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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 232-240, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4877

Antisense Oligodeoxynucleotides Decrease LGL1 mRNA and Protein Levels and Inhibit Branching Morphogenesis in Fetal Rat Lung

Lami Oyewumi, Feige Kaplan, Stéphane Gagnon and Neil B. Sweezey

Lung Biology Research, Research Institute, The Hospital for Sick Children; Department of Physiology, University of Toronto; Departments of Pediatrics and Human Genetics, McGill University-Montreal Children's Hospital Research Institute, McGill University; and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada

Address correspondence to: Neil B. Sweezey, Lung Biology Research, Research Institute, The Hospital for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8 Canada. E-mail: neil.sweezey{at}sickkids.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously described the cloning of the late gestation lung 1 gene (LGL1), a novel glucocorticoid-inducible gene expressed in the mesenchyme of fetal lung. We report here evidence for a role of the LGL1 gene product (lgl1) in fetal rat lung airway branching morphogenesis, temporal and spatial localization of LGL1 mRNA and lgl1 protein in fetal rat lung, and a correction of the previously published LGL1 sequence. Both the mRNA and protein were detected during fetal lung development. LGL1 mRNA was detected from gestational Day 12 by reverse transcriptase–polymerase chain reaction, and from Day 13 by in situ hybridization. lgl1 protein was detected from Day 18 by Western analysis and from Day 16 by immunohistochemistry. The types of cells expressing LGL1 mRNA and lgl1 protein were assessed by immunohistochemical staining of adjacent serial tissue sections for markers of mesenchymal (vimentin) and smooth muscle ({alpha}-actin) cells. As gestation advanced, increasing amounts of mRNA and protein were expressed in these cells. In support of a role for lgl1 in airway branching morphogenesis, antisense (but neither sense nor scrambled) oligodeoxynucleotides directed against LGL1 inhibited airway branching in fetal rat lung buds in explant culture, in a dose- and time-dependent manner. The levels of lgl1 protein and LGL1 mRNA expression were decreased in those explants that had inhibited airway branching, compared with the uninhibited controls. Our findings suggest that lgl1 plays an important role in fetal airway branching morphogenesis.

Abbreviations: cysteine-rich secreted protein, CRISP • normal goat serum, NGS • oligodeoxynucleotide, ODN • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The developing airways of fetal lungs form from epithelial buds derived from foregut endoderm, surrounded by mesenchyme. Precise signaling between mesenchymal cells and epithelial cells regulates cell proliferation, fate, migration, and differentiation (1). Organ-specific humoral and extracellular matrix (ECM) factors determine branching morphogenesis and epithelial differentiation (1, 2). Interactions with specific local populations of mesenchymal cells induce branching by fetal rat lung epithelial rudiments in explant culture (3, 4). However, the mesenchymal factor(s) mediating this effect remain to be identified.

We previously reported the isolation of a novel glucocorticoid-responsive gene, late gestation lung 1 (LGL1), which is differentially expressed in fetal lung mesenchymal cells during development, but is not detectable in lung epithelium (5). LGL1 is well-conserved in mammals, suggesting its gene product is of functional importance (5). We therefore sought to identify its functional role in lung development, initially at its earliest period of expression. In light of the mesenchymal-specific localization of LGL1 mRNA, and homology to known cell adhesion molecules, we hypothesized that LGL1 function is required for normal branching morphogenesis in the fetal lung. We report here detailed spatial and temporal localization of expression of LGL1 mRNA and lgl1 protein in fetal rat lung. A correction of the LGL1 cDNA sequence indicates the deduced protein is much longer (437 amino acid residues) than our previous report (188 amino acids) (6). We also found that antisense, but not sense or scrambled, LGL1 oligodeoxynucleotides (ODNs) decrease expression of LGL1 mRNA and lgl1 protein and inhibit airway branching, suggesting a role for lgl1 protein in airway branching morphogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Drugs and chemicals were obtained from the following sources: culture media (minimal essential medium), nylon (Hybond N) membranes from Amersham (Buckinghamshire, UK); penicillin, streptomycin, urea, agarose, TRIzol, ethidium bromide, random hexanucleotide primers, Taq polymerase, and restriction endonucleases from Gibco/BRL Life Technologies (Burlington, ON, Canada); PCR primers from Sheldon Biotechnology (Montreal, PQ, Canada); Sequenase from Amersham; deoxynucleotides and RNA Guard RNase inhibitor from Pharmacia Biotech Inc. (Baie d'Urfé, PQ, Canada); and 32P{alpha}-dCTP from Dupont Canada (Mississauga, ON, Canada).

