Neuroepithelial Bodies Research Articles

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

Co-expression of Achaete-Scute Homologue-1 and Calcitonin Gene-Related Peptide during NNK-Induced Pulmonary Neuroendocrine Hyperplasia and Carcinogenesis in Hamsters.

Achaete-scute homologue-1 or ASCL1 (MASH1, hASH1) plays roles in neural development and pulmonary neuroendocrine (NE) differentiation, and it is expressed in certain lung cancers. This study was aimed to assess whether and/or how ASCL1 plays a role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced pulmonary NE hyperplasia and carcinogenesis in hamsters. Hamsters were injected 3 times weekly with either NNK or solvent alone (control) for treatment periods of 6 and 24 weeks, both without and with 6-week recovery. Immunohistochemical analysis was carried out to examine the expressions of ASCL1, CGRP (calcitonin gene-related peptide), secretoglobin SCGB1A1 (club [Clara] cell specific 10 kD protein, CC10, CCSP), synaptophysin (SYP), and PCNA (proliferating cell nuclear antigen). The number of ASCL1-expressing NE foci per airway increased from 0.8 in controls to 1.6 and 2.0 during NNK exposure for 6 and 24 weeks, respectively, and the number of cells per foci doubled after NNK exposure. Most ASCL1-expressing cells in NEBs (neuroepithelial bodies) were also CGRP immunoreactive; NNK enhanced this co-expression with CGRP, a NE marker with known proliferation-promoting properties. NNK also increased PCNA expression within NE foci. NNK-induced tumors showed no immunoreactivity for NE markers. This study confirms ASCL1 as an excellent marker for pulmonary NE cells and demonstrates CGRP co-expression in ASCL1-positive NEB cells participating in NNK-induced NE hyperplasia.

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.

Transient acute lung injury induces activation and proliferation of a selective cell population in the neuroepithelial body microenvironment

Transient acute lung injury induces activation and proliferation of a selective cell population in the neuroepithelial body microenvironment

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.

A mitochondrial redox oxygen sensor in the pulmonary vasculature and ductus arteriosus

The mammalian homeostatic oxygen sensing system (HOSS) initiates changes in vascular tone, respiration, and neurosecretion that optimize oxygen uptake and tissue oxygen delivery within seconds of detecting altered environmental or arterial PO2. The HOSS includes carotid body type 1 cells, adrenomedullary cells, neuroepithelial bodies, and smooth muscle cells (SMCs) in pulmonary arteries (PAs), ductus arteriosus (DA), and fetoplacental arteries. Hypoxic pulmonary vasoconstriction (HPV) optimizes ventilation-perfusion matching. In utero, HPV diverts placentally oxygenated blood from the non-ventilated lung through the DA. At birth, increased alveolar and arterial oxygen tension dilates the pulmonary vasculature and constricts the DA, respectively, thereby transitioning the newborn to an air-breathing organism. Though modulated by endothelial-derived relaxing and constricting factors, O2 sensing is intrinsic to PASMCs and DASMCs. Within the SMC's dynamic mitochondrial network, changes in PO2 alter the reduction-oxidation state of redox couples (NAD(+)/NADH, NADP(+)/NADPH) and the production of reactive oxygen species, ROS (e.g., H2O2), by complexes I and III of the electron transport chain (ETC). ROS and redox couples regulate ion channels, transporters, and enzymes, changing intracellular calcium [Ca(2+)]i and calcium sensitivity and eliciting homeostatic responses to hypoxia. In PASMCs, hypoxia inhibits ROS production and reduces redox couples, thereby inhibiting O2-sensitive voltage-gated potassium (Kv) channels, depolarizing the plasma membrane, activating voltage-gated calcium channels (CaL), increasing [Ca(2+)]i, and causing vasoconstriction. In DASMCs, elevated PO2 causes mitochondrial fission, increasing ETC complex I activity and ROS production. The DASMC's downstream response to elevated PO2 (Kv channel inhibition, CaL activation, increased [Ca(2+)]i, and rho kinase activation) is similar to the PASMC's hypoxic response. Impaired O2 sensing contributes to human diseases, including pulmonary arterial hypertension and patent DA.

Hyperplasia of pulmonary neuroendocrine cells in infancy and childhood.

