A transcription factor glial cell missing (Gcm) in the freshwater crayfish Pacifastacus leniusculus.

The transcription factor glial cell missing, Gcm, is known to be an important protein in the determination of glial cell fate as well as embryonic plasmatocyte differentiation in Drosophila melanogaster. So far, no function for Gcm in crustaceans has been reported. In this study, we show the cDNA sequence of a Gcm homologue in the freshwater crayfish Pacifastacus leniusculus. The P. leniusculus Gcm transcript is expressed exclusively in brain and nervous tissue, and by in situ hybridization we show that the expression is restricted to a small number of large cells with morphology similar to neurosecretory cells. Furthermore, we show that the expression of Gcm coincides with the expression of a Repo homologue, that is induced in expression by Gcm in Drosophila. Moreover, the Gcm transcript is increased shortly and transiently after injection of cystamine, a substance that inhibits transglutaminase and also strongly affects the movement behavior of crayfish. This finding of Gcm transcripts in a subpopulation of brain cells in very low numbers may enable more detailed studies about Gcm in adult crustaceans.

Altered secretory and neuroprotective function of the choroid plexus in progressive multiple sclerosis.

The choroid plexus (CP) is a key regulator of the central nervous system (CNS) homeostasis through its secretory, immunological and barrier properties. Accumulating evidence suggests that the CP plays a pivotal role in the pathogenesis of multiple sclerosis (MS), but the underlying mechanisms remain largely elusive. To get a comprehensive view on the role of the CP in MS, we studied transcriptomic alterations of the human CP in progressive MS and non-neurological disease controls using RNA sequencing. We identified 17 genes with significantly higher expression in progressive MS patients relative to that in controls. Among them is the newly described long non-coding RNA HIF1A-AS3. Next to that, we uncovered disease-affected pathways related to hypoxia, secretion and neuroprotection, while only subtle immunological and no barrier alterations were observed. In an ex vivo CP explant model, a subset of the upregulated genes responded in a similar way to hypoxic conditions. Our results suggest a deregulation of the Hypoxia-Inducible Factor (HIF)-1 pathway in progressive MS CP. Importantly, cerebrospinal fluid levels of the hypoxia-responsive secreted peptide PAI-1 were higher in MS patients with high disability relative to those with low disability. These findings provide for the first time a complete overview of the CP transcriptome in health and disease, and suggest that the CP environment becomes hypoxic in progressive MS patients, highlighting the altered secretory and neuroprotective properties of the CP under neuropathological conditions. Together, these findings provide novel insights to target the CP and promote the secretion of neuroprotective factors into the CNS of progressive MS patients.

Local neuropeptide signaling modulates serotonergic transmission to shape the temporal organization of C. elegans egg-laying behavior.

Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act in vivo to alter circuit performance and shape behavior. To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states-short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program.

Molecular mechanisms governing Ca(2+) regulation of evoked and spontaneous release.

The relationship between transmitter release evoked by action potentials and spontaneous release has fascinated neuroscientists for half a century, and separate biological roles for spontaneous release are emerging. Nevertheless, separate functions for spontaneous and Ca(2+)-evoked release do not necessarily indicate different origins of these two manifestations of vesicular fusion. Here we review how Ca(2+) regulates evoked and spontaneous release, emphasizing that Ca(2+) can briefly increase vesicle fusion rates one-millionfold above spontaneous rates. This high dynamic range suggests that docked and readily releasable pool (RRP) vesicles might be protected against spontaneous release while also being immediately available for ultrafast Ca(2+)-evoked release. Molecular mechanisms for such release clamping of highly fusogenic RRP vesicles are increasingly investigated. Thus, we view spontaneous release as a consequence of the highly release-competent state of a standing pool of RRP vesicles, which is molecularly fine-tuned to control spontaneous release.

Glutamate triggers neurosecretion and apoptosis in bovine chromaffin cells through a mechanism involving NO production by neuronal NO synthase activation.

