Neuroendocrine Cell Population Research Articles

Cocaine and amphetamine regulated transcript (CART): a newly characterized neuropeptide in human prostate

ABSTRACT Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.

A somatotroph pituitary adenoma, with signet-ring like cells. A case report and review of the literature

Background: Pituitary adenomas are neoplasms affecting the adenohypophysis and are derived from either early progenitor or fully differentiated hormone secreting cells. Histologically signet-ring like cells in pituitary adenomas are considered a normal morphological variant. This is an extremely rare entity, with four cases reported in the literature to date. Aims: We report a case of a 36-year-old man with symptoms of acromegaly and MRI findings of a 6 mm pituitary adenoma. Methods: A transphenoidal resection of the pituitary tumour was undertaken and submitted for light microscopic, immunohistochemical and electron microscopic assessment Results and conclusions: On histology the adenoma consisted of a monotonous population of neuroendocrine cells diffusely positive for synaptophysin, cytokeratin AE1-3 and Pit-1, with patchy positive growth hormone staining. There were admixed signet-ring like cells in which the cytoplasmic vacuoles were positive for synaptophysin and PAS-diastase and negative for the CD68 macrophages stain. This case describes the rare morphological signet-ring like cell variant in pituitary adenomas as well as highlighting differences between this benign neoplasm and metastatic signet ring cell carcinoma. Reference Trabzonlu L, Trabzonlu TA, Girbuz Y, et al. ACTH-cell pituitary adenoma with signet ring cells: a rare case report and review of the literature. Appl Immunohistochem Mol Morphol 2018; 28: e13–16.

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

GABAA receptor-mediated inhibition of Dahlgren cells electrical activity in the olive flounder, Paralichthys olivaceus

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. We investigated its potential role as a neurotransmitter in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the flounder Paralichthys olivaceus. The application of GABA in vitro resulted in a decrease in electrical activity of Dahlgren cells, followed by an increase of the number of silent cells, together with a decreased firing frequency of all three activity patterns (tonic, phasic, bursting). GABAA receptor agonist etomidate decreased Dahlgren cell firing activity, in a similar way to GABA. The response to GABA was blocked by the GABAA receptor antagonist bicuculline. GABAA receptor gamma2 subunit (Gabrg2) and chloride channel (Clcn2) mRNA expression were significantly upregulated in the CNSS after GABA superfusion. These data suggest that GABA may modulate CNSS activity in vivo mediated by GABAA receptors.

Ion Channel Profiling in Prostate Cancer: Toward Cell Population-Specific Screening.

In the last three decades, a growing number of studies have implicated ion channels in all essential processes of prostate carcinogenesis, including cell proliferation, apoptosis, migration, and angiogenesis. The changes in the expression of individual ion channels show a specific profile, making these proteins promising clinical biomarkers that may enable better molecular subtyping of the disease and lead to more rapid and accurate clinical decision-making. Expression profiles and channel function are mainly based on the tumoral tissue itself, in this case, the epithelial cancer cell population. To date, little data on the ion channel profile of the cancerous prostate stroma are available, even though tumor interactions with the microenvironment are crucial in carcinogenesis and each distinct population plays a specific role in tumor progression. In this review, we describe ion channel expression profiles specific for the distinct cell population of the tumor microenvironment (stromal, endothelial, neuronal, and neuroendocrine cell populations) and the technical approaches used for efficient separation and screening of these cell populations.

SAT-034 Photometry Based Measurement of G Protein-Coupled Receptor-Mediated Signalling

Many hormones and neurotransmitters exert their effects by binding to G protein-coupled receptors (GPCRs). These membrane-spanning receptors couple primarily to heterotrimeric G proteins which can act on effector proteins to modulate cellular functions such as membrane excitability, second messenger production or gene expression. GPCRs represent the most druggable targets in the mammalian genome, however individual receptors can often activate multiple signaling pathways, only some of which may mediate therapeutic effects. Much of what is known about the signaling pathways linked to activation of specific GPCRs comes from studies using heterologous cell models, but evidence now suggests that the specific complement of effectors engaged by a given GPCR is dependent on the cellular and tissue context, such that the same receptor may induce different signaling patterns in different cell types. This phenomenon is particularly evident in the central nervous system which contains hundreds of specialized cell populations whose function are regulated by the combined action of hormones, neurotransmitters and neuromodulators, and disruption of select signaling pathways may contribute to disease. To study GPCR mediated signaling in cells of interest in their native context, we have developed a biosensor-based approach that would allow in vivo expression and recording of fluorescent reporters in behaving animals. Our approach uses adeno-associated viruses (AAV) to express genetically encoded sensors in animals using cell-type selective promoters and Cre-recombinase dependent expression. Biosensor responses are recorded in real time from live animals using a fiber-photometry-based FRET recording platform. Our approach allows simultaneous measurement of cell-specific signaling with behavioral or physiological measurements made from live animal subjects. This approach has wide applicability to the study of neuroendocrine cell populations, hormone action in the brain, and to evaluate drug action on target cells.

