that are down-regulated in these cells during the process. Changes induced by treatments are therefore more likely to be due to altered expression levels. Both glucocorticoid administration and exposure to hyperoxia similarly result in an arrest of alveolar development. Postnatal glucocorticoid treatment induces precocious microvascular lung maturation that is likely to prevent further septation (51), but indeed induces no inflammation, whereas inflammatory response contributes to the deleterious effect of high oxygen on the neonatal lung (64). Although Dex and hyperoxia are therefore likely to operate through quite different mechanisms, they presented very similar consequences for the expression of most of the selected genes. As a common consequence of both treatments, arrested alveolar septation strongly argues in favor of an important role of the selected genes in alveolar formation. Taken together with the characteristic developmental expression profiles of these different genes, this indicates that alveolar septation not only involves up-regulation of specific genes, but also necessitates down-regulation of other sets of genes that are much more strongly expressed during periods that immediately precede and follow the septation period. Because corticosteroids are believed to terminate the process of secondary septation
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through inducing thinning of septa and fusion of capillary vessels (40, 51), Dex treatment is likely to induce precociously those events that occur later in normal development. This assumption is consistent with the inhibition of genes that are up-regulated when alveolar septation initiates, and with enhanced expression of genes that are up-regulated at the end of the process. Because of the antagonistic effects of glucocorticoids and retinoids (37), it is therefore tempting to speculate that, among other possible mechanisms, retinoids trigger the induction of up-regulated genes and repress down-regulated genes in a first step, whereas glucocorticoids repress the former and trigger the induction of the latter in a second step. Imbalance in these chronological regulations may precipitate the disorders that lead to impaired alveolarization in BPD. It is not possible from the present study to determine whether the same or different populations of fibroblasts are involved in either phase. Further investigations should address this question.
In conclusion, present data identify novel genes that are likely to be involved in the regulation of secondary septation, and suggest that alterations of their expression could account for some of the disorders that characterize BPD. Based on information obtained from this study, further investigations will aim at characterizing their functions. This might provide new therapeutic targets for the prevention and care of BPD.
Acknowledgements This work was supported by a grant from the Réseau National des Génopoles (Project #212). O.B. was supported by a Doctoral-fellowship grant from the Fondation pour la Recherche Médicale.
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Legends for Figures
Figure 1. Imunostaining of cells isolated on postnatal day 7. Nuclear labeling was obtained with dapi (blue fluorescence). Almost all the cells isolated from enzymaticallydispersed lung cells after 45-min adherence to plastic were vimentin-positive (red fluorescence in A). Rare cells were cytokeratin positive (green fluorescence, arrow in B).
Figure 2. Clustergrams of genes up -regulated in fibroblasts during alveolarization. Genes selected on the basis of significant change by MAS (see criteria in Material and Methods) were exported to TIGR MeV array tool for hierarchical clustering using Manhattan correlation-based distance and average linkage. Each row represents one particular gene and each column represents expression ratio of that gene between two samples from a same litter at different stages. Data are presented as groups of 3 comparisons: 7d lung fibroblasts compared to 2d lung fibroblasts (first three columns) and 21d lung fibroblasts compared to 7d lung fibroblasts (last three columns). Genes that were present at higher levels in the examined group are shown in progressively brighter shades of yellow, whereas genes that were expressed at lower levels are shown in progressively brighter shades of blue. Genes in black were not different between the two groups. Gene symbols, gi accession numbers and GO molecular function are listed adjacent to each gene. Expressed sequence tags were not included to the list.
Figure 3. Clustergrams of genes down-regulated in fibroblasts during alveolarization. Same as in Figure 1.
Figure 4. Quantitative developmental changes of mRNA expression level in whole lung tissue of a selection of genes shown to be up- or down-regulated during septation in the microarray study. Expression was quantified by real-time PCR from fetal life (canalicular stage of development) to adulthood in three individual lung samples per stage. Relative expression was referred to fetal day-18 level, arbitrary set at 100. (A) Transcription factor genes found to be up-regulated in fibroblasts during alveolar septation by microarray analysis. (B) Genes of signal molecules and receptor found to be up-regulated in fibroblasts during alveolar septation by microarray analysis. (C) Genes found to be down-regulated in fibroblasts during alveolar septation by microarray analysis. The study indicates that major changes were specifically associated with the
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period of alveolarization. Mean ± s.e.m.; absence of an error bar indicates that the corresponding s.e.m. was too small to be represented at this scale; *P