Pulmonary neuroendocrine cells (PNEC) are widely distributed throughout the airway mucosa of mammalian lung as solitary cells and as distinctive innervated clusters, neuroepithelial bodies (NEB). These cells differentiate early during lung development and are more prominent in fetal/neonatal lungs compared to adults. PNEC/NEB cells produce biogenic amine (serotonin) and a variety of peptides (i.e., bombesin) involved in regulation of lung function. During the perinatal period, NEB are thought to function as airway O(2)/CO(2) sensors. Increased numbers of PNEC/NEBs have been observed in a variety of perinatal and postnatal lung disorders. Recent advances in cellular and molecular biology of these cells, as they relate to perinatal and postnatal lung disorders associated with PNEC/NEB cell hyperplasia are reviewed and their possible role in pulmonary pathobiology discussed (WC 125).

Sensory input to the central nervous system from the lungs and airways: A prominent role for purinergic signalling via P2X2/3 receptors.

Specific subpopulations of lung-related primary afferent neurons in dorsal root and vagal sensory ganglia have been reported to express P2X2 and P2X3 receptors both in the neuronal cell bodies and in their peripheral terminals. The afferent innervation of airways and lungs is organised as sensory receptor structures, of which at least seven types with a vagal origin and two with a spinal origin have been reported. In view of the recently suggested therapeutic promise of ATP antagonism - specifically at P2X3 receptor expressing nociceptive fibres - in respiratory disorders, the present work focusses on four distinct populations of pulmonary sensory receptors that have so far been reported to express P2X2/3 receptors. Three of them originate from myelinated nerve fibres that display similar mechanosensor-like morphological and neurochemical characteristics. Two of the latter concern vagal nodose sensory fibres, either related to pulmonary neuroepithelial bodies (NEBs), or giving rise to smooth muscle-associated airway receptors (SMARs); the third gives rise to visceral pleura receptors (VPRs) and most likely arises from dorsal root ganglia. The fourth population concerns C-fibre receptors (CFRs) that also derive from neuronal cell bodies located in vagal nodose ganglia. Although the majority of the airway- and lung-related sensory receptors that express P2X2/3 receptors apparently do not belong to accepted nociceptive populations, these data definitely point out that ATP may be an important player in the physiological transduction of different lung-related afferent signals from the periphery to the CNS. The observed variety within the populations of pulmonary sensory receptors that express P2X2/3 receptors argues for a critical and careful interpretation of the functional data.

Murine Pulmonary Slowly‐Adapting Receptors (SARs): Putative links to Neuroepithelial Body (NEB) hypoxia chemoreception and the Calcium Sensing Receptor (CaSR)

Pulmonary SARs are mechanosensitive vagal afferents, which Adriaensen (J Appl Physiol, 2006) proposed to be functionally linked to morphologically‐defined, hypoxia‐sensitive NEBs (Youngson et al. 1993, Nature). Post‐natal NEBs express CaSR (Lembrechts et al. 2013, J Cell Sci), and vagal NEB innervation expresses P2X2 and P2X3 ATP receptors (Brouns et al. 2009, Histochem Cell Biol). We tested in vivo murine SAR responses to quasi‐static inflation (0‐20 cmH2O) in: 1) hypoxia (10% O2) 2) absence of CaSR (CaSR / parathyroid hormone double‐knockout mouse, KO) and 3) P2 purinergic receptor blockade (Suramin: 50 mg/kg). SAR mechanosensitivity was defined as action potential frequency (f) at tracheal pressures of 5, 10, 15, and 20 cmH2O. Hypoxia caused a small but statistically significant increase in f (9.6±2.8% at 20cmH2O; P = 0.002, mean±SEM). Loss of CaSR significantly reduced f at all pressures (‐34.9% and ‐39.5% at 15cmH2O in hyper‐ and hypoxia, respectively; P < 0.001) with no impact of hypoxia within each genotype. Suramin caused a small but significant reduction in f (‐10.6±1.5% at 20cmH2O; P = 0.01). These data suggest that CaSR significantly impacts SAR mechanosensitivity, while purinergic signaling and hypoxia exert modest effects with as yet to be determined physiologic roles.Supported by CIHR MOP81211 (JTF), Spear Endowment for Respiratory Research (Queen's University JTF and RJM), NSERC PostGrad Scholarship (NJD).