Modulatory effect of dopamine receptor 5 on the neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus

A neuromodulatory role for dopamine has been reported for magnocellular neuroendocrine cells in the mammalian hypothalamus. We examined its potential role as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder Paralichthys olivaceus. In vitro application of dopamine (DA) caused an increase in electrical activity (firing frequency, recorded extracellularly) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The dopamine precursor, levodopa (L-DOPA), also increased firing frequency, cell recruitment and enhanced bursting and tonic activity. The effect of dopamine was blocked by the D1, D5 receptor antagonist SCH23390, but not by the D2, D3, D4 receptor antagonist amisulpride. Transcriptome sequencing revealed that all DA receptors (D1, D2, D3, D4, and D5) were present in the flounder CNSS. However, quantitative RT-PCR revealed that D5 receptor mRNA expression was significantly increased in the CNSS following dopamine superfusion. These findings suggest that dopamine may modulate CNSS activity in vivo, and therefore neurosecretory output, through D5 receptors.

Modulatory effect of glutamate GluR2 receptor on the caudal neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus

A neuromodulatory role for glutamate has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined the potential role of glutamate as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) in the euryhaline flounder Paralichthys olivaceus. In pharmacological experiments in vitro, glutamate (Glu) caused an increase in electrical activity of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The glutamate substrate, glutamine (Gln), led to increased firing frequency, cell recruitment and enhanced bursting activity. The glutamate effect was not blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, or the GluR1/GluR3 (AMPA) receptor antagonist IEm1795-2HBr, but was blocked by the broad-spectrum α-amino-3-hydroxy- 5- methyl-4-isoxazo-lepropionic acid (AMPA) receptor antagonist ZK200775. Our transcriptome sequencing study revealed three AMPA receptor (GluR1, GluR2 and GluR3) in the olive flounder CNSS. Quantitative RT-PCR revealed that GluR2 receptor mRNA expression was significant increased following dose-dependent superfusion with glutamate in the CNSS. GluR1 and GluR3 receptor mRNA expression were decreased following superfusion with glutamate. L-type Ca2+ channel mRNA expression had a significant dose-dependent decrease following superfusion with glutamate, compared to the control. In the salinity challenge experiment, acute transfer from SW to FW, GluR2 receptor mRNA expression was significantly higher than the control at 2 h. These findings suggest that GluR2 is one of the mechanisms which can medicate glutamate action within the CNSS, enhancing electrical activity and hence secretory output.

Abstract 3935: Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry

Abstract Introduction: Small cell lung cancer (SCLC) is a high grade neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths. Treatment in SCLC has not changed significantly in the last 20 years. SCLC initially responds well to chemotherapy, but inevitably recurs. Characterization of tumor heterogeneity and changes in SCLC cell signaling and phenotypes after chemotherapy could yield new insights and therapeutic options. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of more than 40 proteins in single cells and offers the opportunity to identify new subpopulations including potential cancer stem cell populations as well as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with one cycle of carboplatin/etoposide or saline injection. Tumors were harvested at ~2000mm3, disaggregated, and cryopreserved. PDX samples were stained with a 22 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. ViSNE analysis and biaxial gating were used to identify major subpopulations of interest. Results: PDX tumors released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. ACK buffer and enzymatic dissociation yielded the best quality cells by depleting red blood cells. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and iridium intercalator was used to identify nucleated cells. Single cell protein expression and phosphorylation was analyzed using viSNE and yielded at least 9 distinct subpopulations based on density islands with neuroendocrine (CD56+) and non-neuroendocrine (CD56-) populations. Chemotherapy treated cells had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included 2-3 fold expansion of SOX2+, CD117+, and pSTAT3+ populations with chemotherapy treatment. A small CD44+ tumor subpopulation identified in the chemotherapy treated cells was not present in the matched mock treated tumor suggesting a potential chemotherapy resistant/ stem- like subpopulation. Kinase activity showed stable p-AKT overall, but increased p-S6 in the chemotherapy treated cells. Conclusions: Mass cytometry was able to identify multiple neuroendocrine and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of multiple stem-like signaling factors. This work demonstrates the utility of mass cytometry and viSNE as novel techniques to identify subpopulations associated with chemotherapy resistance for future targeting and demonstrates the feasibility of this technique for characterizing signaling heterogeneity in human SCLC tumors. Citation Format: Jonathan M. Lehman, Nalin Leelatian, Bradford Harris, Megan Hoeksema, Zou Yong, Deon B. Doxie, Jonathan M. Irish, Pierre P. Massion. Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2017-3935

Merkel Cell Polyomavirus Small T Antigen Initiates Merkel Cell Carcinoma-like Tumor Development in Mice.

