Secretory Cell Types Research Articles

Advances in Ca2+ modulation of gastrointestinal anion secretion and its dysregulation in digestive disorders (Review).

Intracellular calcium (Ca2+) is a critical cell signaling component in gastrointestinal (GI) physiology. Cytosolic calcium ([Ca2+]cyt), as a secondary messenger, controls GI epithelial fluid and ion transport, mucus and neuropeptide secretion, as well as synaptic transmission and motility. The key roles of Ca2+ signaling in other types of secretory cell (including those in the airways and salivary glands) are well known. However, its action in GI epithelial secretion and the underlying molecular mechanisms have remained to be fully elucidated. The present review focused on the role of [Ca2+]cyt in GI epithelial anion secretion. Ca2+ signaling regulates the activities of ion channels and transporters involved in GI epithelial ion and fluid transport, including Cl- channels, Ca2+-activated K+ channels, cystic fibrosis (CF) transmembrane conductance regulator and anion/HCO3- exchangers. Previous studies by the current researchers have focused on this field over several years, providing solid evidence that Ca2+ signaling has an important role in the regulation of GI epithelial anion secretion and uncovering underlying molecular mechanisms. The present review is largely based on previous studies by the current researchers and provides an overview of the currently known molecular mechanisms of GI epithelial anion secretion with an emphasis on Ca2+-mediated ion secretion and its dysregulation in GI disorders. In addition, previous studies by the current researchers demonstrated that different regulatory mechanisms are in place for GI epithelial HCO3- and Cl- secretion. An increased understanding of the roles of Ca2+ signaling and its targets in GI anion secretion may lead to the development of novel strategies to inhibit GI diseases, including the enhancement of fluid secretion in CF and protection of the GI mucosa in ulcer diseases.

Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis.

SARS‐CoV‐2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

The SARS‐CoV‐2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID‐19 pathogenesis, especially host factors facilitating virus infection and replication. SARS‐CoV‐2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS‐CoV‐2 infection. Our data provide a rich resource for future investigations of COVID‐19 infection and pathogenesis.

Salmonella biases epithelial differentiation in murine enteroids through Wnt and Notch signaling, which may contribute to diarrheal pathogenesis

Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.

The Ldb1 Transcriptional Co‐Regulator is Required for Establishment, Development, and Survival of the Pancreatic Endocrine Lineage

Pancreatic islets are comprised of hormone secreting cell types that are vital regulators of glucose metabolism. Specifically, the pancreatic β cell is indispensable for glucose control and its dysfunction is central to diabetes mellitus. β cell development is regulated by transcription factor (TF) cascades, mediating differentiation of progenitors into mature insulin producing β cells. Our prior studies show that TF, Islet1 (Isl1), interacts with a transcriptional co‐regulator, Ldb1, to regulate β cell maturation from embryonic day (E) 18.5 onward. However, Ldb1 is also expressed in early stages of pancreas development, before Isl1 is present (as early as E10.5). The earlier Ldb1+ cell types include Pdx1+ multipotent progenitor cells (MPCs) and endocrine progenitors expressing the TF Neurogenin3 (Ngn3). MPC progeny will populate the entire pancreas (endocrine and exocrine), while Ngn3+ endocrine progenitors (Isl1−) are fated to become islet cells. Our hypothesis is that Ldb1 has Isl1 independent roles in maintaining progenitor identity in these requisite populations during early pancreatic development. To test this we generated a whole pancreas knockout of Ldb1 (Ldb1Δpanc) and observed severe developmental and postnatal phenotypes. At E13.5, Ldb1Δpanc mice exhibit disorganized progenitor pools, suggesting early defects in endocrine identity. At E15.5, Ldb1Δpanc mice had a significant reduction of Ngn3+ progenitors and Pdx1HI immunoreactivity, a mark of presumptive β cells. Ldb1Δpanc mice die by postnatal day 7 (P7) with severe hyperglycemia and hypoinsulinemia due to drastic islet hormone cell reduction. Interestingly, total pancreatic mass remained unchanged in Ldb1Δpanc neonates, suggesting that Ldb1 impacts are islet specific in the pancreas. Considering these observations, we generated a new model of Ldb1 loss specifically in Ngn3+ islet progenitors, termed Ldb1Δendo. We confirmed loss of Ldb1 in endocrine clusters and observed postnatal hyperglycemia, with a reduction of islet cells in neonates, similar to that seen in Ldb1Δpanc mice. Chromatin immunoprecipitation in vivo and in vitro highlights that Ldb1 imparts control on Pdx1 through occupation of the Pdx1 Area I–II regulatory domains, and that Ngn3 control also occurs via direct occupation. We are now further assessing the developmental phenotype, examining markers of proliferation, apoptosis, and altered cell identity through lineage tracing in the Ldb1Δendo model. Concomitantly, our published work revealed that Ldb1 and Isl1 interact in complex with the single stranded DNA binding protein co‐regulator, SSBP3, which helps stabilize this transcriptional complex. To examine the functional relationship between SSBP3, Isl1, and Ldb1 in vivo, we developed a new SSBP3 floxed mouse model in order to generate pancreas wide loss of SSBP3 (SSBP3Δpanc). We observed preliminary reductions in key islet mRNAs along with hyperglycemia in neonates at P1. Our work provides insight into the transcriptional complexes dictating how islet progenitors adopt their cell fate. Enhancing our understanding of this differentiation process can contribute to therapeutic β and islet cell generation strategies to help the growing diabetic population.Support or Funding InformationNIH‐NIDDK R01DK111483‐01NIH‐F31DK120217NIH‐NIGMS T32GM008111