Previous work from our group stated that nitric oxide (NO), via cytokines, induces apoptosis in chromaffin cells by a mechanism involving iNOS, nNOS, and NF-κB. In this paper the involvement of glutamate as a possible intracellular trigger of neurosecretion and NO-mediated apoptosis has been evaluated. We show that chromaffin cells express different ionotropic and metabotropic glutamate receptors, this exerting different effects on the regulation of basal and glutamate-induced catecholamine secretion, via NO/cGMP. In addition, we studied the effects of endogenously generated NO, both basal and glutamate-stimulated, on apoptosis of chromaffin cells. Our results show that glutamate agonists are able to induce cell death and apoptosis in bovine chromaffin cells, parallel to an increase in NO production. Such effects were reversed by NOS inhibitors and glutamate receptor antagonists. Under basal conditions, iNOS inhibitors did not have any effect on apoptosis, whereas nNOS inhibitors induced apoptosis, indicating a neuroprotective effect of constitutive nNOS-generated NO. In contrast, glutamate-induced apoptosis was strongly reversed by nNOS inhibitors and weakly by iNOS inhibitors, thus indicating nNOS involvement in glutamate-mediated apoptosis. These results were confirmed by the fact that nNOS expression, but not iNOS, is specifically activated by glutamate. Finally, our results suggest the participation of PKG, PKA, PKC, and MAPK pathways in glutamate-mediated nNOS activation in chromaffin cells and point out the involvement of both PKA and PKC signaling pathways in the apoptotic effect of glutamate.

Structure and expression of a shrimp prohormone convertase 2.

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P<0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.

A signaling loop of REST, TSC2 and ß-catenin governs proliferation and function of PC12 neural cells.

The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-ß-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active ß-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

HID-1, a new component of the peptidergic signaling pathway.

hid-1 was originally identified as a Caenorhabditis elegans gene encoding a novel conserved protein that regulates the decision to enter into the enduring dauer larval stage. We isolated a novel allele of hid-1 in a forward genetic screen for mutants mislocalizing RBF-1 rabphilin, a RAB-27 effector. Here we demonstrate that HID-1 functions in the nervous system to regulate neuromuscular signaling and in the intestine to regulate the defecation motor program. We further show that a conserved N-terminal myristoylated motif of both invertebrate and vertebrate HID-1 is essential for its association with intracellular membranes in nematodes and PC12 cells. C. elegans neuronal HID-1 resides on intracellular membranes in neuronal cell somas; however, the kinesin UNC-104 also transports HID-1 to synaptic regions. HID-1 accumulates in the axons of unc-13 and unc-31 mutants, suggesting it is associated with neurosecretory vesicles. Consistent with this, genetic studies place HID-1 in a peptidergic signaling pathway. Finally, a hid-1 null mutation reduces the levels of endogenous neuropeptides and alters the secretion of fluorescent-tagged cargos derived from neuronal and intestinal dense core vesicles (DCVs). Taken together, our findings indicate that HID-1 is a novel component of a DCV-based neurosecretory pathway and that it regulates one or more aspects of the biogenesis, maturation, or trafficking of DCVs.

In PC12 cells, expression of neurosecretion and neurite outgrowth are governed by the transcription repressor REST/NRSF.

A rapid drop of the transcription repressor REST/NRSF during precursor differentiation into nerve cells is known to release the repression of hundreds of specific genes and thus to orchestrate the acquisition of the specific phenotype. REST, however, is important not only for differentiation, but also for the maintenance of key properties in mature nerve cell. The PC12 line is uniquely favorable for studying REST because, in addition to the wild-type, low REST neurosecretory cells, it includes spontaneously defective clones lacking neurosecretion, where REST is as high as in non-nerve cells. In this article, we summarize our cell biologic studies of two nerve cell-specific processes dependent on REST, neurosecretion and neurite outgrowth. We demonstrate that, in wild-type PC12 transfected with REST constructs, expression of genes encoding proteins of dense-core and synaptic-like vesicles is decreased, though, to different extents, with chromogranins being the most and the SNAREs (except SNAP25) the least affected. Concomitantly, dense core-vesicles decrease markedly in size but can still be discharged by regulated exocytosis. When, in contrast, dominant-negative constructs of REST are transfected in high-REST PC12, and the main effector enzymes of REST, histone deacetylases, are blocked, dense-core vesicles reappear and are discharged upon stimulation. In high-REST PC12, also neurite outgrowth is inhibited by down regulation of the NGF receptor. Concomitantly, however, high REST induces the expression of proteins and of an exocytic organelle, the enlargeosome, which sustain a Rac1-dependent form of neurite outgrowth, unknown until now, operative in PC12, in neuroblastoma SH-SY5Y cells, and also in neurons.

