Endocrine Cell Populations Research Articles

Abstract A22: Molecular subtypes and resistance programs in pancreatic ductal adenocarcinoma elucidated with single-nucleus RNA-seq

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease as existing molecular subtypes are insufficient and do not currently inform clinical decisions. Rare cell types, including those responsible for resistance, are difficult to detect with bulk transcriptomic profiling. Indeed, several previously identified transcriptomic subtypes of PDAC are unintentionally driven by “contaminating” stromal components. Single-cell transcriptomics provides an unprecedented degree of resolution into the properties of individual cells. However, RNA extraction from RNase- and stroma-rich pancreatic tissue is difficult and prior single-cell efforts have been limited by suboptimal dissociation/RNA quality. We developed a robust single-nucleus RNA-seq (sNuc-seq) technique compatible with frozen archival PDAC specimens and computational techniques to identify the transcriptomic programs driving tumor subtypes and therapeutic resistance. Methods: Patients with localized PDAC undergoing surgical resection with or without neoadjuvant chemoradiotherapy were consented for this IRB-approved study. Specimens were screened for RNA Integrity Number >6. Single nuclei suspensions were extracted from flash-frozen primary PDAC specimens and organoids. Approximately 8,000 nuclei were loaded on the 10x Genomics Chromium platform per sample to generate and sequence 3’ gene expression libraries (Illumina HiSeq 2500, 125 bp paired-end reads). sNuc-seq derived reads were processed using the 10X CellRanger v3.0.2 pipeline. Unsupervised clustering was utilized to identify different cell populations and known marker genes from literature were used to label cell types. Results: Both treatment-naïve (n=12) and treatment-resistant (n=11) specimens yielded high-quality sNuc-seq data (>1,000 nuclei per sample, >1,000 median genes per nucleus). In each tumor, distinct clusters with gene expression profiles consistent with ductal, fibroblast, endothelial, endocrine, lymphocyte, and myeloid cell populations were identified. Malignant cells were confirmed by inferred copy number variation analysis (InferCNV v3.9) and segregated into several distinct clusters for each individual patient highlighting intratumoral heterogeneity. While some malignant clusters corresponded to previously identified basal-squamous and classical-progenitor bulk subtypes, others featured expression profiles distinct from known subtypes, including cells with upregulation of hypoxia-associated or cytoskeletal genes. Conclusions: Applying sNuc-seq to treatment-naïve and pretreated PDAC specimens, we uncovered significant intratumoral heterogeneity in the malignant and stromal compartments and identified malignant cells featuring transcriptomic programs that do not fit previously identified bulk subtypes. Characterization of therapeutic resistance programs, spatial relationships among cell types, and association with clinical outcomes is ongoing. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Orr Ashenberg, Eugene Drokhlyansky, George Eng, Nicholas Van Wittenberghe, William Freed-Pastor, Clifton Rodriguez, Danielle Dionne, Julia Waldman, Michael Cuoco, Alexander Tsankov, Connor Lambden, Caroline Porter, Jason Schenkel, Laurens Lambert, Debora Ciprani, Andrew J. Aguirre, Mari Mino-Kenudson, Theodore S. Hong, Orit Rozenblatt-Rosen, Carlos Fernandez-del Castillo, Andrew S. Liss, Aviv Regev, Tyler E. Jacks. Molecular subtypes and resistance programs in pancreatic ductal adenocarcinoma elucidated with single-nucleus RNA-seq [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A22.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

SAT-547 Pituitary Thyrotrophs: A Generator of Repetitive Calcium Waves in Freely-Moving Animal Models

Hypothyroidism refers to the pathological state of thyroid hormone (TH) deficiency. It affects between 0.3% and 3.7% of the general population in the USA and between 0.2% and 5.3% in Europe. Hypothyroid patients suffer from extreme fatigue, altered heart rate, depression, weight gain, and difficulty in conceiving. Also, maternal hypothyroidism, which affects 0.5% of all pregnant women, is the leading preventable cause of mental retardation in offspring. Importantly, diagnosis of hypothyroidism is based on a single thyroid-stimulating-hormone (TSH) measurement. TSH secretion by pituitary thyrotrophs is under the positive control of hypothalamic thyrotropin-releasing-hormone (TRH) and negative feedback exerted by THs. Despite the undeniable implication of thyrotrophs in the hypothalamus-pituitary-thyroid (HPT) axis regulation, little is known about the mechanisms underlying adaptive TSH secretion. Being the smallest endocrine cell population of the pituitary (2-4%), thyrotrophs display high plasticity in cell shape, number and network topology throughout development and in disease. Moreover, enzymatically-dispersed thyrotrophs lose their ability to express and secrete TSH in both basal and TRH-stimulated conditions. Altogether, these findings led us to the hypothesis that thyrotrophs form a very finely regulated and highly plastic functionally-organized cell population capable of adapting to physiological (i.e. pregnancy) and pathological (i.e. hypothyroidism) demand. The aim of this study was to investigate how thyrotrophs function in their native environment. Using gradient-index (GRIN) lenses implanted at the pituitary level and a 2g head-mounted miniscope, multicellular calcium activities of the thyrotroph population were monitored in freely-moving TSHβ-crexR26fl-flGCaMP6f mice. Strikingly, thyrotrophs displayed both short-lived calcium spikes of high amplitude, and slowly-evolving calcium waves propagating among the thyrotroph population (n=3). Such calcium waves recurred every 80min during episodes lasting 3min and were interspaced by short-lived activity/silent periods. As TSH displays a 2.722min half-life (n=6), repetitive bouts of calcium-dependent TSH exocytosis would lead to cumulative increase in circulating TSH levels. Hence, the thyrotroph population functions in vivo as a robust generator of repetitive calcium waves which would orchestrate the generation of ultradian TSH fluctuations. Such signaling events could be monitored during longitudinal studies (weeks to months) in which individual animals could be their own controls.

