Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics.

BACKGROUND: The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. METHODS: A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. RESULTS: Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. CONCLUSIONS: This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.

Uroplakin 3a+ Cells Are a Distinctive Population of Epithelial Progenitors that Contribute to Airway Maintenance and Post-injury Repair.

There is evidence that certain club cells (CCs) in the murine airways associated with neuroepithelial bodies (NEBs) and terminal bronchioles are resistant to the xenobiotic naphthalene (Nap) and repopulate the airways after Nap injury. The identity and significance of these progenitors (variant CCs, v-CCs) have remained elusive. A recent screen for CC markers identified rare Uroplakin3a (Upk3a)-expressing cells (U-CCs) with a v-CC-like distribution. Here, we employ lineage analysis in the uninjured and chemically injured lungs to investigate the role of U-CCs as epithelial progenitors. U-CCs proliferate and generate CCs and ciliated cells in uninjured airways long-term and, like v-CCs, after Nap. U-CCs have a higher propensity to generate ciliated cells than non-U-CCs. Although U-CCs do not contribute to alveolar maintenance long-term, they generate alveolar type I and type II cells after Bleomycin (Bleo)-induced alveolar injury. Finally, we report that Upk3a+ cells exist in the NEB microenvironment of the human lung and are aberrantly expanded in conditions associated with neuroendocrine hyperplasias.

Directed Migration of Pulmonary Neuroendocrine Cells toward Airway Branches Organizes the Stereotypic Location of Neuroepithelial Bodies.

The airway epithelium consists of diverse cell types, including neuroendocrine (NE) cells. These cells are thought to function as chemoreceptors and as a component of the stem cell niche as well as the cells of origin in small-cell lung cancer. NE cells often localize at bifurcation points of airway tubes, forming small clusters called neuroepithelial bodies (NEBs). To investigate NEB development, we established methods for 3D mapping and ex vivo 4D imaging of developing lungs. We found that NEBs localize at stereotypic positions in the bifurcation area irrespective of variations in size. Notch-Hes1 signaling contributes to the differentiation of solitary NE cells, regulating their number but not localization. Live imaging revealed that individual NE cells migrate distally to and cluster at bifurcation points, driving NEB formation. We propose that NEB development is a multistep process involving differentiation of individual NE cells and their directional migration to organize NEBs.

Formation of a Neurosensory Organ by Epithelial Cell Slithering.

Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic.

Different assemblies of Notch receptors coordinate the distribution of the major bronchial Clara, ciliated and neuroendocrine cells.

In the developing lung, it is thought that the terminal buds of elongating airways contain a population of multipotent epithelial progenitors. As the bronchial tree extends, descendants of these cells give rise to lineage-restricted progenitors in the conducting airways via Notch signaling, which is involved in the establishment of epithelial Clara, ciliated and pulmonary neuroendocrine (NE) cell populations. However, the precise molecular details of this selection process are still emerging. Our stepwise removal of the three Notch receptors from the developing lung epithelium reveals that, whereas Notch2 mediates the Clara/ciliated cell fate decision with negligible contributions from Notch1 and Notch3, all three Notch receptors contribute in an additive manner to regulate the abundance of NE cells and the size of the presumptive pulmonary neuroepithelial body (pNEB) as a result of mutual interactions between NE cells and the Notch-dependent, SSEA-1(+), CC10(-) cell population surrounding the pNEB (SPNC cells). Ectopic expression of the Notch1 or Notch2 intracellular domain was sufficient to induce SSEA-1(+) cells and to suppress pNEB formation without expending Clara cells. We provide evidence that the additive functions of Notch receptors, together with other signaling pathways, maintains the expression of Hes1, a key regulator of NE cell fate, and that maintenance of Hes1 expression in epithelial cells is key to the regulation of pNEB size. These results suggest that two different assemblies of Notch receptors coordinate the numbers and distribution of the major epithelial cell types in the conducting airway during lung organogenesis.

The simultaneous presence of neuroepithelial cells and neuroepithelial bodies in the respiratory gas bladder of the longnose gar, Lepisosteus osseus, and the spotted gar, L. oculatus.