Munc18-1 as a key regulator of neurosecretion.

Munc18-1 plays essential roles in neurosecretion by interacting with syntaxin-1 and controlling the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18-1 have been proposed: (i) molecular chaperone of syntaxin-1 for appropriate localization and expression of syntaxin-1, (ii) priming/stimulation of the SNARE-mediated membrane fusion, and (iii) docking of large dense-core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18-1 and syntaxin-1: (i) binary binding to a 'closed' conformation of syntaxin-1, and (ii) binding to the N-terminal peptide of syntaxin-1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18-1 and closed syntaxin-1. Recent structural analyses have identified critical Munc18-1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18-1 null neurons, chromaffin cells and Munc18-1/-2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18-1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18-1 in neuroexocytosis.

Transmembrane-domain determinants for SNARE-mediated membrane fusion.

Neurosecretion involves fusion of vesicles with the plasma membrane. Such membrane fusion is mediated by the SNARE complex, which is composed of the vesicle-associated protein synaptobrevin (VAMP2), and the plasma membrane proteins syntaxin-1A and SNAP-25. Although clearly important at the point of membrane fusion, the precise structural and functional requirements for the transmembrane domains (TMDs) of SNAREs in bringing about neurosecretion remain largely unknown. Here, we used a bimolecular fluorescence complementation (BiFC) approach to study SNARE protein interactions involving TMDs in vivo. VAMP2 molecules were found to dimerise through their TMDs in intact cells. Dimerisation was abolished when replacing a glycine residue in the centre of the TMD with residues of increasing molecular volume. However, such mutations still were fully competent in bringing about membrane-fusion events, suggesting that dimerisation of the VAMP2 TMDs does not have an important functional role. By contrast, a series of deletion or insertion mutants in the C-terminal half of the TMD were largely deficient in supporting neurosecretion, whereas mutations in the N-terminal half did not display severe secretory deficits. Thus, structural length requirements, largely confined to the C-terminal half of the VAMP2 TMD, seem to be essential for SNARE-mediated membrane-fusion events in cells.

Differential repression by the transcription factor REST/NRSF of the various Ca2+ signalling mechanisms in pheochromocytoma PC12 cells.

Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+](i) changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation ( approximately 15%) of the defective, high REST cells responded to depolarization (Ca(V) expression approximately 10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated.

[Neuroendocrine system in Drosophila melanogaster lifespan control].

Drosophila is used as a model organism to review the mechanisms of neuroendocrine system involvement in lifespan control. The role of neuron specific expression of genes participating in antioxidative system in lifespan control is described. Data on endocrine function of the nervous system in lifespan control are discussed. The participation of genes involved in the regulation of nervous system development and function in lifespan control is contemplated. Based on the data available, hypotheses considering assembly of neurons as the lifespan limiting tissue and (or) the tissue providing regulation of lifespan via systemic effects on other cell types are evaluated.

Exon expression profiling reveals stimulus-mediated exon use in neural cells.

BACKGROUND: Neuronal cells respond to changes in intracellular calcium ([Ca2+]i) by affecting both the abundance and architecture of specific mRNAs. Although calcium-induced transcription and transcript variation have both been recognized as important sources of gene regulation, the interplay between these two phenomena has not been evaluated on a genome-wide scale. RESULTS: Here, we show that exon-centric microarrays can be used to resolve the [Ca2+]i-modulated gene expression response into transcript-level and exon-level regulation. Global assessments of affected transcripts reveal modulation within distinct functional gene categories. We find that transcripts containing calcium-modulated exons exhibit enrichment for calcium ion binding, calmodulin binding, plasma membrane associated, and metabolic proteins. Additionally, we uncover instances of regulated exon use in potassium channels, neuroendocrine secretory proteins and metabolic enzymes, and demonstrate that regulated changes in exon expression give rise to distinct transcript variants. CONCLUSION: Our findings connect extracellular stimuli to specific exon behavior, and suggest that changes in transcript and exon abundance are reflective of a coordinated gene expression response to elevated [Ca2+]i. The technology we describe here lends itself readily to the resolution of stimulus-induced gene expression at both the transcript and exon levels.

Involvement of genes required for synaptic function in aging control in C. elegans.

OBJECTIVE: To identify new genes required for neurosecretory control of aging in C. elegans. METHODS: In view of the importance of nervous system in aging regulation, we performed the screen for genes involved in the aging regulation from genetic loci encoding synaptic proteins by lifespan assay and accumulation of lipofuscin autofluorescence. We further investigated the dauer formation phenotypes of their corresponding mutants and whether they were possibly up-regulated by the insulin-like signaling pathway. RESULTS: The genetic loci of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4, nrx-1, unc-13, sbt-1 and unc-64 might be involved in the aging control. In addition, functions of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4 and nrx-1 in regulating aging may be opposite to those of unc-13, sbt-1 and unc-64. The intestinal autofluorescence assay further indicated that the identified long-lived and short-lived mutants were actually due to the suppressed or accelerated aging. Among the identified genes, syd-2, hlb-1, mkk-4, scd-2, snb-1, ric-4 and unc-64 were also involved in the control of dauer formation. Moreover, daf-2 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, ric-4, sbt-1, rpm-1, unc-10, dlk-1 and unc-13. The daf-16 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, sbt-1, rpm-1, unc-10, dlk-1 and unc-13. CONCLUSION: These data suggest the possibly important status of the synaptic transmission to the animal's life-span control machinery, as well as the dauer formation control.

Squeeze involvement in the specification of Drosophila leucokinergic neurons: Different regulatory mechanisms endow the same neuropeptide selection.

One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. We have addressed this question by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. We show that all leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on LK expression of loss- and gain-of-function mutations, we can describe three types of Lk regulation. In the brain LHLK cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the suboesophageal SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors.

A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory-deficient PC12 cells.

The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down-regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1-silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant-negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the beta3 subunit of the voltage-gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re-expressed. Granule and synaptic vesicle proteins were not re-expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post-transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory-deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

The transcriptional repressor REST is a critical regulator of the neurosecretory phenotype.

Release of distinct cellular cargoes in response to specific stimuli is a process fundamental to all higher eukaryotes and controlled by the regulated secretory pathway (RSP). However, the mechanism by which genes involved in the RSP are selectively expressed, leading to the establishment and appropriate functioning of regulated secretion remaining largely unknown. Using the rat pheochromocytoma cell line PC12, we provide evidence that, by controlling expression of many genes involved in the RSP, the transcriptional repressor REST can regulate this pathway and hence the neurosecretory phenotype. Introduction of REST transgenes into PC12 cells leads to the repression of many genes, the products of which are involved in regulated secretion. Moreover, chromatin immunoprecipitation assays show that many of the repressed genes recruit the recombinant REST protein to RE1 sites within their promoters and abrogation of REST function leads to reactivation of these transcripts. In addition to the observed transcriptional effects, PC12 cells expressing REST have fewer secretory granules and a reduction in the ability to store and release noradrenaline. Furthermore, an important trigger for synaptic release, influx of calcium through voltage-operated calcium channels, is compromised. This is the first demonstration of a transcription factor that directly controls expression of many major components of the RSP and provides further insight into the function of REST.

Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice.

The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.