Targeted Disruption of NeuroD, a Proneural Basic Helix-Loop-Helix Factor, Impairs Distal Lung Formation and Neuroendocrine Morphology in the Neonatal Lung^*,S⃞

EXPERIMENTAL PROCEDURES

Mice—Fibrillin-1-deficient mice and NeuroD-deficient mice were bred and maintained as described (1, 10). We used the NeuroD-deficient mice maintained in 129/SvJ background (11, 12). Progeny from heterozygote matings were used for the described studies. These mice were housed in a facility accredited by the American Association of Laboratory Animal Care, and the animal studies were reviewed and approved by the institutional animal care and use committee of The Johns Hopkins School of Medicine.

Expression Profiling Analysis—Lungs from fibrillin-1-deficient mice and littermate controls at postnatal day 1 (PD1) and postnatal day 5 (PD5) were harvested and quickly frozen. Lungs from two groups of mice (wild-type and fibrillin-1-deficient) at each time point were pooled in groups of two or three. Total RNA was extracted using TRIZOL reagent (Invitrogen). Expression profiling was done with Affymetrix A734 GeneChips (48 chips for this study), containing ∼7000 full-length genes and ∼1000 expressed sequence tags. Biotinylated cRNAs prepared from total RNAs were hybridized to U34A GeneChips in duplicate. Fluorescent signals were measured with a Hewlett Packard G2500A Gene Array Scanner. Data were analyzed with Affymetrix suite 5.0, corrected for saturation, and evaluated with GeneSpring 5.0 software. The primary gene expression data can be found on the World Wide Web at the NCBI GEO data base (GDS242).

Morphology and Histology—Three to five mice of each genotype were studied at the noted ages. For histologic and morphometric analyses, mouse lungs were inflated at 25 cm of H₂O and fixed with 4% paraformaldehyde in low melt agarose. The lungs were equilibrated in cold 4% paraformaldehyde overnight, sectioned, and then embedded in paraffin wax. Sections were cut at 5 μm and either stained with hematoxylin and eosin or processed for immunohistochemistry.

Morphometry—Measurements were performed on hematoxylin and eosin-stained sections taken at intervals throughout both lungs. Slides were coded, captured by an observer, and masked for identity. Ten to fifteen images per slide were acquired at ×20 magnification. Mean chord lengths and mean linear intercepts were assessed by automated morphometry with a macro operation performed by Metamorph Imaging Software (Universal Imaging, Molecular Devices, Downingtown, PA).

Semiquantitative PCR—Total RNAs were isolated from mouse lung by homogenization in TRIZOL reagent (Invitrogen). First-strand cDNA was prepared from 1 μg of each RNA sample using the Invitrogen Superscript II RT kit and random hexamer primers. The primers for NeuroD amplification were 5′-ATCGTCACTATTCAGAACCTT-3′ (forward) and 5′-TTCCTCGTCCTGAGAACTGAG-3′ (reverse). The primers for β-actin amplification were 5′-TTGCTGACAGGATGCAGAAG-3′ (forward) and 5′-ACATCTGCTGGAAGGTGGAC-3′ (reverse). All samples were tested in the absence of reverse transcriptase to control for DNA contamination. PCR products were analyzed on agarose gels. All results were confirmed with at least three different RNA samples.

Western Blot Analysis—Total cell lysates were extracted in M-Per buffer from Pierce. Protein concentrations were determined using the Bio-Rad Protein Assay. Aliquots of 30-50 μg of protein were boiled and then loaded onto Tris-HCl gels and transferred electrophoretically to nitrocellulose membranes. Membranes were incubated with the primary antibody for 1 h at room temperature. Detection was by the Pierce West Dura ECL detection system. Primary antibodies and dilutions were as follows: β-actin (1:1000; Abcam), c-Myc (1:1000; Abcam), NCAM (1:500; Chemicon), NCAM (DHSB (13); 1:500), ACTH (1:100; Abcam), Hes-1 (1:200; Chemicon), Mash1 (1:250; Abcam), Gfi-1 (1:250; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and chromogranin A (1:250; Santa Cruz Biotechnology).