Anatomical and functional studies on the autonomic innervation as well as the location of airway receptors in the air-bladder of lepisosteids are very fragmentary. These water-breathing fishes share in common with the bichirs the presence of a glottis (not a ductus pneumaticus) opening into the esophagus. In contrast to a high concentration of neuroepithelial cells (NECs) contained in the furrowed epithelium in the lung of Polypterus, these cells are scattered as solitary cells in the glottal epithelium, and grouped to form neuroepithelial bodies (NEBs) in the mucociliated epithelium investing the main trabeculae in the air-bladder of Lepisosteus osseus and L. oculatus. The present immunohistochemical studies also demonstrated the presence of nerve fibers in the trabecular striated musculature and a possible relation to NEBs in these species, and identified immunoreactive elements of this innervation. Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), 5-HT and neuropeptide immunoreactivities were detected in the intramural nerve fibers. 5-HT and VIP immunopositive nerve fibers are apparently associated with NEBs. TH, VIP and SP immunoreactivities are also present in nerve fibers coursing in the radially arranged striated muscle surrounding the glottis and its submucosa. 5-HT positive neurons are also found in submucosal and the muscle layers of the glottis. The physiological function of the adrenergic and inhibitory innervation of the striated muscle as well as the neurochemical coding and morphology of the innervation of the NEBs are not known. Future studies are needed to provide evidence for these receptors with the capacity of chemoreceptors and/or mechanoreceptors.

The role of NOX2 and "novel oxidases" in airway chemoreceptor O(2) sensing.

In pulmonary neuroepithelial bodies (NEB), presumed airway chemoreceptors, classical NADPH oxidase (gp91 phox, NOX2) is co-expressed with O(2) sensitive K(+) channels (K(+)O(2)) and functions as an O(2) sensor. Here we examined related NADPH oxidase homologues "novel oxidases "(NOX 1, 3&4) and their possible involvement in O(2) sensing. For immunolocalization we used specific antibodies against various NADPH components and K(+) (O(2)) subunits to label NEB in rat /rabbit lung and NEB related H146 tumor cell line. For gene expression profiling of NEB cells microdissected from human lung, and H146 cells, we used custom MultiGene-12TM RT-PCR array that included NADPH oxidase components and homologues /accessory proteins (NOX1-4, phox-p22, p40, p47, p67, Rac1, NOXO1 and NOXA1) and K(+)O(2) channels (Kv -1.2, 1.5, 2.1, 3.1, 3.3, 3.4, 4.2, 4.3;TASK1-3). In rat lung, NOX2, NOX4, p22phox, Kv3.3 (and Kv3.4 in rabbit) and TASK1 localized to the apical plasma membrane of NEB cells, and membrane or sub-membrane regions in H146 cells. NEB and H146 cells expressed all NOX proteins except NOX3, as well as all K(+)O(2) channels, except Kv1.5 and Kv4.3. Co-immunoprecipitation using Western blot multicolor Quantum dot labeling showed NOX2 molecular complexes with Kv but not with TASK, while NOX4 associated with TASK1 but not with Kv channel proteins. Hypoxia -induced serotonin release was inhibited in H 146 cells by siRNA to NOX2, while siRNA to NOX4 had only a partial effect, implicating NOX 2 as the predominant NEB cell O(2) sensor. Present findings support NEB cell specific plasma membrane model of O(2) sensing, and suggest unique NOX/K(+)O(2) channel combinations for diverse physiological NEB functions.

Recruitment of GABA(A) receptors in chemoreceptor pulmonary neuroepithelial bodies by prenatal nicotine exposure in monkey lung.

Pulmonary neuroepithelial bodies (NEB) act as airway oxygen sensors and produce serotonin, a variety of neuropeptides and are involved in autonomic nervous system control of breathing, especially during the neonatal period. We now report that NEB cells also express a GABAergic signaling loop that is increased by prenatal nicotine exposure. In this study, cultured monkey NEB cells show hypoxia-evoked action potentials and hypoxia-sensitive K(+) current. As shown by both immunofluorescence and RT-PCR, monkey NEB cells synthesize and contain serotonin. The monkey NEB cells express the beta2 and beta3 GABA_A receptor subunits, GAD and also express alpha7, alpha4 and beta4 nicotinic receptor (nAChR) subunits. The alpha7 nAChR is co-expressed with GAD in NEB. The numbers of NEB and beta3 GABA_A receptor subunits expressed in NEB cells in lungs from control newborn monkeys were compared to lungs from animals that received nicotine during gestation. Prenatal nicotine exposure increased the numbers of NEB by 46% in lung and the numbers of NEB cells expressing GAD and GABA_A beta3 receptors increased by 67% and 66%, respectively. This study suggests that prenatal nicotine exposure can modulate NEB function by increasing the numbers of NEB cells and by increasing both GAD expression and beta3 GABA_A receptor subunit expression. The interaction of the intrinsic GABAergic system in the lung with nicotinic receptors in PNEC/NEB may provide a mechanism to explain the link between smoking during pregnancy and SIDS.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

Pulmonary neuroendocrine cell system in pediatric lung disease-recent advances.