Reverse Transcriptase–Polymerase Chain Reaction Analysis
Total (nuclear and cytoplasmic) RNA was prepared from fetal rat lung tissue using the TRIzol reagent, according to manufacturer's instructions. Briefly, whole rat lungs were homogenized in TRIzol reagent and RNA extracted with chloroform. RNA was ethanol precipitated, collected by centrifugation, lyophilized, and dissolved in RNase-free water. Three micrograms of total RNA from explants was used for reverse transcription–polymerase chain reaction (RT-PCR). RT-PCR reactions were performed using 27 cycles of PCR for LGL1 and 25 cycles for ß-actin, to control for loading and integrity of samples. Each cycle consisted of denaturation (45 s at 94°C), annealing (45 s at 55°C), and extension (4 min at 72°C). Synthetic oligodeoxynucleotide pairs were designed corresponding to conserved sequences of rat LGL1, yielding a 472-bp product, and rat ß-actin, yielding a 515-bp product. The following sets of PCR primers were used: LGL1 forward primer, 5'-ATG CTG CAC AAC AAG GCT GCG-3'; reverse primer, 5'-GCT CTG AGT GTC CGT CCA GCT-3'; ß-actin forward primer, 5'-GTG GGC CGC TCT AGG CAC CAA-3'; ß-actin reverse primer, 5'-CTC TTT GAT GTC ACG CAG CAT TTC-3'. DNA contamination was excluded by performing PCR of each sample without first transcribing mRNA with MuLV reverse transcriptase.

RT-PCR products were separated on 1.5% (wt/vol) agarose gels and visualized by ethidium bromide staining.

In Situ Hybridization
Nonradioactive in situ hybridization (ISH) was preformed as described by Moorman and coworkers (7), using a 1.4-kb LGL1 digoxigenin-labled RNA probe. Rat LGL1 cDNA flanked by Kpn I and Sma I sites, subcloned into pBluescript KS, was used as a template for in vitro transcription. Riboprobes were generated after linearization and in vitro transcription by T3 or T7 polymerase labeled in the presence of dig-UTP. Briefly, tissue sections were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS). Pretreatment included proteinase K digestion (20 µg/ml, 15 min at 37°C), termination in glycine (0.2% in PBS, 15 min, room temperature), and postfixation in 4% paraformaldehyde/0.2% gluteraldehyde (15 min, room temperature). Sections were then washed in PBS and prehybridized for 1 h at 70°C. Riboprobes were added to freshly prepared hybridization solution (50% formamide, 5x SSC pH 4.5, 1% Boehringer block [Boehringer Mannheim, Laval, QC, Canada], 1 mg/ml yeast tRNA, 5 mM EDTA, 0.1% Tween-20, and 0.1% CHAPS) at a concentration of 1.5 ng/µl. Following denaturation at 100°C, the probe was incubated with tissue sections at 70°C for 18 h. Tissues were then washed with 50% formamide and 2x SSC at 65°C. The dig nucleic acid detection kit (Boerhinger Mannheim) was used for immunologic detection of the hybridized probe. Tissues were then dehydrated and mounted with xylene. Images were captured using a Leica digital imaging system.