Immunohistochemistry—Tissue sections were deparaffinized and rehydrated in an ethanol series. Sections were blocked for nonspecific binding with 3% normal serum from chicken and incubated with the primary antibodies for 1 h at room temperature. For immunofluorescence, sections were then incubated with secondary antibodies at 1:200 for 30 min at room temperature (Molecular Probes). Sections were counter-stained with 4′,6-diamidinio-2-phenylindole (DAPI) and mounted with Vectashield hard set mounting medium (Vector Laboratories). For immunohistochemistry, following incubation with the primary antibody overnight at 4 °C, slides were washed with PBST, incubated with an appropriate biotinylated secondary antibody (Jackson ImmunoResearch), and developed by using ABC protocol. 5-bromo-4-chloro-3-indolyl phosphate, and 3,3′-diaminobenzidine detection reagents (Vector Laboratories). Antibodies were used at the following concentrations: NeuroD (1:100; Santa Cruz Biotechnology), PGP9.5 (1:500; Dako), synaptophysin (1:100; Zymed Laboratories Inc.), calcitonin gene-related peptide (CGRP; Sigma; 1:3000), proliferating cell nuclear antigen (1:50; Santa Cruz Biotechnology), Ki67 (1:50; Santa Cruz Biotechnology), NCAM (1:250; Chemicon), CC10 (1:1500; gift from G. Singh), TTF-1 (1:500; Dako), and active caspase-3 (1:25; Abcam).

Neuroendocrine Cell Quantitation—For details of the protocol, refer to Ref. 14. To characterize NE differentiation in fibrillin-deficient mice and wild-type littermates, we counted the total number of synaptophysin, CGRP, and PGP9.5 immunoreactive solitary PNECs and NEBs (neuroendocrine foci) in formaldehyde-fixed, paraffin-embedded lung sections. This was correlated to the number of airways in the same sections (foci/airway). To determine the size of NEBs in these same mice, pictures of each NEB were acquired using MetaMorph® software (Universal Imaging/Molecular Devices, Downingtown, PA). The immunoreactive area was manually outlined and measured using the same software, and the size in μm² for each individual NEB was tabulated. Statistical analysis was performed using the Mann-Whitney test (SigmaStat).

For differential measurement of solitary PNECs versus NEBs, lung tissue sections were co-stained with PGP9.5 (Dako) and DAPI (Molecular Probes) as described under “Immunohistochemistry.” The total number of airway cells was determined by counting cells per 20× field staining positive for DAPI in the airway. Neuroendocrine cells were quantified based on the total number of cells staining positive for PGP9.5. Those cells positive for PGP9.5 were characterized as either components of NEBs (cluster of ≥3 neuroendocrine cells) or solitary neuroendocrine cells based on morphology. These two neuroendocrine cell types were then quantified and normalized to airway basement membrane length in μm × 10⁴.

Cell Culture and Transfection—The MLE12 cell line, an immortalized mouse lung epithelial cell line that maintains characteristics of type II alveolar epithelial cells (15), was a kind gift from Dr. Jeffrey Whitsett (University of Cincinnati) and was maintained in Dulbecco's minimal essential medium/F-12 medium (1:1) supplemented with 2% fetal calf serum. Subconfluent cells were transfected with Lipofectamine Plus (Invitrogen) according to the manufacturer's protocol and harvested after 24 or 48 h. All transfections were performed in triplicate. The Myc-tagged murine NeuroD cDNA construct was a gift from Drs. David Turner (University of Michigan) and Jacqueline Lee (University of Colorado).

For conditioned media experiments, medium was removed from transiently transfected cells after 24 h and replaced with serum-free medium. Supernatants were harvested after 24 and 48 h, and cells were removed by low speed centrifugation. After washing with phosphate-buffered saline, serum-starved MLE12 cells were treated with supernatants from transfected cells. For proliferation assays, cells were incubated for the designated time periods after treatment or transfection and then assayed per protocol with the Promega CellTitre 96 nonradioactive proliferation assay.

Data Analysis—Absolute analysis of Affymetrix image data were done using Affymetrix Microarray Suite 4.0. Values of intensity differences as well as ratios of each probe pair are used for determination of whether a transcript is called “present” or “absent.” The differentially expressed gene lists for each time point were generated using GeneSpring software (Silicon Genetics, CA). In GeneSpring, those genes showing at least four Affymetrix present calls were selected prior to further data processing. Welch's t test was performed to calculate the probabilities of significant gene expression changes. Since the probe sets were tested multiple times, we used a highly stringent p value cut-off (p < 0.001) to reduce the number of false positives to less than 1 in 1000.