Vagal Sensory Neuron Subtypes that Differentially Control Breathing

Breathing is essential for survival and under precise neural control. The vagus nerve is a major conduit between lung and brain required for normal respiration. Here, we identify two populations of mouse vagus nerve afferents (P2ry1, Npy2r), each a few hundred neurons, that exert powerful and opposing effects on breathing. Genetically guided anatomical mapping revealed that these neurons densely innervate the lung and send long-range projections to different brainstem targets. Npy2r neurons are largely slow-conducting C fibers, while P2ry1 neurons are largely fast-conducting A fibers that contact pulmonary endocrine cells (neuroepithelial bodies). Optogenetic stimulation of P2ry1 neurons acutely silences respiration, trapping animals in exhalation, while stimulating Npy2r neurons causes rapid, shallow breathing. Activating P2ry1 neurons did not impact heart rate or gastric pressure, other autonomic functions under vagal control. Thus, the vagus nerve contains intermingled sensory neurons constituting genetically definable labeled lines with different anatomical connections and physiological roles.

Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing.

Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H(+)) is presently lacking. We tested the effects of CO2/H(+) alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca(2+) imaging method was used to assess CO2/H(+)-evoked changes in intracellular Ca(2+). Exposure to 10 and 20% CO2 or pH 6.8-7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca(2+) in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca(2+). Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H(+)-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H(+) sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.

Augmented 5-HT Secretion in Pulmonary Neuroepithelial Bodies from PHD1 Null Mice.

Sustained exposure to low oxygen concentration leads to profound changes in gene expression to restore oxygen homeostasis. Hypoxia-inducible factors (HIFs) comprise a group of transcription factors which accumulate under hypoxia and contribute to the complex changes in gene expression. Under normoxic conditions HIFs are degraded by prolyl-hydroxylases (PHD), however during hypoxia this degradation is inhibited causing HIF accumulation and subsequent changes in gene expression. Pulmonary neuroepithelial bodies (NEB) are innervated serotonin (5-HT)-producing cells distributed throughout the airway epithelium. These putative O(2) sensors are hypothesized to contribute to the ventilatory response to hypoxia. NEB dysfunction has been implicated in several paediatric lung diseases including neuroendocrine cell hyperplasia of infancy and sudden infant death syndrome, both characterized by a marked NEB hyperplasia with unknown functional significance. We have previously reported striking NEB hyperplasia in PHD1(-/-) mice making these mice a potential model to study the role of NEBs in paediatric lung diseases. Here we report in vitro studies on 5-HT release from NEB using this model.

Neuroepithelial Bodies in the Lungs in Rats

We report here our studies of neuroepithelial bodies (NEB) in the lungs in healthy Wistar rats (n = 12). Immunocytochemical reactions for synaptophysin identified NEB and similar immunopositive nerve terminals. NEB in the lungs in rats were found to be consistently present elements. In contrast to diffuse neuroendocrine elements, these structures were characterized by grouped cells. Some NEB lacked efferent innervation.

Immunohistochemical characterization of the chemosensory pulmonary neuroepithelial bodies in the naked mole-rat reveals a unique adaptive phenotype.

The pulmonary neuroepithelial bodies (NEBs) constitute polymodal airway chemosensors for monitoring and signaling ambient gas concentrations (pO2, pCO2/H+) via complex innervation to the brain stem controlling breathing. NEBs produce the bioactive amine, serotonin (5-HT), and a variety of peptides with multiple effects on lung physiology and other organ systems. NEBs in mammals appear prominent and numerous during fetal and neonatal periods, and decline in the post-natal period suggesting an important role during perinatal adaptation. The naked mole-rat (NMR), Heterocephalus glaber, has adapted to the extreme environmental conditions of living in subterranean burrows in large colonies (up to 300 colony mates). The crowded, unventilated burrows are environments of severe hypoxia and hypercapnia. However, NMRs adjust readily to above ground conditions. The chemosensory NEBs of this species were characterized and compared to those of the conventional Wistar rat (WR) to identify similarities and differences that could explain the NMR’s adaptability to environments. A multilabel immunohistochemical analysis combined with confocal microscopy revealed that the expression patterns of amine, peptide, neuroendocrine, innervation markers and chemosensor component proteins in NEBs of NMR were similar to that of WR. However, we found the following differences: 1) NEBs in both neonatal and adult NMR lungs were significantly larger and more numerous as compared to WR; 2) NEBs in NMR had a more variable compact cell organization and exhibited significant differences in the expression of adhesion proteins; 3) NMR NEBs showed a significantly greater ratio of 5-HT positive cells with an abundance of 5-HT; 4) NEBs in NMR expressed the proliferating cell nuclear antigen (PCNA) and the neurogenic gene (MASH1) indicating active proliferation and a state of persistent differentiation. Taken together our findings suggest that NEBs in lungs of NMR are in a hyperactive, functional and developmental state, reminiscent of a persistent fetal state that extends postnatally.