FISHing for β Cells

β cell heterogeneity has emerged as an important contributor to islet function, with potential implications for diabetes. Using an optimized smFISH technique in intact islets, Farack etal. (2018) identify in the islet core an endocrine cell population of "extreme" β cells with distinct molecular properties.

Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3' transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

Neurog3 misexpression unravels mouse pancreatic ductal cell plasticity.

In the context of type 1 diabetes research and the development of insulin-producing β-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene Neurog3 in ductal cells in vivo. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary β-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new β-like cells and that strategies aiming at controlling the expression of Neurog3, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.

The phytoestrogen genistein prevents trabecular bone loss and affects thyroid follicular cells in a male rat model of osteoporosis.

As a major phytoestrogen of soy, genistein effectively prevents bone loss in both humans and rat models of osteoporosis. However, although the bone-sparing effects of genistein are achieved directly through estrogen receptors, its mode of action on bone by modulation of other endocrine functions is not entirely clear. Thus, thyroid hormones and calcitonin (CT) have an essential influence on bone metabolism. Besides its action on bones, in this study we examined the effect of genistein on the activity of two different endocrine cell populations, thyroid follicular and C-cells. Fifteen-month-old Wistar rats were either bilaterally orchidectomized (Orx) or sham-operated (SO). Two weeks after surgery, half of the Orx rats were treated chronically with 30mgkg-1 b.w. genistein (Orx+G) subcutaneously (s.c.) every day for 3weeks, while the remaining Orx rats and the SO rats were given the same volume of sterile olive oil to serve as controls. For histomorphometrical analysis of the trabecular bone microarchitecture an ImageJ public domain image processing programme was used. Thyroid sections were analysed histologically and stereologically after visualization of follicular and C-cells by immunohistochemical staining for thyroglobulin and CT. Thyroid follicular epithelium, interstitium, colloid and CT-immunopositive C-cells were examined morphometrically. Serum concentrations of osteocalcin (OC), triiodothyronine (T3 ), thyroxine (T4 ) and CT were determined as well as urinary calcium (Ca2+ ) concentrations. Genistein treatment significantly increased cancellous bone area (B.Ar), trabecular thickness (TbTh) and trabecular number (TbN) (P<0.05), but trabecular separation (Tb.Sp) was decreased (P<0.05) compared with control Orx rats. In the thyroid, genistein treatment significantly elevated the relative volume density (Vv) of the follicular cells (P<0.05) compared with Orx, whereas Vv of the colloid was lower (P<0.05) than in the Orx. Evaluation of the biochemical parameters showed significant reductions in serum OC, T3 , T4 and urinary Ca2+ concentrations (P<0.05), compared with Orx rats. These data indicate that genistein treatment improves the trabecular microarchitecture of proximal tibia, induces histomorphometrical changes in thyroid glands, and decreases circulating thyroid hormone levels in orchidectomized rat model of male osteoporosis.

Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

Is Ghrelin Synthesized in the Central Nervous System?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas.

BackgroundReports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas.MethodsTranscriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student’s t-test and ANOVA.ResultsApproximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA.ConclusionsIn the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0146-x) contains supplementary material, which is available to authorized users.