Statistical Analysis—Analysis of variance and Student's t-tests were used to determine differences between groups. In the morphometric study, statistical differences were determined by the unpaired Student's t test for comparison of septal measurements. Values for all measurements were expressed as means ± S.E., and p values for significance were designated at <0.05.

RESULTS

Fibrillin-1 Deficiency Is Associated with Airspace Defects and Reduced Expression of NeuroD—Neonatal mice deficient in fibrillin-1 display increased airspace caliber at PD5, a time point that coincides with the initiation of alveolar septation in the murine lung (Fig. 1, A and B). In order to identify candidate mediators of distal lung development, whole organ gene expression profiling was performed on wild-type and fibrillin-1-deficient mice at PD1 and PD5. Upon pairwise comparison, we reasoned that candidate genes would be (a) down-regulated in the mutant lung compared with wild-type lung on PD5 and (b) increased in the wild-type PD5 lung compared with the wild-type PD1 lung (Fig. 1C). Interestingly, NeuroD, a basic helix-loop-helix protein, demonstrated a 25-fold induction in the wild-type PD5 lung compared with the wild-type PD1 lung and a 5-fold reduction in the fibrillin-1 PD5 lung compared with wild-type littermates. These results were confirmed by semiquantitative RT-PCR (Fig. 1D). Since transforming growth factor-β-neutralizing antibody restores normal septation in fibrillin-deficient mice, we determined whether NeuroD levels responded to this maneuver. We found a 10-fold induction of NeuroD expression in the wild-type lung upon treatment with antibody treatment but only a 1.5-fold induction in the mutant lung. This finding suggests that although NeuroD deficiency may contribute in a minor way to the lung phenotype in the fibrillin-1-deficient mouse, it is not a predominant mechanism. Since fibrillin-1 deficiency is associated with complex disturbances involving tissue structure, cytokine dysregulation, and recently angiotensin signaling, the lack of an intimate mechanistic association between NeuroD signaling and the fibrillin-1 phenotype is not unexpected (1, 16-19). Nonetheless, we surmised, NeuroD might play a role in lung maturation distinct from the context of fibrillin-1 deficiency.

NeuroD-deficient Mice Have Impaired Alveolar Formation Secondary to Reduced Airspace Epithelial Proliferation—Our expression profiling strategy was designed to identify determinants of distal lung formation. In order to establish whether NeuroD is critically involved in distal lung morphogenesis, we examined the lung phenotype of mice deficient in NeuroD. These mice had known defects in pancreatic, enteroendocrine, cerebellar, and hippocampal morphogenesis, but their lung phenotype has not been examined. The homozygous mutant mice had a marked increase in distal airspace caliber at PD5 and PD10 compared with littermate controls, similar to our findings in fibrillin-1-deficient mice (Fig. 2, A and B). By adulthood, the airspace caliber remained significantly larger than the wild type controls but showed no progression compared with the PD10 mutant lung. Using markers of alveolar epithelial cells (TTF-1) and airway epithelial cells (CC10), we found preserved epithelial differentiation in the neonatal mutant lung (Fig. 2, C and D). Since airspace simplification can result from reduced proliferation or increased cell death in the lung parenchyma, we examined distal lung proliferation and apoptosis in the developing and adult lungs. We found a dramatic reduction (27%) in proliferation of airway and alveolar epithelial cells in the developing airspace and airway of the mutant lung (Fig. 3, A and B) but no difference in apoptosis by terminal dUTP nick-end labeling and active caspase 3 staining (data not shown). This profile contrasts with our finding of increased apoptosis but normal proliferation in fibrillin-1-deficient murine lungs. Importantly, the mitotic index falls to trivial levels in the mutant and wild-type lung by adulthood (Fig. 3B) and is probably responsible for the inability to correct the airspace enlargement after lung maturation. Taken together, these data suggest that the early airspace phenotype of these mice is not attributable to an altered epithelial differentiation program but rather to reduced epithelial proliferation.