Merkel cell carcinoma (MCC) tumor cells express several markers detected in normal Merkel cells, a nonproliferative population of neuroendocrine cells that arise from epidermis. MCCs frequently contain Merkel cell polyomavirus (MCPyV) DNA and express viral transforming antigens, sT and tLT, but the role of these putative oncogenes in MCC development, and this tumor's cell of origin, are unknown. Using a panel of preterm transgenic mice, we show that epidermis-targeted coexpression of sT and the cell fate-determinant atonal bHLH transcription factor 1 (ATOH1) leads to development of widespread cellular aggregates, with histology and marker expression mimicking that of human intraepidermal MCC. The MCC-like tumor phenotype was dependent on the FBXW7-binding domain of sT, but not the sT-PP2A binding domain. Coexpression of MCPyV tLT did not appreciably alter the phenotype driven by either sT or sT combined with ATOH1. MCPyV sT, when coexpressed with ATOH1, is thus sufficient to initiate development of epidermis-derived MCC-like tumors in mice. Cancer Res; 77(12); 3151-7. ©2017 AACR.

AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.

Fasting evokes a homeostatic response that maintains circulating levels of energy-rich metabolites and increases the drive to eat. Centrally, this reflex activates a small population of hypothalamic neurons that are characterized by the expression of AgRP, a neuropeptide with an extremely restricted distribution. Apart from the hypothalamus, the only other site with substantial expression is the adrenal gland, but there is disagreement about which cells synthesize AgRP. Using immunohistochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, we show AgRP is present in the mouse adrenal medulla and is expressed by neuroendocrine chromaffin cells that also synthesize the catecholamines and neuropeptide Y. Short-term fasting led to an increase in adrenal AgRP expression. Because AgRP can act as an antagonist at MC3/4 receptors, we tested whether melanotan II, an MC3/4 receptor agonist, could regulate pre- and postsynaptic signaling within the adrenal medulla. Melanotan II decreased the paired-pulse ratio of evoked synaptic currents recorded in chromaffin cells; this effect was blocked by exogenous AgRP. In contrast, neither melanotan II nor AgRP altered the optogenetically evoked release of catecholamines from isolated chromaffin cells. These results are consistent with the idea that AgRP regulates the strength of the sympathetic input by modulation of presynaptic MC3/4 receptors located on preganglionic neurons. We conclude that a small population of neuroendocrine cells in the adrenal medulla, and the arcuate nucleus of the hypothalamus, express AgRP and neuropeptide Y and are functionally involved in the systemic response to fasting.

PanIN Neuroendocrine Cells Promote Tumorigenesis via Neuronal Cross-talk

Abstract Nerves are a notable feature of the tumor microenvironment in some epithelial tumors, but their role in the malignant progression of pancreatic ductal adenocarcinoma (PDAC) is uncertain. Here, we identify dense innervation in the microenvironment of precancerous pancreatic lesions, known as pancreatic intraepithelial neoplasms (PanIN), and describe a unique subpopulation of neuroendocrine PanIN cells that express the neuropeptide substance P (SP) receptor neurokinin 1-R (NK1-R). Using organoid culture, we demonstrated that sensory neurons promoted the proliferation of PanIN organoids via SP-NK1-R signaling and STAT3 activation. Nerve-responsive neuroendocrine cells exerted trophic influences and potentiated global PanIN organoid growth. Sensory denervation of a genetically engineered mouse model of PDAC led to loss of STAT3 activation, a decrease in the neoplastic neuroendocrine cell population, and impaired PanIN progression to tumor. Overall, our data provide evidence that nerves of the PanIN microenvironment promote oncogenesis, likely via direct signaling to neoplastic neuroendocrine cells capable of trophic influences. These findings identify neuroepithelial cross-talk as a potential novel target in PDAC treatment. Cancer Res; 77(8); 1868–79. ©2017 AACR.

PanIN Neuroendocrine Cells Promote Tumorigenesis via Neuronal Cross-talk.

Nerves are a notable feature of the tumor microenvironment in some epithelial tumors, but their role in the malignant progression of pancreatic ductal adenocarcinoma (PDAC) is uncertain. Here, we identify dense innervation in the microenvironment of precancerous pancreatic lesions, known as pancreatic intraepithelial neoplasms (PanIN), and describe a unique subpopulation of neuroendocrine PanIN cells that express the neuropeptide substance P (SP) receptor neurokinin 1-R (NK1-R). Using organoid culture, we demonstrated that sensory neurons promoted the proliferation of PanIN organoids via SP-NK1-R signaling and STAT3 activation. Nerve-responsive neuroendocrine cells exerted trophic influences and potentiated global PanIN organoid growth. Sensory denervation of a genetically engineered mouse model of PDAC led to loss of STAT3 activation, a decrease in the neoplastic neuroendocrine cell population, and impaired PanIN progression to tumor. Overall, our data provide evidence that nerves of the PanIN microenvironment promote oncogenesis, likely via direct signaling to neoplastic neuroendocrine cells capable of trophic influences. These findings identify neuroepithelial cross-talk as a potential novel target in PDAC treatment. Cancer Res; 77(8); 1868-79. ©2017 AACR.

Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty.

Mucosal immunology: Neuroendocrine cells regulate lung inflammation.

A rare population of neuroendocrine cells in the lung epithelium can shape immune responses and tissue remodelling in the lungs.

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis.

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

Sa2018 INSL5 Indicates a Unique Population of Neuroendocrine Cells in the Colorectal Epithelium

Sa2018 INSL5 Indicates a Unique Population of Neuroendocrine Cells in the Colorectal Epithelium

A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.

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.