Adjacent serial sections were kept for immunostaining with anti-vimentin and anti–{alpha}-actin antibodies, to identify the positively stained LGL1 cells.

Fetal Rat Lung Explant Culture
Time-mated Wistar rats (Charles River, St. Constant, PQ, Canada) were killed by diethylether excess on Days 12 or 13 of gestation. Embryos were aseptically removed from uterine deciduas, and fetal lung rudiments were isolated from the embryos by microdissection. Lung rudiments were cultured in serum-free, chemically defined medium (DMEM/F12 [1:1 mix, Gibco] supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 mg/ml ascorbic acid). Four to five lungs, gestational Day 13, were placed on each porous membrane insert (0.8 µm pore size, 25 mm diameter; Nucleopore, Whatman, NY) in each of six separate wells of a culture plate (Costar, Corning, NY) containing 800 µl of culture medium. Explants were incubated as floating cultures with an air–liquid interface for 48 h at 37°C in a humidified atmosphere of 5% CO2 in 3% O2. Lung explants were treated to either 30 or 40 µM of antisense, sense, or scrambled ODNs. Untreated explants (incubated in serum-free media alone) served as additional controls.

LGL1 Antisense Oligodeoxynucleotides
Phosphorothioate ODNs of 18 base pairs, targeted against sequences adjacent to the ATG initiation codon of LGL1 mRNA, were synthesized (DNA synthesis facility, HSC, Toronto, ON, Canada). The antisense (As1) LGL1 oligodeoxynucleotide was: 5'-GTT GTG CAG CAT GAG GAT-3'. The corresponding control sense (S1) was 5'-ATC CTC ATG CTG CAC AAC-3'. The scrambled sequence (Sc1) had the same nucleotides as As1, but with a randomly scrambled sequence: 5'-GTA CTT GAC GCT TGA GAA-3'. ODN sequences were modified by phosphothiolation to improve stability against nuclease degradation in culture (8, 9). The ODNs were purified by high-performance liquid chromatography, dissolved in double-distilled water, and quantified by ultraviolet spectrometry at a wavelength of 260 nm.

ODNs can be used to specifically inhibit the translation of gene products in the fetal rat lung (10, 11). Antisense (As) ODNs targeted to sequences adjacent to initiation codons (ATG) are very efficient in inhibiting translation, due to their ability to interfere with ribosome binding (12).

Quantification of Branching Morphogenesis
Lung explants (Days 12 or 13) were monitored daily by phase contrast microscopy. To quantitatively assess branching morphogenesis, the terminal airway buds were counted after 0, 24, and 48 h of incubation. Representative explants were photographed after 48 h in culture.

Western Blot Immunoanalysis
We determined the LGL1 protein content of Day 13 fetal lung explants, cultured for 48 h in the presence of ODNs and in rat lung rudiments from gestational ages 13–21. Briefly, lungs were homogenized in RIPA buffer containing triton X-100, sodium deoxycholate, sodium dodecyl sulfate, NaCl, Tris pH 8.0, and protease inhibitors. The total protein concentration was determined according to Bradford (13). Fifty micrograms of protein diluted with sample buffers was loaded in each well on a 10% (wt/vol) sodium dodecyl sulfate polyacrylamide gel. Transfer efficiency was determined by ponceau staining. A rabbit polyclonal lgl1 antibody raised against a synthetic peptide corresponding to amino acid residues 417–434 of rat lgl1 was generated (Medicorp, Montreal, PQ, Canada). Immunodetection was done according to standard protocol (14) with few modifications. Briefly, nonspecific binding was blocked by incubation with 2% normal goat serum (NGS) in TBS-T at room temperature for 90 min. The membrane was then incubated with rabbit anti-lgl1 at room temperature for 90 min, washed 4 times with TBS-T, and then incubated with horseradish peroxidase–conjugated goat anti rabbit IgG (1:10,000) in TBS-T containing 2% NGS. After TBS-T washes, blots were developed with an enhanced chemiluminescence kit. The films were quantified by laser densitometry.

The lgl1 antibody binds to a 52-kD band, consistent with the deduced size of the lgl1 protein. This band was substantially eliminated by preabsorbtion of the antibody with a 5-fold excess of the peptide used in generation of the antibody.

Immunohistochemistry
Rat lung tissue from gestational ages 16, 19, and 22 were fixed overnight at 4°C in Bouin's fixative, embedded in paraffin, and cut into 5-µm sections. Tissue sections were immunostained according to Hsu and coworkers (15), with few modifications. In short, antigen was retrieved by incubation in boiling 0.1 M sodium citrate (pH 6) for 10 min. Endogenous peroxidase activity was quenched with 1.5% (vol/vol) hydrogen peroxide in methanol for 45 min. Nonspecific binding sites were blocked using 5% (vol/vol) NGS and 1% (wt/vol) bovine serum albumin in PBS for 1 h at room temperature. Preliminary experiments determined optimal antibody concentrations. Rabbit antibody against lgl1 was used at 1:400 dilution, and mouse antibody against vimentin and {alpha}-actin were used at 1:250 and 1:1,000, respectively. Sections were incubated with primary antibodies overnight at 4°C, washed in PBS, and then incubated with secondary antibody, either biotinylated anti rat IgG at 1:500 or biotinylated anti mouse IgG at 1:400, for 2 h at room temperature. After washing with PBS, slides were incubated with ABC for 1 h at room temperature and developed using diaminobenzidine substrate (Vector Laboratories, Burlingame, CA). Tissues were then counterstained with hematoxylin and prepared for viewing.

Statistical Analysis
All data are presented as mean values ± SEM. Statistical significance was determined by two-way analysis of variance. Pairwise group comparisons were then assessed using Student-Neuman-Keuls test. Significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LGL1 mRNA in the Developing Lung
The developmental expression of LGL1 during lung development was examined using RT-PCR (Days 12–15) and Northern analysis (Days 16–21; Figure 1). LGL1 mRNA was detected as early as Day 12, corresponding to the onset of epithelial branching morphogenesis, with maximal expression at Day 21 of gestation.



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  		Figure 1. Temporal expression of LGL1 mRNA in fetal rat lung. (Left panel) Ethidium bromide–stained agarose gels showing LGL1 RT-PCR products (472-bp bands) from Days 12–15 of gestation after 27 cycles of amplification. Control ß-actin RT-PCR (515 bp; 25 cycles). (Right panel) Northern blots for LGL1 and control GAPDH at Days 16–21 gestation. LGL1 expression is evident from the earliest day tested (Day 12), and maximal at gestational Day 21.

 
By ISH, LGL1 mRNA was localized exclusively in the mesenchyme of Day 13 fetal rat lung, with a homogeneous distribution (Figure 2A), but with no detectable expression in the epithelium. This was confirmed by immunostaining of immediately adjacent serial sections with anti-vimentin antibodies, a mesenchymal marker (Figure 2B). This mesenchyme specific pattern of expression is still conserved later in gestation. In the cannicular (Day 18) stage of rat lung development, LGL1 mRNA is still found throughout the mesenchyme, but there is also an increasing population of positively stained LGL1 cells that localize in smooth muscle cells surrounding large and small blood vessels and large airways (Figure 2C). This was confirmed by immunostaining of immediately adjacent sections with antibody against {alpha}-actin, a smooth muscle cell marker (Figure 2D). During the saccular stage of rat lung development (Days 20–22), LGL1 mRNA spreads throughout the mesenchyme, with patchy expression in the smooth muscle cells of blood vessels and large airways (Figure 2E).