NeuroD-deficient mice in the Sv/129 background have ∼30-40% perinatal mortality, strictly observed within the first week of life (12). Between the time points of PD5 and adulthood, we observed no mutant pup loss. Since PD10 is beyond the point of expected pup loss but still within the terminal phase of lung alveolar development, our demonstration of persistent airspace enlargement suggests that the PD5 phenotype did not merely reflect mice that would ultimately die before adulthood (Fig. 2, A and B). However, in order to further establish that the PD5 and PD10 mice were comparable, we evaluated Mendelian ratios of respective genotypes. Assessment of 40+ mice at each time point revealed 26, 52, and 22% versus 23, 57, and 20% (NeuroD^+/+, NeuroD^+/-, and NeuroD^-/- genotypes), respectively, at PD5 and PD10. We also found no reduction in weight in the mutant mice at either PD5 or PD10, suggesting that mice assessed at these two time points were comparable (see supplemental materials).

NeuroD Is Expressed in Pulmonary Neuroendocrine Cells, and Fibrillin-1-deficient Mice Have Altered Neuroendocrine Cell Morphology—Mice with a targeted deletion in Mash1, another proneural bHLH factor, do not develop pulmonary neuroendocrine cells and die in the neonatal period (20). Because neuroendocrine cells probably perform an endocrine function for the developing lung by secreting growth factors that are instructive for distal lung morphogenesis (10, 11), we considered whether NeuroD might be involved in neuroendocrine cell differentiation and function (21, 22). We used the panneuroendocrine marker PGP9.5 to assess the morphology and abundance of neuroendocrine cells. Immunohistochemical studies employing antibodies against NeuroD and PGP9.5 revealed that NeuroD is expressed in pulmonary neuroendocrine cells within the distal airway (Fig. 4A). To explore the possibility that the reduction in NeuroD expression in the fibrillin-1-deficient lung was secondary to an abnormality in the neuroendocrine compartment, we examined the quotient of neuroendocrine cells in fibrillin-1-deficient lungs. We have previously reported that reduced neuroendocrine cell number and/or mass area correlate with altered neuroendocrine maturation (14). The total number of neuroendocrine foci (composed of NEBs and PNECs) was significantly higher in PD5 fibrillin-deficient mice compared with wild-type littermates (Fig. 4B). However, the average size of NEBs in fibrillin-deficient animals was about 30% smaller than that in control mice (p = 0.056) (Fig. 4C). Thus, although there was a modest increase in neuroendocrine foci in the fibrillin-1-deficient lung (Fig. 4B), we found a trend toward reduction in the size of NEBs in the NeuroD mutant lung (Fig. 4C), consistent with impaired neuroendocrine maturation.

NeuroD-deficient Mice Have an Alteration in Pulmonary Neuroendocrine Morphology—Given that inactivation of the proneural bHLH factor Mash1 prevents pulmonary neuroendocrine differentiation (20), we hypothesized that a similar impairment might exist in NeuroD-deficient mice. Surprisingly, we found that neuroendocrine cells were present throughout airway and airspace development in the mutant lung (Fig. 5A). Pulmonary neuroendocrine cells exist in two distinct morphologies: solitary PNECs and NEBs. The former subserves a primary secretory function, liberating mitogenic and vasoactive amines, and the latter is thought to act as a hypoxia and/or chemoreceptor (23, 24). When we quantified the number of solitary PNECs versus NEBs in NeuroD-deficient mice, we found (a) a significant increase in the number of NEBs in the mutant lung, similar to the fibrillin-1 deficient lung, but (b) a striking reduction in solitary PNECs in the mutant lung (Fig. 5, B-D). Neuroendocrine hyperplasia attends several inflammatory and fibrotic lung disorders, such as bronchopulmonary dysplasia and cystic fibrosis, and is thought to contribute to these conditions via the liberation of profibrotic cytokines. Because solitary PNECs are thought to function primarily as endocrine cells without neural innervation, we considered whether the marked reduction in the number of these cells correlated with impaired liberation of mitogenic amines and reduced distal airspace cell proliferation.

To determine whether the time course of the altered neuroendocrine morphology correlated with the evolution of airspace lesion, we quantified neuroendocrine cells during murine lung development (PD5 and PD10) and early adulthood (6 weeks). We found a persistent increase in NEBs and reduction in solitary PNECs during the first 2 weeks of postnatal life but a full normalization by adulthood (Fig. 5, E and F). Thus, by temporal criteria, the altered neuroendocrine morphology may partially contribute to the airspace lesion, plausibly via suboptimal liberation of mitogenic amines by solitary PNECs.