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

Islet amyloid with macrophage migration correlates with augmented β-cell deficits in type 2 diabetic patients

Aims: Islet amyloid is a hallmark in type 2 diabetic subjects, but its implication in clinical features and development of islet pathology is still unclear.Methods: From 118 autopsy cases with type 2 diabetes, 26 cases with islet amyloid deposition (DA+) were selected. Twenty diabetic subjects without obvious amyloid deposition (DA−) matched for the age and diabetes duration and 20 non-diabetic subjects (ND) served for comparison. We examined the severity of amyloid deposition and its relationships with population of endocrine cells, expression of cell damage markers or macrophage infiltration. Correlation of clinical profile with islet pathology was also sought on the subset of the investigated patients.Results: β-Cell volume density was nearly 40% less in DA+ and 20% less in DA− when compared to ND. Severity of amyloid deposition correlated with reduced volume densities of β-cell and α-cell, and increased body mass index (BMI), but not with duration of diabetes, age or HbA1c. Amyloid-rich islets contained an increased number of macrophages mixed with β-cells with oxidative stress-related DNA damage, characterized by γH2AX expression, and suppressed (pro)insulin mRNA expression.Conclusions: In Japanese type 2 diabetic patients, islet amyloid was more common with severe β-cell loss and high BMI, associated with macrophage infiltration.

Morpho-functional characteristics of rat fetal thyroid gland are affected by prenatal dexamethasone exposure

Thyroid hormones (TH) and glucocorticoids strongly contribute to the maturation of fetal tissues in the preparation for extrauterine life. Influence of maternal dexamethasone (Dx) administration on thyroid glands morpho-functional characteristics of near term rat fetuses was investigated applying unbiased stereology. On the 16th day of pregnancy dams received 1.0mg/Dx/kg/b.w., followed by 0.5mg/Dx/kg/b.w. on the 17th and 18th days of gestation. The control females received the same volume of saline. The volume of fetal thyroid was estimated using Cavalieri’s principle; the physical/fractionator design was applied for the determination of absolute number of follicular cells in mitosis and immunohistochemically labeled C cells; C cell volume was measured using the planar rotator. The functional activity of thyroid tissue was provided from thyroglobulin (Tg) and thyroperoxidase (TPO) immunohistochemical staining. Applying these design-based modern stereological methods it was shown that Dx treatment of gravid females led to a significant decrease of fetal thyroid gland volume in 19- and 21-day-old fetuses, due to decreased proliferation of follicular cells. The Tg and TPO immunohistochemistry demonstrated that intensive TH production starts and continues during the examined period in control and Dx-exposed fetuses. Under the influence of Dx the absolute number of C cells was lower in both groups of near term fetuses, although unchanged relation between the two populations of endocrine cells, follicular and C cells suggesting that structural relationships within the gland are preserved. In conclusion maternal glucocorticoid administration at the thyroid gland level exerts growth-inhibitory and maturational promoting effects in near term rat fetuses.

Role of Calcium and EPAC in Norepinephrine-Induced Ghrelin Secretion

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to β1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.

Nkx2.2:Cre knock‐in mouse line: A novel tool for pancreas‐ and CNS‐specific gene deletion

Nkx2.2 is a homeodomain-containing transcriptional regulator necessary for the appropriate differentiation of ventral neuronal populations in the spinal cord and hindbrain, and endocrine cell populations in the pancreas and intestine. In each tissue, Nkx2.2 inactivation leads to reciprocal cell fate alterations. To confirm the cell fate changes are due to respecification of Nkx2.2-expressing progenitors and to provide a novel tool for lineage tracing in the pancreas and CNS, we generated an Nkx2.2:Cre mouse line by knocking in a Cre-EGFP cassette into the Nkx2.2 genomic locus and inactivating endogenous Nkx2.2. The R26R-CAG-LSL-tdTomato reporter was used to monitor the specificity and efficiency of Nkx2.2:Cre activity; the tomato reporter faithfully recapitulated endogenous Nkx2.2 expression and could be detected as early as embryonic day (e) 9.25 in the developing CNS and was initiated shortly thereafter at e9.5 in the pancreas. Lineage analyses in the CNS confirmed the cell populations thought to be derived from Nkx2.2-expressing progenitor domains. Furthermore, lineage studies verified Nkx2.2 expression in the earliest pancreatic progenitors that give rise to all cell types of the pancreas; however they also revealed more robust Cre activity in the dorsal versus ventral pancreas. Thus, the Nkx2.2:Cre line provides a novel tool for gene manipulations in the CNS and pancreas.

α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistent depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of β-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of β-cells.

Regulated secretory cells go to the BAR for a bud

Syndromes caused by insufficient secretion of peptide hormones originate either from an inadequate population of endocrine secretory cells or from deficiencies in the secretory process. Two remarkable new studies highlight the importance of PICK1 and ICA69 in creating the protein storage organelles needed for regulated exocytosis, independent of stimulus-secretion coupling.

Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss

The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, Aristaless-related homeobox gene (Arx) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of Arx in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of Arx, we have generated mice in which the Arx gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of Arx in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express β-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.

Smad signaling pathways regulate pancreatic endocrine development

Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-β) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-β signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for β-cell development.