The Distribution and Frequency of Pulmonary Neuroendocrine Cells in Down Syndrome Fetal Lungs

The pulmonary neuroendocrine cells (PNEC) are located in the epithelial lining of the airways and consist of solitary neuroendocrine cells (NEC) and NEC clusters, the neuroepithelial bodies (NEB). During fetal life, PNEC are the first to differentiate within the primitive airway epithelium, and bombesin expression favors branching of the respiratory tree.We investigated PNEC in Down syndrome (DS), where the lungs often show enlarged and reduced number of alveoli. Immunohistochemistry for bombesin and synaptophysin, PNEC markers, was evaluated in fetal lungs from 15 cases of DS and 11 age-matched controls from the 17th to 23rd week of gestation.Morphometric analysis assessed PNEC in the mucosal lining of each lung, expressed as number/mm. Nonparametric Mann-Whitney U test showed no statistical difference in frequency of PNEC in DS and controls. Our findings suggest that, at least in late second trimester, the distribution and frequency of PNEC in DS fetuses is not altered.

Expression of BMP‐2 in the pulmonary neuroepithelial body microenvironment (869.7)

Recently, members of the bone morphogenetic protein (BMP) family, in particular BMP‐2, have been reported to promote adult lung tissue repair after injury. Following severe experimental intrapulmonary airway epithelial damage, the neuroepithelial body (NEB) microenvironment (ME) has been shown to be involved in epithelial regeneration, and today may be regarded as an airway stem cell niche. The NEB ME consists of highly innervated groups of pulmonary neuroepithelial endocrine cells, which are specifically covered by Clara‐like cells with supposed stem cell characteristics.Here we studied the expression of BMP‐2 in the NEB ME of healthy postnatal mouse lungs. After collection of ‘NEB sample’ by selective isolation of GFP‐fluorescent NEBs via laser microdissection from frozen sections of GAD67‐GFP mouse lungs, (q)RT‐PCR revealed a significantly higher expression of BMP‐2 in the NEB ME, compared to control airway epithelium. Localization of the BMP‐2 protein in postnatal mouse lungs by multiple immunofluorescence, revealed that BMP‐2 was expressed by vagal sensory nerve fiber endings that selectively contact NEB cells.The presented data reveal a strong expression of BMP‐2 in the NEB ME and strengthen the suggestion of an important role in postnatal airway epithelial repair after injury.Grants: FWO (G.0589.11), Hercules Foundation (AUAH‐09‐001); UA (GOA BOF 2007)

Neuroepithelial bodies as airway hypoxia sensors: re‐evaluation of the concept in different mouse live cell imaging models (869.6)

In pulmonary neuroepithelial bodies (NEBs), acute hypoxia has been suggested to close O2‐sensitive K+ channels, resulting in membrane depolarization, voltage‐gated Ca2+ entry and subsequent transmitter release.To visualize this mechanism in NEBs, our established live cell imaging (LCI) model, based on lung vibratome slices, was applied. Real‐time changes in different cellular processes were monitored during hypoxic challenge in NEBs of postnatal, prenatal and GAD67‐GFP mice.As a proof of concept, LCI experiments were performed on isolated mouse carotid body, proving that our LCI set up is indeed capable of visualizing hypoxia‐induced changes. However, in none of the NEBs studied in the lung slice models, severe (2% O2), mild (12% O2) or intermittent hypoxia, resulted in a rise in intracellular Ca2+ ([Ca2+]i) or subsequent neurotransmitter release. Moreover, low O2 levels appeared to decrease NEB cell excitability. The observed hypoxic mitochondrial membrane depolarization and increased ROS production rather seemed to be part of a general cellular defense mechanism, as supported by the non‐selective and non‐functional expression of the key O2 sensing protein NADPH oxidase.All together, our data argue for a reconsideration of the proposed straightforward hypoxic signal transduction pathway in pulmonary NEBs.Grants: FWO (G.0589.11), Hercules Foundation (AUAH‐09‐001); UA (GOA BOF 2007)