Since NeuroD-deficient mice were maintained in the Sv/129 strain but the fibrillin-1-deficient mice were maintained in a mixed Sv/129:C57Bl/6 background, we wanted to establish that the baseline relative neuroendocrine profile was comparable in these two strains. We quantified solitary PNECs, NEBs, and total neuroendocrine cells in PD5 mice of each background strain and found a reduced proportion of NEBs in both strains (18 ± 5% NEB/PNEC ratio in Sv/129 strain and 30 ± 2% in Sv/129/C57Bl/6 background). Accordingly, the reversal of this ratio in the NeuroD-deficient mice has broad relevance to both background strains.

NeuroD Overexpression Confers a Neuroendocrine Phenotype—NeuroD family members promote progenitor cell differentiation in neural and endocrine tissues. Although neuroendocrine cells are present in the primitive vertebrate lung, their resident precursor has not been identified. Notably, lineage tracing of surfactant protein C-expressing (SPC⁺) progenitors in the developing murine lung did not demonstrate CGRP⁺ neuroendocrine progeny in the perinatal period (25). By contrast, Wuenschell et al. (26) characterized a pluripotent cell type in the developing lung that expressed neuroendocrine, airway, and alveolar epithelial markers, suggesting that a common precursor might exist. In order to determine whether the overexpression of NeuroD in a nonneuroendocrine-derived cell line was sufficient to induce the neuroendocrine phenotype, we overexpressed a Myc-tagged NeuroD in a nonneuroendocrine mouse lung alveolar epithelial cell line (MLE12). Remarkably, NeuroD expression induced robust expression of chromogranin A and ACTH, both neuroendocrine markers and p21 expression (Fig. 6A). We did not find repression of surfactant protein B or TTF-1, markers of lung epithelial cells (data not shown). Furthermore, using two different antibodies for NCAM detection, we found that NeuroD expression induced high molecular weight NCAM140 expression (Fig. 6B). Since Ito described opposing effects of Mash1 and Hes-1 in neuroendocrine maturation, we surveyed both of these candidate mediators after NeuroD overexpression (27). Although we observed no change in the levels of Hes-1, we did see an induction of Mash1 at 24 h, consistent with its critical early role in neuroendocrine differentiation (Fig. S2). We also observed an induction of Gfi-1, a recently identified neuroendocrine determinant, in NeuroD-overexpressing cells (28). In summary, the whole cell studies suggest that NeuroD overexpression is sufficient to confer a neuroendocrine phenotype to lung epithelial cells.

NeuroD Overexpression Induces Autocrine and Paracrine Proliferation in Lung Epithelial Cells—Although they represent a relatively sparse cell population in the lung, neuroendocrine cells can alter geographic proliferative and morphogenic programs via autocrine and paracine effects on local epithelial cells. Notably, we have recently shown that mice that are deficient in Gfi1, a determinant of neuroendocrine cell morphogenesis, display reduced airway cell proliferation after naphthalene injury (29). In order to connect the altered neuroendocrine developmental phenotype we observed in NeuroD deficient lungs with reduced epithelial proliferation, we examined whether overexpression of NeuroD in lung epithelial cells increased proliferation. We found a significant increase in proliferation in cells transfected with NeuroD compared with cells transfected with a control vector (Fig. 7A). Since we observed reduced proliferation in cells that did not express NeuroD in the airway and epithelium of the distal lung, we examined whether NeuroD-transfected cells secreted mitogenic factors (a known property of neuroendocrine cells) that induced proliferation in cells not expressing NeuroD. Conditioned medium from cells transfected with NeuroD promoted a >55% increase in proliferation compared with medium from vector-transfected cells (Fig. 7B). These data suggest that NeuroD expression supports autocrine and paracrine proliferative signaling in lung epithelial cells. This mechanism may account for the widespread reduction in proliferation in the NeuroD-deficient lung.

DISCUSSION

Although neuroendocrine cells represent less than 1% of epithelial cells in the vertebrate lung, they are active participants in lung repair and malignant transformation (reviewed in Ref. 30). Consistent with previous reports of NeuroD expression in neuroendocrine tumors (31), we show here that NeuroD is expressed in the neuroendocrine cells of the developing lung. We also describe for the first time a temporal association between the maturation of the pulmonary neuroendocrine compartment and distal lung morphogenesis. Reminiscent of its role in the gastrointestinal tract and the pancreas, one of the pulmonary effects of NeuroD deficiency is an alteration in the morphology of the pulmonary neuroendocrine compartment. However, it follows that for these relatively sparse PNECs to participate in global morphogenic functions, paracrine effects mediated by secreted factors must be operative. We show here that a disturbance in PNEC morphology conferred by a deficiency in NeuroD may contribute to a more global morphogenic perturbation. Several investigators have shown that mitogens (bombesin-like peptides, gastrin-releasing peptide, CGRP, etc.) synthesized and secreted by PNECs induce airway and alveolar epithelial cell proliferation in explant and whole cell systems (32-34). Sunday et al. (35) have also demonstrated adverse effects of exaggerated pulmonary neuropeptide secretion in the neonatal setting, possibly contributing to bronchopulmonary dysplasia and other lung diseases of prematurity. Cutz and co-workers (36) have shown that the lung parenchymal phenotype of cystic fibrosis transmembrane conductance regulator-deficient mice includes disturbances in PNEC and NEB morphogenesis. Taken together, the mitogenic and morphogenic consequences of PNEC dysregulation probably contribute to parenchymal lung disease. However, because of the paucity of reagents that alter local neuropeptide secretion and function in the lung, a direct confirmation of the in vivo consequences of altered neuroendocrine system morphology has yet to be provided.

The pulmonary phenotype of NeuroD-targeted mice recapitulates selective aspects of the nonneuronal phenotypes observed in other organs. In the small intestine and pancreas, NeuroD deficiency promotes cell autonomous apoptotic cell loss and reduced proliferation. In this report, although we show compromised proliferation, we do not detect enhanced apoptosis in the lung parenchyma of mutant mice. However, we cannot rule out the possibility that apoptotic loss of PNECs precedes the observed antiproliferative phenotype.

Despite the fact that NEBs and solitary PNECs are highly conserved phylogenetically, the physiological significance and the developmental ontogeny of these two morphologies remain unclear (37). Additionally, the genetic and biochemical pathways that instruct neuroendocrine differentiation are unknown. Apart from the complete absence of pulmonary neuroendocrine cells in Mash1-deficient mice, no published genetically targeted model has shown alterations of neuroendocrine morphology using the most PNEC-inclusive marker, PGP9.5. Mice deficient for Gfi-1 have reduced CGRP-expressing solitary PNECs and NEBs but preserved abundance of PGP9.5-expressing cells (28). The combination of a marked reduction in solitary PNECs and expansion in NEBs that we show in the NeuroD-deficient lung supports integrative developmental regulation of these two structures. Two models reconcile this dual compartment mechanism. First, solitary PNECs may originate in the NEB niche, but at an early developmental time point, separate from the cluster. Accordingly, it remains possible that the lack of solitary PNECs in the NeuroD-deficient lung reflects an as yet undefined defect in the NEB niche. A second model is that solitary PNECs and NEBs may have a common resident precursor but develop as distinct, non-overlapping entities, with only the former requiring NeuroD expression for terminal differentiation. The development of methods to reliably isolate these low abundance cells from rodent lungs would greatly facilitate our dissection of their differentiation programs.

The finding of impaired alveolar septation in the NeuroD-deficient lung seems discordant, given that NeuroD is not expressed in the airspace. The markedly reduced airway and airspace proliferation implicate a global perturbation in mitogenesis. Two mechanisms are plausible. First, since neuroendocrine cells liberate mitogenic amines that act in a local paracrine manner and which are known to be involved in lung morphogenesis, the reduced solitary neuroendocrine cell compartment might compromise the liberation of requisite promorphogenic factors during alveolar development. Our finding of NeuroD-induced proliferation in transfected MLE12 cells supports this mechanism. Second, the known diabetic phenotype of these mice could exert antimorphogenic effects on the lung, an interaction that has been observed in human and animal models (38, 39). Although the precise basis for the airspace phenotype is unclear, the finding supports an intimate interaction between lung morphogenesis and overall neuroendocrine function.