The copulatory apparatus of the marine gastropod Haminella solitaria (Heterobranchia: Cephalaspidea) and its phylogenetic relevance

AbstractThe hermaphroditic marine snail species Haminella solitaria was formerly included in the genus Haminoea, but it was recently assigned to the genus Haminella. The copulatory apparatus in H. solitaria was investigated by light and transmission electron microscopy to obtain additional information about this apparatus in cephalaspidean gastropods and to evaluate the taxonomic relevance of its morphofunctional features in the framework of a new phylogenetic tree of the family Haminoeidae. The copulatory apparatus in H. solitaria consisted of the atrium with a muscular wall and papilla, a seminal duct, and a single‐lobed prostate. Epithelial and subepithelial secretory cells were detected in the proximal and middle region of the atrium wall, and a third type of secretory cell occurred in the distal region of the muscular papilla. The seminal duct was lined by ciliated cells and its muscular wall included some vacuolar cells. The prostate in H. solitaria consisted of lateral pouches surrounding a large central lumen that was filled with spermatozoa. A single type of secretory cell intermingled with ciliated cells formed the epithelium of the prostate. A histological comparison between the copulatory apparatus in H. solitaria and Haminoea navicula revealed substantial differences that support the placement of these two species in different genera, as established by recent molecular studies.

Detailed comparative anatomy of the Pinnidae (Mollusca, Bivalvia) reveals further unusual mantle specializations

Pinnids are semi-infaunal bivalves with several unusual morphological features, some of them regarded as adaptations to environments with high concentration of suspended particles. The presence of a waste canal on the mantle wall, for example, helps rejecting surplus particles from the mantle cavity. Despite being such anatomically intriguing bivalves, detailed comparative morphology studies on pinnids are still scarce. To gain further insights into the functional anatomy of pinnids, we have thoroughly investigated the mantle of Pinna carnea Gmelin, 1791 by means of scanning electron and light microscopy, including the application of histochemical staining methods. Additionally, the mantle was analyzed in ten pinnids from the genera Atrina, Pinna, and Streptopinna obtained in museum collections. Comparative morphology revealed a uniform structural organization of the mantle in Pinnidae, but a pronounced variation in pigmentation, presence of commarginal folds, and papillae. Our results also revealed intense secretory activity in the inner mantle fold, inner mantle epithelium, and waste canal, including at least three secretory cell types. Secretions are diverse and comprise mainly acidic mucosubstances on the mantle margin, while the waste canal secretes abundant acidophilic material apparently composed of basic proteins, and also possibly lipid content. Based on these results, we hypothesize that efficient mucociliary transportation is achieved in a hydrophobic environment provided by the waste canal, rapidly agglutinating and removing large amounts of particles from the mantle. Discoid glands secreting protein-rich content were found on the middle mantle fold of Pinna carnea and Streptopinna saccata (Linnaeus, 1758). Surprisingly, these mantle glands have hitherto not been recorded in the Bivalvia, representing, therefore, a new morphological feature in the Pinnidae.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Comparative histomorphology and histochemistry of exocrine pancreas in Deccani sheep and Bidri goat

The present study on comparative histological study of exocrine pancreas in six adult Deccani sheep and Bidri goat to know the structural differences between the small ruminants. Histologically, in both species exocrine pancreas forms bulk of gland and was tubuloacinar in nature. Tubular secreting units were dominating type as compared to acinar secreting units. In present study secretory cells were pyramidal in shape. They were arranged in single layer and two zones in each secretory cell. Three types of secretory cells viz., active, exhausted and resting cells were recorded in present study in both species. Centroacinar cells were observed at lumen of secretory units which continues with intercalated ducts. Histochemically, secretory cells in both species showed moderate (++) PAS positive reaction towards the apical part.

BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions

Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. Rapidly evolving and molecularly diverse, they derive from multiple male secretory cells and tissues. In Drosophila melanogaster, most SFPs are produced in the accessory glands, which are composed of ∼1,000 fertility-enhancing "main cells" and ∼40 more functionally cryptic "secondary cells." Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might therefore make highly specific contributions to the seminal proteome and ejaculate function; alternatively, they might regulate more global-but hitherto undiscovered-SFP functions and proteome composition. Here, we present data that support the latter model. We show that in addition to previously reported phenotypes, secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, we reveal changes to the seminal proteome that surprisingly encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. Our results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands.

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

External Morphology and Ultrastructure of Tegumental Glands of Aegla platensis (Crustacea, Anomura, Aeglidae) Pleopods: Might They Play A Role in Egg Attachment?

Egg incubation on the female abdomen is the parental care behavior observed in aeglids, in which eggs are kept adhered to maternal pleopods and maintained, cleaned and aerated. In A. platensis, egg attachment occurs with the aid of pleopodal setae, which are twisted around their axis in the distal region, forming the funiculus, and pleopodal glands, which are responsible for the production of the adhesive substance that seems to be involved in egg fixation to pleopodal setae. Those glands are acini formed by secretory cells arranged concentrically around a central duct, giving them a rosette appearance. Two types of secretory cells were observed, those that produce electron-lucid vesicles and those having electron-dense ones. Both kinds of vesicles are released in a duct whose opening pore is located on the pleopodal surface and constitute the adhesive substance that coats eggs and pleopodal setae, ensuring egg fixation to the female body and maternal care maintenance. This study investigates the internal and external morphology of Aegla platensis pleopods, to understand the egg attachment process and identify the structures involved in this phenomenon. Three microscopy techniques are used: scanning electron microscopy (SEM), transmission electron microscopy (TEM), and optical microscopy (OM).

Abstract 467: Deep single-cell RNA-seq of the putative cell of origin revealed a novel molecular subtype of high-grade serous ovarian cancer with poor prognosis

Abstract Accurate molecular classification in cancer is essential for rationalized therapy. However, achieving stable subtyping is highly challenging due to the underlying genomic complexity of tumors. An example of the most successful classification is in breast cancer where it is possible to link individual tumor types, basal vs luminal, to individual cell types of origin. However, the generalization of such an approach has not been easy because of the lack of knowledge about subtypes of the putative cells of origin. For instance, it has been difficult to establish a stable molecular classification of high-grade serous ovarian cancer (HGSOC). More recent evidence strongly supports the notion that HGSOC originates from the secretory cells in fallopian tube epithelium (FTE). However, whether or not there are multiple subtypes of secretory cells and how such subtypes are linked to tumor molecular subtypes have remained elusive. To address this issue, we applied single-cell RNA sequencing (Smart-Seq2) on around 4000 FTE cells from patients with ovarian or endometrial cancers. We developed and employed an in-house differential-expression-based clustering method, termed ClinCluster, for clinical samples that takes into account inter-patient variability. Apart from the established secretory and ciliated cell types, we discovered four novel subtypes of secretory cells and a rare intermediate cell type that co-expresses secretory and ciliated markers. We termed these four novel secretory subtypes as cell cycle, progenitor, epithelial-mesenchymal transition (EMT) and immunoreactive subtypes. Based on fifty-three biomarkers of these subtypes, we designed a panel of transcriptomic signatures and exploited them to deconvolute two bulk expression datasets of ovarian cancer: the RNA-seq dataset of TCGA (The Cancer Genome Atlas) and the microarray dataset of AOCS (Australian Ovarian Cancer Study). The deconvolution analysis revealed a poor-prognostic EMThigh subtype of HGSOC, which was not driven by copy number aberrations. Cox regression analysis indicated that the EMThigh tumors were strongly associated with poor overall survival in the two independent datasets tested (hazard ratio = 2.7, 95% confidence interval = 1.4 - 5.1, P = 0.002, n = 307 for TCGA; hazard ratio = 3.2, 95% confidence interval = 1.7 - 6.1, P = 0.0002, n = 253 for AOCS). Importantly, we found that the PI3K-Akt signaling pathway is upregulated in the EMThigh tumors (FDR < 0.002 for the two independent datasets) giving a strong potential for effective rationalized therapies. In summary, our work portrays a new model for the landscape of fallopian tube epithelium, the putative origin of HGSOC, and illustrates that the cellular subtypes in FTE are related to the HGSOC tumor types with important prognostic and therapeutic implications. Citation Format: Zhiyuan Hu, Abdulkhaliq Alsaadi, Nina Wietek, Laura Santana González, Christopher Yau, Ahmed Ashour Ahmed. Deep single-cell RNA-seq of the putative cell of origin revealed a novel molecular subtype of high-grade serous ovarian cancer with poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 467.