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  		Figure 2. LGL1 mRNA localization by non-radioactive in situ hybridization. Mesenchyme-specific labeling is present on gestational Days 13 (A), 20 (C), and 22 (E) (purple staining). Mesenchymal cells were identified by immunochemistry for vimentin (brown) on adjacent serial tissue sections (B). During the pseudoglandular stage of lung development (A), LGL1 mRNA is widespread in the mesenchyme. Later in gestation (C, E), the mRNA is primarily localized to cells positive for smooth muscle {alpha}-actin (brown, D, F) found in close proximity to airways and blood vessels (v). (E, inset) Negative control in situ using sense LGL1 probe. Arrows point to the presence, and arrowheads to the absence, of the signal. Magnification is x250.

 
lgl1 Protein in the Developing Lung
lgl1 protein is expressed in fetal lung as early as Day 18 by Western blotting (Figure 3A), and on Day 16 by immunohistochemistry (Figure 4A), the earliest stages studied by either method. Both methods showed that lgl1 protein levels in the lung gradually increase, reaching a maximum on Day 21.



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  		Figure 3. Temporal expression of lgl1 protein in embryonic rat lung. Western blot analysis on whole lung tissue extracts in the fetal rat lung (upper panel) and lgl1 peptide competition (bottom panel). At all gestational ages tested (Days 18–21), lgl1 antibody identifies a band at the expected 52 kD. Detection of lgl1 protein is substantially prevented by adding a 5-fold molar excess of the peptide used in the generation of the antibody.

 


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  		Figure 4. Immunostaining for lgl1 protein, using a polyclonal rabbit anti rat lgl1 antibody. In Day 16 (A), Day 20 (C), and Day 22 (E) fetal rat lung sections, lgl1 protein (brown) is localized to the mesenchyme in a patchy fashion. Immunohistochemistry for vimentin (brown) labeled all mesenchymal cells of the lung in adjacent serial tissue sections (B, D, F). No background staining was observed in tissue sections incubated with nonimmune rabbit IgG (not shown). Arrows point to the presence of the signal. Magnification is x400.

 
lgl1 protein, like the LGL1 mRNA, was localized to mesenchymal cells at all gestational ages tested, but the lgl1 positively stained cell populations were not spread homogeneously throughout the mesenchyme. Rather, "patchy" clumps of positively stained cells, localized in the mesenchyme, were observed (Figures 4A, 4C, and 4E). Earlier in gestation (Day 16), positively stained clumps of cells are adjacent to small airways and large vessels (Figure 4A). Later in gestation (Day 22), positively stained cells are still adjacent to small airways (Figure 4E).

Disruption of Endogenous lgl1 Activity Results in Impaired Branching Morphogenesis
Untreated fetal rat lungs isolated at gestational Day 13 have right and left mainstem bronchi (Figure 5A) and the beginnings of lobar bronchi. After 2 d in explant culture in medium only (Figure 5B, M), branching of the bronchi has formed a respiratory tree and the lungs have grown, but less than in the in vivo situation at gestational Day 15. To assess the biologic function of lgl1 in lung development, Day 13 embryonic lung rudiments (littermates of the M group) were also cultured in the presence of sense (Figure 5C, S), scrambled (Figure 5D, SC) or antisense (Figure 5E, As) LGL1 ODNs. After 2 d, As-treated explants showed significant airway dilatation, decreased septation, and reduction in number of terminal airway buds (Figure 5E) compared with all other groups. There were no corresponding changes in morphology in explants exposed to S or Sc ODNs compared with M.



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  		Figure 5. ODNs antisense (As) to LGL1 specifically inhibit branching morphogenesis in fetal rat lung explant cultures. (A) Gestational Day 13 lung rudiment immediately after dissection. Trachea and left and right mainstem bronchi are present. (B) Day 13 lung, showing normal branching morphogenesis after culture for 48 h in serum-free medium. Lungs were maintained in explant culture for 48 h in 3% O2/N2 in the presence of 30 µM LGL1 ODNs: (C) Sense, (D) random "Scrambled," or (E) Antisense sequence.

 
The amount of branching was quantified by counting the number of terminal airway buds at the end of 48 h. Explants exposed to 30 or 40 µM antisense ODNs displayed branching that was decreased by 47 and 57%, respectively (Figure 6), suggesting that inhibition of terminal branching by LGL1 antisense ODNs is dose dependent. The inhibitory effect of As LGL1 ODNs on branching morphogenesis is more pronounced when lung rudiments are cultured from Day 12 (Figure 7). At this stage, the tracheal epithelium is just starting to bud and form mainstem bronchi. The airways of Day 12 As-treated (40 µM) explants do not branch, but simply dilate over time, whereas progressive branching occurs in all control-treated explants (Figures 8B–8G).



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  		Figure 6. LGL1 As ODNs decrease the number of terminal airway buds. Lung rudiments were maintained in serum-free medium for 48 h in the presence of either 30 µM or 40 µM ODNs, antisense (lightly shaded bars), sense (black bars), or scrambled (darkly shaded bars). Medium-shaded bars, medium. Results are the sum of four lungs/condition, in each of three independent experiments. *P < 0.02, **P < 0.001, compared with controls.

 


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  		Figure 7. Quantitation of branching over time. Day 12 lung rudiments were cultured for 48 h in the presence of 40 µM of As (black lines), S (pale lines), Sc ODNs (darkly shaded lines), or medium only (lightly shaded lines). Terminal airway buds were counted every 24 h. Results are based on four lungs/condition, in each of three independent experiments. *P < 0.05, compared with controls.

 


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  		Figure 8. Effect of antisense LGL1 ODNs on branching morphogenesis over time. Day 12 lungs were maintained for 48 h in the presence of 40 µM LGL1 ODNs. (A) Freshly isolated Day 12 fetal rat lung rudiment. Explants treated to antisense (D, G), sense (C, F), or medium only (B, E). Progressive branching over time is evident in sense, scrambled (not shown) and medium controls, but not in antisense-treated lungs. The difference is more pronounced after 48 h than after 24 h.

 
Effect of Antisense LGL1 ODN Treatment on LGL1 mRNA and lgl1 Protein Expression in Explanted Rat Lungs
The specificity of the reduction in LGL1 mRNA levels in response to treatment with anti-LGL1 antisense ODN was confirmed by the lack of response to sense ODN, and by the lack of alteration in ß-actin mRNA levels (Figure 9A). To determine the degree of inhibition of endogenous lgl1 protein production by antisense LGL1 ODNs, the lgl1 protein content of Day 13 explants cultured for 48 h was assessed by Western blotting and quantified by densitometry. The treatment with LGL1 antisense ODNs significantly reduced the total amount of lgl protein in cultured explants by 51% (Figure 9B). No reduction of lgl1 protein content was observed in any control explants (Figure 9B).



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  		Figure 9. Antisense ODNs inhibit LGL1 mRNA expression (A) and lgl1 protein (B and C) in fetal rat lung. Day 13 lung rudiments were cultured for 48 h in the absence (M, medium alone) or presence of 30 µM ODNs: antisense (As), sense (S), or scrambled (Sc). (A) LGL1 mRNA levels, as assessed by low-cycle RT-PCR, were abrogated by exposure to As, but not S or Sc ODNs. Control ß-actin mRNA levels were unaltered by all ODNs. (B) lgl1 protein expression was detected using a polyclonal anti-lgl1 antibody. (C) Bands were quantified by densitometric analysis and normalized to control cultures exposed to medium only. lgl1 protein levels in As-treated explants are reduced by 51 ± 2% relative to controls (mean ± SEM; P < 0.05; n = 3 independent experiments, four lungs/condition).

 
Correction of LGL1 Sequence
Trexler and coworkers (6) recently reported a correction to our published LGL1 sequence (5), extending the open reading frame of LGL1 in the 3' direction to include two copies of a module they named LCCL. Independently, we had carefully repeated sequencing of our original LGL1 cDNA, identifying the same single nucleotide omission in the 185th amino acid codon. We confirm the same pair of LCCL modules in the extended 3' portion of the predicted lgl1 protein sequence.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present report, we provide evidence implicating lgl1 protein in the regulation of airway branching morphogenesis of developing fetal rat lung. The previously reported high degree of homology between human and rodent LGL1 suggested that this protein serves one or more biologically important functions. We initially sought to identify its function during its earliest period of expression in developing lung. Incubation of gestational Day 12 and 13 embryonic rat lung rudiments with antisense LGL1 ODNs specifically inhibited LGL1 expression at both protein and mRNA levels, while significantly inhibiting airway branching morphogenesis. Antisense-treated explants had dilated terminal airway buds and a decrease in the number of secondary and tertiary branches relative to a variety of controls.

The magnitude of the effects of antisense inhibition of lgl1 expression was dependent on both the dose (concentration) of ODN and on the duration of exposure. At the highest doses and durations employed, lgl1 protein levels were cut in half, not eliminated. Our finding of such a profound derangement in bronchial tree anatomy in the ongoing presence of 50% of normal lgl1 protein levels suggests the formation of new airway branches must be very sensitive to lgl1. This might suggest a requirement for local interaction of lgl1 with other protein(s) in a specific concentration or molar ratio to be effective. Alternatively, local lgl1 protein concentrations in the cellular microenvironment at the precise sites of epithelial branching may have been more profoundly reduced than reflected by the overall explant concentrations. The fact that virtually complete abrogation of LGL1 message results in only a 50% reduction in lgl1 protein on Western analysis suggests that lgl1 protein must have a relatively long half-life.

We provide the first evidence of the mesenchymal specificity of lgl1 protein expression in fetal rat lung using immunohistochemistry, and extend localization of LGL1 mRNA using ISH in the fetal period of rat lung development. LGL1 mRNA was found exclusively in mesenchyme, with no observed epithelial localization. Maximal LGL1 expression occurs late in gestation (Days 20–21), when the branching of the conducting airways is complete, and the formation of alveolar (gas-exchange) units is beginning in earnest (16). During this developmental stage, the mRNA and protein become progressively restricted to mesenchymal cells, positive for smooth muscle actin, which are in close proximity to airway epithelial cells. Interestingly, these same actin-positive cells have been reported to regulate the formation of new alveolar units (17). Our data, taken together with these findings, suggest lgl1 may also have a role in the regulation of alveolarization. The period of late gestation in which LGL1 expression is maximal is marked not only by the process of alveolarization, but also by the onset of surfactant production and rapid epithelial cell proliferation. We speculate that lgl1 may also serve additional, as yet unreported function(s) during late fetal lung development.

Branching morphogenesis, proliferation, and cytodifferentiation of the fetal lung airway epithelium are controlled locally by the surrounding mesenchyme through the integrated action of a range of secreted growth factors, ECM components, and paracrine/autocrine factors. These regulatory molecules include transcription factors acting upstream (2, 18, 19) as well as other growth and differentiation factors (19–23) that are involved in the complex network of reactions essential to terminal lung organogenesis. These factors act through incompletely understood mechanisms (1, 24). Communication between mesenchyme and epithelium are known to influence the branching capabilities of the primitive lung epithelium. Recombination studies have shown that tracheal mesenchyme inhibits branching of tracheal or normally branching bronchial epithelium, whereas bronchial mesenchyme is able to induce epithelial branching and differentiation to a more distal phenotype (4). Soluble and diffusible factors mediate this instructive ability of lung mesenchyme on epithelium (3, 4).

