Molecular phenotyping reveals the identity of Barrett's esophagus and its malignant transition.

The origin of human metaplastic states and their propensity for cancer is poorly understood. Barrett's esophagus is a common metaplastic condition that increases the risk for esophageal adenocarcinoma, and its cellular origin is enigmatic. To address this, we harvested tissues spanning the gastroesophageal junction from healthy and diseased donors, including isolation of esophageal submucosal glands. A combination of single-cell transcriptomic profiling, in silico lineage tracing from methylation, open chromatin and somatic mutation analyses, and functional studies in organoid models showed that Barrett's esophagus originates from gastric cardia through c-MYC and HNF4A-driven transcriptional programs. Furthermore, our data indicate that esophageal adenocarcinoma likely arises from undifferentiated Barrett's esophagus cell types even in the absence of a pathologically identifiable metaplastic precursor, illuminating early detection strategies.

Immunoreactivities of AR, ERα, ERß and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period.

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of AR, ERα, ERß and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERß and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERß protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.

The Influence of Pregnancy on Female Prostate Morphophysiology in Gerbils (Meriones unguiculatus).

Morphophysiological changes of the female prostate during pregnancy are still little known. Considering that this gland is highly influenced by steroid hormones, the aim of this study was to evaluate the impact of the pregnancy on female prostate morphophysiology in gerbils. Pregnant females were timed, and the prostates were analyzed at pregnancy days 6 (P6), 12 (P12), 18 (P18), and 24 (P24). Virgin females were used as the control group (C). We observed a profound change in the hormonal profile during gestation, which was marked by a high oscillation of the progesterone (P4) hormone. P4 serum levels increased, peaking at the middle of gestation, and decreased to the end of the pregnancy. The morphology of the gland in pregnant females also changed, being marked by an increase of acini lumen, and a decrease in stroma. Indeed, the acinar changes during pregnancy were followed by a significant reduction of the epithelial height, besides a change of the smooth muscle cells' morphology that became more relaxed. The number of progesterone receptor (PR) and androgen receptor (AR)-positives cells decreased with the increase of progesterone serum levels, showing an inverse relationship. Finally, we observed a reduction of epithelial proliferation and a significant increase of gland PAS-positive secretion at the end of pregnancy. Altogether, these results showed, for the first time, that the female prostate morphophysioloy is profoundly influenced by the gestational period, suggesting that the fluctuation of the P4 serum levels is the main factor influencing the gland during this period.

Ca2+ Signaling in Exocrine Cells.

Calcium (Ca2+) and cyclic AMP (cAMP) signaling cross talk and synergize to stimulate the cardinal functions of exocrine cells, regulated exocytosis, and fluid and electrolyte secretion. This physiological process requires the organization of the two signaling pathways into complexes at defined cellular domains and close placement. Such domains are formed by membrane contact sites (MCS). This review discusses the basic properties of Ca2+ signaling in exocrine cells, the role of MCS in the organization of cell signaling and in cross talk and synergism between the Ca2+ and cAMP signaling pathways and, finally, the mechanism by which the Ca2+ and cAMP pathways synergize to stimulate epithelial fluid and electrolyte secretion.

Myoepithelial Cells of Submucosal Glands Can Function as Reserve Stem Cells to Regenerate Airways after Injury.

Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice.

A promising approach to new diabetes therapies is to generate ß cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into ß cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into ß cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Morphological characterization of the glandular system in the salamander Plethodon shermani (Caudata, Plethodontidae).

Amphibians have evolved a wide variety of mechanisms that provide a certain degree of protection against predators, including camouflage, tail autonomy, encounter behavior and noxious or toxic skin secretions. In addition to these strategies, some amphibians release a glue-like secretion onto the surface of their skin when threatened. While some information regarding the origin and production of these adhesive secretions is available for frogs such as Notaden bennetti, these aspects are only partially understood in salamanders. We contribute to an earlier study and provide additional information regarding the origin, production, and characterization of the adhesive secretion in the red-legged salamander (Plethodon shermani) at a microanatomical level. When stressed, this salamander secretes a milky, viscous liquid from its dorsal and ventral skin. This secretion is extremely adhesive and hardens within seconds upon exposure to air. This study describes two cutaneous gland types (mucous and granular) in the dorsal and ventral epithelial tissue that differ considerably in their secretory content. While the smaller mucous glands contains flocculent to granular material, mostly acidic glycoproteins, the granular glands synthesize various granules of differing size and density that consist of basic proteinaceous material. The results strongly indicate that the secretions of both gland types from the dorsal as well as the ventral side form the adhesive mucus in Plethodon shermani, consisting of basic and acidic glycoproteins, glycoconjugates with mannose and α-L-fucose residues as well as lipid components.

Development of glandular models from human nasal progenitor cells.