Murine airway slowly‐adapting receptor responses to lung inflation: a role for serotonin? (879.19)

The mechanical phenotype of murine airway slowly‐adapting receptors (SARs), which are mechanosensitive afferents physiologically characterized by maintained discharge to sustained transmural pressure, is only partially defined. Adriaensen et al. suggest that densely‐innervated pulmonary neuroepithelial bodies (NEBs) are a morphological equivalent of SARs (Adriaensen et al. 2006, J Appl Physiol). Since NEBs release serotonin (5‐HT) in response to stretch in vitro (Pan et al. 2006, Am J Physiol Lung Cell Mol Biol), we characterized the mechanosensitivity of SARs during quasi‐static inflation to 20 cmH2O in male C57BL/6 mice and tested the impact of 5‐HT3 receptor blockade (i.v. tropisetron, Trop; 4.5mg/kg). SAR (n=8) discharge frequency (f; impulses/s) mechanosensitivity was proportional to tracheal pressure. Trop caused a small (3‐8%) but significant decrease in f vs. control (P=0.005; 2‐way ANOVA‐RM; mean±SEM (imp/s): 5cmH2O ‐ con: 91±13, Trop: 87±13; 10cmH2O ‐ con: 153±25, Trop: 143±22; 15cmH2O ‐ con: 192±29, Trop: 180±28; 20cmH2O ‐ con: 176±14, Trop: 164±15). It remains to be seen whether such a modest reduction in SAR discharge has a physiologic impact on the reflex effects of SAR medullary input.Grant Funding Source: Supported by: CIHR (JTF; MOP81211) & NSERC PostGrad Scholarship (NJD)

Analysis of Notch Signaling-Dependent Gene Expression in Developing Airways Reveals Diversity of Clara Cells

Clara cells (CCs) are a morphologically and operationally heterogeneous population of Secretoglobin Scgb1a1-expressing secretory cells that are crucial for airway homeostasis and post-injury repair. Analysis of the extent and origin of CC diversity are limited by knowledge of genes expressed in these cells and their precursors. To identify novel putative markers of CCs and explore the origins of CC diversity, we characterized global changes in gene expression in embryonic lungs in which CCs do not form due to conditional disruption of Notch signaling (RbpjkCNULL). Microarray profiling, Real Time PCR (qRT-PCR), and RNA in situ hybridization (ISH) identified eleven genes downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. ISH revealed that several genes had overlapping but distinct domains of expression of in the normal developing lung (E18.5). Notably, Reg3g, Chad, Gabrp and Lrrc26 were enriched in proximal airways, Hp in the distal airways and Upk3a in clusters of cells surrounding Neuroepithelial Bodies (NEBs). Seven of the eleven genes, including Reg3g, Hp, and Upk3a, were expressed in the adult lung in CCs in a pattern similar to that observed in the developing airways. qRT-PCR-based analysis of gene expression of CCs isolated from different airway regions of B1-EGFP reporter mice corroborated the spatial enrichment in gene expression observed by ISH. Our study identifies candidate markers for CC-precursors and CCs and supports the idea that the diversification of the CC phenotype occurs already during embryonic development.

Neuroepithelial body increases in bleomycin-treated mice

Neuroepithelial bodies (NEBs) serve a niche for lung stem cells and proliferate in a variety of pulmonary diseases. We hypothesize that NEBs play an important role in lung injury repair processes, such as during pulmonary fibrosis. To test this hypothesis, we examined NEBs in a bleomycin-induced lung fibrosis mouse model. We divided FVB/NJ mice into bleomycin-treated (BL) and normal saline-treated (NS) groups. Two weeks after intravenous treatment, we immune-stained NEBs with anti-calcitonin gene-related peptide (CGRP) in whole mount preparations and found that the number of NEBs per unit area of airway almost tripled in the BL group (1.11±0.28 number/mm2; n=5) compared with the NS group (0.32±0.14 number/mm2; n=4, p=0.001). The size of NEBs increased significantly in the BL group. Our findings support that NEBs play an important role in the pathogenesis of pulmonary fibrosis.