Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

An active tethering mechanism controls the fate of vesicles.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress.

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

Maturing secretory granules: Where secretory and endocytic pathways converge.

Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.

Membrane traffic related to endosome dynamics and protein secretion in filamentous fungi.

In eukaryotic cells, membrane-surrounded organelles are orchestrally organized spatiotemporally under environmental situations. Among such organelles, vesicular transports and membrane contacts occur to communicate each other, so-called membrane traffic. Filamentous fungal cells are highly polarized and thus membrane traffic is developed to have versatile functions. Early endosome (EE) is an endocytic organelle that dynamically exhibits constant long-range motility through the hyphal cell, which is proven to have physiological roles, such as other organelle distribution and signal transduction. Since filamentous fungal cells are also considered as cell factories, to produce valuable proteins extracellularly, molecular mechanisms of secretory pathway including protein glycosylation have been well investigated. In this review, molecular and physiological aspects of membrane traffic especially related to EE dynamics and protein secretion in filamentous fungi are summarized, and perspectives for application are also described.

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to monitor compensatory endocytosis in chromaffin cells and present an image-based analysis to quantify endocytic vesicles distribution.

Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox.

Up-to-date imaging approaches were used to address the spatiotemporal organisation of the endomembrane system in secretory cells of Dionaea muscipula. Different 'slice and view' methodologies were performed on resin-embedded samples to finally achieve a 3D reconstruction of the cell architecture, using ultrastructural tomography, array tomography, serial block face-scanning electron microscopy (SBF-SEM), correlation, and volume rendering at the light microscopy level. Observations of cryo-fixed samples by high-pressure freezing revealed changes of the endomembrane system that occur after trap activation and prey digestion. They provide evidence for an original strategy that adapts the secretory machinery to a specific and unique case of stimulated exocytosis in plant cells. A first secretion peak is part of a rapid response to deliver digestive fluids to the cell surface, which delivers the needed stock of digestive materials 'on site'. The second peak of activity could then be associated with the reconstruction of the Golgi apparatus (GA), endoplasmic reticulum (ER) and vacuolar machinery, in order to prepare for a subsequent round of prey capture. Tubular continuum between ER and Golgi stacks observed on ZIO-impregnated tissues may correspond to an efficient transfer mechanism for lipids and/or proteins, especially for use in rapidly resetting the molecular GA machinery. The occurrence of one vacuolar continuum may permit continuous adjustment of cell homeostasy. The subcellular features of the secretory cells of Dionaea muscipula outline key innovations in the organisation of plant cell compartmentalisation that are used to cope with specific cell needs such as the full use of the GA as a protein factory, and the ability to create protein reservoirs in the periplasmic space. Shape-derived forces of the pleiomorphic vacuole may act as signals to accompany the sorting and entering flows of the cell.

Subcellular structure and behaviour in fungal hyphae.

This work briefly surveys the diversity of selected subcellular characteristics in hyphal tip cells of the fungal kingdom (Mycota). Hyphae are filamentous cells that grow by tip extension. It is a highly polarised mechanism that requires a robust secretory system for the delivery of materials (e.g. membrane, proteins, cell wall materials) to sites of cell growth. These events result it the self-assembly of a Spitzenkörper (Spk), found most often in the Basidiomycota, Ascomycota, and Blastocladiomycota, or an apical vesicle crescent (AVC), present in the most Mucoromycota and Zoopagomycota. The Spk is a complex apical body composed of secretory vesicles, cytoskeletal elements, and signaling proteins. The AVC appears less complex, though little is known of its composition other than secretory vesicles. Both bodies influence hyphal growth and morphogenesis. Other factors such as cytoskeletal functions, endocytosis, cytoplasmic flow, and turgor pressure are also important in sustaining hyphal growth. Clarifying subcellular structures, functions, and behaviours through bioimagining analysis are providing a better understanding of the cell biology and phylogenetic relationships of fungi. LAY DESCRIPTION: Fungi are most familiar to the public as yeast, molds, and mushrooms. They are eukaryotic organisms that inhabit diverse ecological niches around the world and are critical to the health of ecosystems performing roles in decomposition of organic matter and nutrient recycling (Heath, 1990). Fungi are heterotrophs, unlike plants, and comprise the most successful and diverse phyla of eukaryotic microbes, interacting with all other forms of life in associations that range from beneficial (e.g., mycorrhizae) to antagonistic (e.g., pathogens). Some fungi can be parasitic or pathogenic on plants (e.g., Cryphonectria parasitica, Magnaporthe grisea), insects (e.g., Beauveria bassiana, Cordyceps sp.), invertebrates (e.g., Drechslerella anchonia), vertebrates (e.g., Coccidioides immitis, Candia albicans) and other fungi (e.g., Trichoderma viride, Ampelomyces quisqualis). The majority of fungi, however, are saprophytes, obtaining nutrition through the brake down of non-living organic matter.

Tetanus insensitive VAMP2 differentially restores synaptic and dense core vesicle fusion in tetanus neurotoxin treated neurons.

The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. Here we use tetanus neurotoxin (TeNT) light chain, which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. Expression of tetanus insensitive (TI)-VAMP2 restored SV fusion in the presence of TeNT, but not DCV fusion. Expression of TI-VAMP1 or TI-VAMP3 also failed to restore DCV fusion. Co-transport assays revealed that both TI-VAMP1 and TI-VAMP2 are targeted to DCVs and travel together with DCVs in neurons. Furthermore, expression of the TeNT-cleaved VAMP2 fragment or a protease defective TeNT in wild type neurons did not affect DCV fusion and therefore cannot explain the lack of rescue of DCV fusion by TI-VAMP2. Finally, to test if two different VAMPs might both be required in the DCV secretory pathway, Vamp1 null mutants were tested. However, VAMP1 deficiency did not reduce DCV fusion. In conclusion, TeNT treatment combined with TI-VAMP2 expression differentially affects the two main regulated secretory pathways: while SV fusion is normal, DCV fusion is absent.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Plant Golgi ultrastructure.

The plant Golgi apparatus (sensu lato: Golgi stack + Trans Golgi Network, TGN) is a highly polar and mobile key organelle lying at the junction of the secretory and endocytic pathways. Unlike its counterpart in animal cells it does not disassemble during mitosis. It modifies glycoproteins sent to it from the endoplasmic reticulum (ER), it recycles ER resident proteins, it sorts proteins destined for the vacuole from secretory proteins, it receives proteins internalised from the plasma membrane and either recycles them to the plasma membrane or retargets them to the vacuole for degradation. In functional terms the Golgi apparatus can be likened to a car factory, with incoming (COPII traffic) and returning (COPI traffic) railway lines at the entry gate, and a distribution centre (the TGN) at the exit gate of the assembly hall. In the assembly hall we have a conveyor belt system where the incoming car parts are initially assembled (in the cis-area) then gradually modified into different models (processing of secretory cargo) as the cars pass along the production line (cisternal maturation). After being released the trans-area, the cars (secretory cargos) are moved out of the assembly hall and passed on to the distribution centre (TGN), where the various models are placed onto different trains (cargo sorting into carrier vesicles) for transport to the car dealers. Cars with motor problems are returned to the factory for repairs (endocytosis to the TGN). This simple analogy also incorporates features of quality control at the COPII entry gate with defective parts being returned to the manufacturing center (the ER) via the COPI trains (vesicles). In recent years, numerous studies have contributed to our knowledge on Golgi function and structure in both animals, yeast and plants. This review, rather than giving a balanced account of the structure as well as of the function of the Golgi apparatus has purposely a marked slant towards plant Golgi ultrastructure integrating findings from the mammalian/animal field.

Ultrastructural Correlates of Presynaptic Functional Heterogeneity in Hippocampal Synapses.

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases.

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

Immunogold labeling of synaptic vesicle proteins in developing hippocampal neurons.

Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 µm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.

Linkage between growth retardation and pituitary cell morphology in a dystrophin-deficient pig model of Duchenne muscular dystrophy.

OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.

Class V chitin synthase and ß(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.

The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and ß(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the ß(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkörper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.