Hyperplasia/hypertrophy of submucosal glands contributes to mucus overproduction in chronic diseases of the upper and lower respiratory tracts, especially in adult and pediatric chronic rhinosinusitis. Mechanisms that lead to glandular hyperplasia/hypertrophy are markedly understudied, reflecting a lack of in vitro model systems wherein airway epithelial progenitor cells differentiate into glandular cells. In this study, we developed and compared several in vitro three-dimensional systems using human nasal epithelial basal cells (HNEBCs) cultured by different methods on two types of extracellular matrices. We demonstrate that HNEBCs cultured on Matrigel (Corning, Tewksbury, MA) form glandular acini-like structures, whereas HNEBCs embedded in a collagen type I matrix form a network of tubules. Fibroblast-conditioned medium increases tubule formation in collagen type I. In contrast, HNEBCs cocultured with fibroblasts self-aggregate into organotypic structures with tubules and acini. These observations provide morphological evidence that HNEBCs are pluripotent and retain the capacity to differentiate into structures resembling specific structural components of submucosal glands depending on the extracellular matrices and culture conditions. The resultant models should prove useful in targeting cross-talk between epithelial cells and fibroblasts to decipher molecular mechanisms and specific signals responsible for the development of glandular hyperplasia/hypertrophy, which in turn may lead to new therapeutic strategies for chronic rhinosinusitis and other inflammatory respiratory diseases characterized by glandular hyperplasia/hypertrophy.

Alterations in the mantle epithelium during transition from hatching gland to adhesive organ of Idiosepius pygmaeus (Mollusca, Cephalopoda).

Epithelial gland systems play an important role in marine molluscs in fabricating lubricants, repellents, fragrances, adhesives or enzymes. In cephalopods the typically single layered epithelium provides a highly dynamic variability and affords a rapid rebuilding of gland cells. While the digestive hatching gland (also named Hoyle organ) is obligatory for most cephalopods, only four genera (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions by means of glandular cells in an adhesive area on the mantle or tentacles. In Idiosepius this adhesive organ is restricted to the posterior part of the fin region on the dorsal mantle side and well developed in the adult stage. Two gland cell types could be distinguished, which produce different contents of the adhesive. During the embryonic development the same body area is occupied by the temporary hatching gland. The question arises, in which way the hatching gland degrades and is replaced by the adhesive gland. Ultrastructural analyses as well as computer tomography scans were performed to monitor the successive post hatching transformation in the mantle epithelium from hatching gland degradation to the formation of the adhesive organ. According to our investigations the hatching gland cells degrade within about 1 day after hatching by a type of programmed cell death and leave behind a temporary cellular gap in this area. First glandular cells of the adhesive gland arise 7 days after hatching and proceed evenly over the posterior mantle epithelium. In contrast, the accompanying reduction of a part of the dorsal mantle musculature is already established before hatching. The results demonstrate a distinct independence between the two gland systems and illustrate the early development of the adhesive organ as well as the corresponding modifications within the mantle.

Ca²âº signaling and fluid secretion by secretory cells of the airway epithelium.

Cytoplasmic Ca(2+) is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca(2+) in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca(2+). Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca(2+) as a second messenger. Changes in intracellular Ca(2+) concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca(2+)-activated K(+) channels and Cl(-) channels. We also review evidence of interactions of Ca(2+) signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca(2+) signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.

Epithelial stem cells in adult skin.

The skin is the first line of defense against dehydration and external environmental aggressions. It constantly renews itself throughout adult life mainly due to the activity of tissue-specific stem cells. In this review, we discuss fundamental characteristics of different stem cell populations within the skin and how they are able to contribute to normal skin homeostasis. We also examine the most recent results regarding the cell-intrinsic and -extrinsic components of the stem cell niche within the adult skin epithelium. Finally, we address the recent efforts to understand how abnormal regulation of stem cell activity contributes to the initiation and progression of skin-associated cancers.

[The structure of the esophageal glands proper in the individuals of elderly and senile age].

Esophageal submucosal glands (esophageal glands proper) were studied in 19 individuals of elderly and senile age (without subdivision according to gender), as well as in persons of I period of mature age (6 to 7 cases in each group). It was shown that the glandular acini contained thin, elongated cells and cuboidal cells between the mucocytes surrounded by the myoepithelial cells. Around the acini, the myofibroblasts were found. The number of fibroblasts and lymphocytes in glandular stroma increased with age. The number of plasma cells was increased with age in the stroma of the glands of the upper part of the esophagus. Small excretory ducts were arranged in groups, often surrounded by the clusters of lymphoid cells. The number of lymphoid cells and fibroblasts around the large excretory ducts was found to increase with age.

Tubulogenesis.

Metazoans require epithelial and endothelial tubes to transport liquids and gasses throughout their bodies. Although biological tubes may look relatively similar at first glance, there are multiple and distinct mechanisms by which tubes form and even more regulatory events driving the cell shape changes that produce tubes of specific dimensions. An overview of the current understanding of the molecular processes and physical forces involved in tubulogenesis is presented in this review and the accompanying poster.

Morphology and composition of the spider major ampullate gland and dragline silk.

Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

Isolation of basal cells and submucosal gland duct cells from mouse trachea.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Why mouse airway submucosal gland serous cells do not secrete fluid in response to cAMP stimulation.

Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl(-)) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl(-) permeability, but they lack cAMP-activated calcium (Ca(2+)) signaling observed in human cells. Similar to human cells, basal K(+) conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca(2+) signaling in mouse cells results in the absence of K(+) conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K(+) channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca(2+) signaling to activate basolateral membrane K(+) conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human glands.

The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila.

The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity.

Chloride secretion by cultures of pig tracheal gland cells.

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.