Our induction of neuroendocrine marker expression in murine lung epithelial cells suggests that NeuroD expression is sufficient to confer a neuroendocrine phenotype. Leiter and co-workers (8) showed that NeuroD expression in HeLa cells promoted the expression of p21 and secretin. NeuroD overexpression in non-islet-derived pancreatic cell lines triggered a beta cell differentiation program exemplified by NKX2.2, pax4, and insulin induction (40, 41). Our results demonstrate that nonneuroendocrine lung epithelial cells harbor the requisite machinery to drive differentiation into neuroendocrine cells upon overexpression of NeuroD. We observed that NeuroD induced a restricted repertoire of neuroendocrine markers. Therefore, multiple transcription factors may be required for the expression of the full repertoire of panneuroendocrine markers in a given lung epithelial cell. Alternatively, a given bHLH protein may activate the differentiation of a restricted subset of pulmonary neuroendocrine cells that express a defined repertoire of markers, a pattern observed in the enteroendocrine compartment (42, 43). Ito et al. (27) demonstrated that NeuroD expression in the postnatal lung temporally coincided with Mash1 mediated neuroendocrine cell differentiation, suggesting some measure of cooperativity among these proneural factors.

Lung alveolarization is a dynamic and highly regulated process that integrates proliferative, apoptotic, and morphogenic cues. Both fibrillin-1 deficiency and NeuroD deficiency show impaired alveolar septation in the immediate postnatal period. However, meaningful differences in these phenotypes are evident. In fibrillin-1-deficient mice, the airspace lesion is progressive throughout postnatal life, whereas in the NeuroD-deficient lung, the airspace lesion improves but does not fully correct over time. These findings suggest that NeuroD may participate in the onset of alveolar septation but not the maintenance of the airspace architecture. By contrast, fibrillin-1 deficiency, in affecting the structural integrity of the airspace as well as the cytokine milieu, affects both the establishment and the maintenance of airspace architecture (1, 17, 19, 44). We reported that not only does fibrillin-1 deficiency compromise airspace formation and maintenance, but it also contributes to late onset inflammatory emphysema that recapitulates acquired airspace enlargement. Simply stated, isolated NeuroD deficiency versus relative NeuroD deficiency in the context of fibrillin-1 deficiency leads to distinct airspace sequelae. In this view, our data suggest that NeuroD deficiency is not the sole or predominant determinant of the airspace enlargement observed in fibrillin-1-deficient mice; rather, primary structural distortions and secondary alterations in cytokines are the critical determinants of the more severe and sustained lesion evident in these mice.

There are significant differences between the lung phenotypes of the fibrillin-1-deficient and the NeuroD-deficient mice. First, the airspace defect in the fibrillin-1-deficient mice is attributable to enhanced apoptosis conferred by increased transforming growth factor-β signaling. By contrast, the airspace defect in the NeuroD-deficient mice is a result of reduced proliferation. Second, the airspace lesion in fibrillin-1-deficient mice is progressive, whereas the airspace phenotype in the NeuroD-deficient mice remains stable throughout early adulthood, suggesting that the critical insult occurs during a discrete phase of development without ongoing perturbations. Thus, the down-regulation of NeuroD in fibrillin-1-deficient mice might well be a secondary effect of altered lung maturation and not specifically referable to fibrillin-1 biology.

We conclude that NeuroD, a proneural bHLH factor, contributes to early airway and airspace homeostasis. In the airspace, NeuroD must utilize cell nonautonomous pathways to maintain the proliferative milieu required for normal alveolar morphogenesis. Similarly, dysregulated expression of NeuroD may contribute to pathologic proliferation, as is observed in lung malignancies that overexpress NeuroD (31). In keeping with this previously unrecognized capacity, we report increased proliferation in alveolar epithelial cells overexpressing NeuroD and in epithelial cells exposed to supernatants from NeuroD expressing cells. In the airway, NeuroD is a primary determinant of NE morphology, facilitating the generation of solitary PNECs plausibly from preexistent NEBs. Since one or both of these morphologies may be the cellular substrate for the development of neuroendocrine-type lung malignancies, a better understanding of the specific contextual functions of these cells is needed.

Supplementary Material