LGL1 mRNA is glucocorticoid-inducible and conserved in rat, mouse, and human. The deduced open reading frame predicts an lgl1 protein of 497 amino acid residues, belonging to the Cysteine RIch Secreted Protein (CRISP) family. CRISP proteins have a common carboxy terminal cysteine motif (CX8-CX8-CX8-CX8-CX8) and a 22 amino acid hydrophobic signal sequence at the amino terminus. Various CRISP proteins function as cell adhesion molecules during development (25–30). CRISP 1/AEG is necessary for gamete binding and fusion, CRISP 2/Tpx1 is required for binding of sertoli cells and spermatocytes of the developing testes, and a more recent addition to this diverse family (cocoacrisp) is believed to be involved in septation of the midbrain during development (31). Other CRISP proteins, such as p25Ti, are believed to function as serine protease inhibitors, which play important roles in regulating ECM proteolysis (32).

lgl1 may exert its action through direct action(s) of its own on airway epithelial cells, through interactions with ECM components, or with other factors, or a combination of these effects. lgl1 may play a direct role in regulating lung branching morphogenesis by mediating adhesion between mesenchyme and epithelium, in turn enabling the passage of materials and information between the two cell types. Alternatively, the protease-inhibitory activity of lgl1 may modulate the local expression/concentration of growth factors in the microenvironment of the precise sites of epithelial branching. lgl1 may also directly interact with these growth factors to influence lung branching morphogenesis and epithelial cell differentiation. Finally, lgl1 may directly act on airway epithelial lung cell proliferation and/or apoptosis to regulate growth and patterning.

Clues to possible lgl1 interactions come from observations that disruption of fetal lung expression of other known growth factors results in explant phenotypes similar to that resulting from disruption of LGL1. When hepatocyte growth factor or keratinocyte growth factor (of mesenchymal origin) are disrupted by incubation with antisense ODNs, branching morphogenesis is inhibited. Airways become dilated and the terminal buds form fluid-filled cyst-like lesions (19, 22, 33). Another growth factor with related activity, fibroblast growth factor-10, is chemotactic for growing epithelial cells, enhancing the formation of epithelial buds in explant lung cultures (34, 35). Other growth factors implicated in airway branching morphogensis and/or alveolarization include platelet-derived growth factor, transforming growth factor-ß2 and -ß3, epidermal growth factor, and tumor necrosis factor-{alpha}.

Glucocorticoids have a profound effect on all aspects of lung development (36). Treatment in early gestation of fetal lung explants with pharmacologic doses of glucocorticoid causes a distorted branching pattern characterized by dilated lung tubules and a decrease on the number of terminal airway buds (21). Such a pattern is more consistent with that seen following disruption of LGL1 expression, rather than due to the enhanced lgl1 effects one might expect in response to glucocorticoid exposure. This underlines the point that the final outcome of fetal lung development is the result of the integrated effects of a variety of factors. Moreover, this observation suggests that other glucocorticoid-responsive growth factors (such as transforming growth factor-ß3 or tumor necrosis factor-{alpha}) known to modulate the morphology and differentiation of the developing lung may be prime candidates for interactions with lgl1.

Our original published LGL1 cDNA sequence omitted a nucleotide, which both we and Trexler and coworkers (6) confirm. Correction of this error led them to identify two LCCL modules in the newly extended lgl1 predicted sequence, which they have proposed may confer certain functional, potentially antimicrobial, properties on lgl1. This proposal awaits empirical testing.

In conclusion, we report the localization of LGL1 mRNA and protein expression during the pseudoglandular stage of fetal rat lung development, the period of airway branching morphogenesis. Antisense LGL1 ODNs specifically inhibit branching while decreasing lgl1 protein content in these explants, suggesting that lgl1 plays a role in the normal branching morphogenesis of the fetal lung. We speculate that lgl1 may act as a cell adhesion molecule, regulating mesenchymal–epithelial interactions that direct airway branching morphogenesis. lgl1 may also act to regulate protease activity, modulating the control of branching exerted by ECM components. Further studies will be necessary to elucidate the mechanism(s) by which LGL1 regulate(s) branching morphogenesis during lung development.

Received in original form April 15, 2002

Received in final form October 3, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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