The airway epithelium of human and animal lungs contains highly specialized pulmonary neuroendocrine cells (PNEC), distributed as solitary cells and as innervated clusters, neuroepithelial bodies (NEB). The designation "PNEC system" stems from the expression of both neural and endocrine cell phenotypes, including the synthesis and release of amine (serotonin, 5-HT) and a variety of neuropeptides (that is, bombesin). The role and function of PNEC in the lung have remained a subject of speculation for many years. During the last decade, studies using modern techniques of cellular and molecular biology revealed a complex functional role for PNEC, beginning during the early stages of lung development as modulators of fetal lung growth and differentiation and at the time of birth as airway O2 sensors involved in neonatal adaptation. Postnatally and beyond, PNEC/NEB are providers of a lung stem cell niche that is important in airway epithelial regeneration and lung carcinogenesis. The focus of this review is to present and discuss recent findings pertaining to the responses of PNEC to intrauterine environmental stimuli, ontogeny and molecular regulation of PNEC differentiation, innervation of NEB, and their role as airway chemoreceptors, including mechanisms of O2 sensing and chemotransmission of hypoxia stimulus. Abnormalities of PNEC/NEB have been reported in a variety of pediatric pulmonary disorders but the clinical significance or the mechanisms involved are unknown. The discussion on the possible role of PNEC/NEB in the pathogenesis and pathobiology of pediatric lung diseases includes congenital lung disorders, bronchopulmonary dysplasia, disorders of respiratory control, neuroendocrine hyperplasia of infancy, cystic fibrosis, bronchial asthma, and pulmonary hypertension.

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine.

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.

Functional facets of the pulmonary neuroendocrine system.

Pulmonary neuroendocrine cells (PNECs) have been around for 60 years in the scientific literature, although phylogenetically they are ancient. Their traditionally ascribed functions include chemoreception and regulation of lung maturation and growth. There is recent evidence that neuroendocrine (NE) differentiation in the lung is regulated by genes and pathways that are conserved in the development of the nervous system from Drosophila to humans (such as achaete-scute homolog-1), or implicated in the carcinogenesis of the nervous or NE system (such as the retinoblastoma tumor suppressor gene). In addition, complex neural networks are in place to regulate chemosensory and other functions. Even solitary PNECs appear to be innervated. For the first time ever, we have mouse models for lung NE carcinomas, including the most common and virulent small cell lung carcinoma. Moreover, PNECs may be important for inflammatory responses, and pivotal for lung stem cell niches. These discoveries signify an exciting new era for PNECs and are likely to have therapeutic and diagnostic applications.

Neurochemical characterisation of sensory receptors in airway smooth muscle: comparison with pulmonary neuroepithelial bodies.

Descriptions of morphologically well-defined sensory airway receptors are sparse, in contrast to the multiplicity of airway receptors that have been identified electrophysiologically. The present study aimed at further determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. The results were compared with those obtained for pulmonary neuroepithelial bodies (NEBs), which are complex intraepithelial sensory airway receptors. Multiple immunocytochemical staining showed branching laminar subepithelial receptor-like endings, which were found to intercalate in the smooth muscle layer of intrapulmonary conducting airways in rats. Because of the consistent intimate association with the airway smooth muscle, the laminar terminals will further be referred to as 'smooth muscle-associated airway receptors (SMARs)'. SMARs were characterised by their Na(+)/K(+)-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2-immunoreactivity, expression of the ATP receptor P2X(3), and the presence of calcium-binding proteins. Nerve fibres giving rise to SMARs were shown to be myelinated and to have a vagal origin. Interestingly, the neurochemical coding and receptor-like appearance of SMARs appeared to be almost identical to at least part of the complex vagal sensory terminals in NEBs. Intraepithelial nerve endings in pulmonary NEBs were indeed also shown to originate from myelinated vagal afferent nerve fibres, and to express Na(+)/K(+)-ATPase alpha3, VGLUT1, VGLUT2, P2X(3) and calcium-binding proteins. Since several of the latter proteins have been reported as markers for mechanoreceptor terminals in other organs, both SMARs and the vagal nodose nerve terminals in NEBs seem good candidates to represent the morphological counterparts of at least subsets of the extensive population of physiologically characterised myelinated vagal airway mechanoreceptors. The observation that SMARs and NEBs are regularly found in each other's immediate neighbourhood, and the very similar characteristics of their nerve terminals, point out that the interpretation of electrophysiological data based on 'local' stimuli should be made with great caution.