Raising the steaks ‐ a taste of what's to come

Raising the steaks ‐ a taste of what's to come

Autophagy proteins are required for club cell structure and function in airways.

Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1T300A/T300A, which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types.

Targeting Wnt Signaling via Notch in Intestinal Carcinogenesis.

Proliferation and differentiation of intestinal epithelial cells is assisted by highly specialized and well-regulated signaling cascades. The Wnt pathway, which is one of the fundamental pathways in the intestine, contributes to the organization of proliferative intestinal crypts by positioning and cycling of intestinal stem cells and their derivatives. The Wnt pathway promotes differentiation of intestinal secretory cell types along the crypt-plateau and crypt-villus axis. In contrast to the Wnt pathway, the intestinal Notch cascade participates in cellular differentiation and directs progenitor cells towards an absorptive fate with diminished numbers of Paneth and goblet cells. Opposing activities of Notch and Wnt signaling in the regulation of intestinal stem cells and the enterocytic cell fate have been elucidated recently. In fact, targeting Notch was able to overcome tumorigenesis of intestinal adenomas, prevented carcinogenesis, and counteracted Paneth cell death in the absence of caspase 8. At present, pharmacological Notch inhibition is considered as an interesting tool targeting the intrinsic Wnt pathway activities in intestinal non-neoplastic disease and carcinogenesis.

Sprouty2 restricts colonic tuft and goblet cell numbers by repressing epithelial IL‐33 expression

OBJECTIVETuft and goblet cells are secretory cell types of the intestinal epithelium that serve key roles in sensing and responding to inflammation. However, the intracellular mechanisms controlling their numbers and activity are incompletely defined. Here, we investigated the role of Sprouty2 (Spry2), an intracellular modulator of PI3K and MAPK signaling, in tuft and goblet cell development. Both PI3K and MAPK are implicated in control of differentiation in the intestine. Furthermore, both pathways have been linked to expression of IL‐33, a cytokine that indirectly influences secretory differentiation by stimulating IL‐13 release from immune cell populations. We tested the hypothesis that Spry2 controls IL‐33 production in the colon, thus altering intestinal secretory cell development.METHODSMucosal scrapings from wild type mice given dextran sodium sulphate (DSS) colitis, and colonoids treated with TNF, were subjected to qPCR analysis for Spry2 expression. Colons from mice with intestinal epithelial‐specific Spry2 deletion (Spryflox/flox;Villin‐Cre, here called Spry2KOIE) and littermate controls were collected at 8 weeks of age. Cytokine levels and epithelial marker expression were determined by immunostaining and qPCR. Signaling pathway alterations were assessed by dot blot array and confirmed by western blot. In vitro, murine colonoids were treated with IL‐33 or IL‐13 +/− signaling pathway inhibitors (LY294002, PI3K; CHIR99021, GSK3β) to determine the effects on cytokine and epithelial marker expression.RESULTSSpry2 levels were decreased in inflamed colons (59% decrease, p<0.05) and TNF‐treated colonoids (45% decrease, p<0.05), demonstrating regulation by inflammation. Spry2KOIE mice displayed increased colonic IL‐33 expression (80% increase, p<0.05), more mesenchymal IL‐13+ cells, more Dclk1+ tuft cells (2.2‐fold, p<0.01), and more Muc2+ goblet cells (1.57‐fold, p<0.01). These mice also had elevated GSK3β phosphorylation (88% increase, p<0.01) on Ser9 (an inhibitory site phosphorylated by Akt), and increased phospho‐Akt (p<0.01). In vitro, inactivation of GSK3β in murine colonoids with CHIR99021 mimicked the increased IL‐33 observed in vivo, but interestingly did not alter Dclk1 or Muc2 expression. This effect was attenuated by PI3K inhibition. IL‐13, but not IL‐33, induced Dclk1 and Muc2 in colonoids, suggesting an epithelial‐mesenchymal crosstalk circuit drives alterations in tuft and goblet cells following Spry2 loss.CONCLUSIONInflammation‐driven loss of colonic epithelial Spry2 de‐represses PI3K/Akt signals that lead to elevated IL‐33 expression. IL‐33 may act, in turn, to promote mesenchymal IL‐13 release that drives a protective expansion of tuft and goblet cells in the epithelium.Support or Funding InformationNIH R01DK095004 (Frey); Crohn's and Colitis Foundation Career Development Award (Schumacher)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunohistochemical localization of estrogen receptor alpha (ERα) in the oviduct of Indian buffalo during follicular and luteal phases of estrous cycle.

The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.

The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides.

ABSTRACTThe disk-shaped millimeter-sized marine animal, Trichoplax adhaerens, is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies.