Tert-expressing cells contribute to salivary gland homeostasis and tissue regeneration after radiation therapy.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

Depletion of lipid storage droplet-1 delays endoreplication progression and induces cell death in Drosophila salivary gland.

Perilipins are evolutionarily conserved from insects to mammals. Drosophila lipid storage droplet-1 (LSD-1) is a lipid storage droplet membrane surface-binding protein family member and a counterpart to mammalian perilipin 1 and is known to play a role in lipolysis. However, the function of LSD-1 during specific tissue development remains under investigation. This study demonstrated the role of LSD-1 in salivary gland development. Knockdown of Lsd-1 in the salivary gland was established using the GAL4/UAS system. The third-instar larvae of knockdown flies had small salivary glands containing cells with smaller nuclei. The null mutant Drosophila also showed the same phenotype. The depletion of LSD-1 expression induced a delay of endoreplication due to decreasing CycE expression and increasing DNA damage. Lsd-1 genetically interacted with Myc in the third-instar larvae. These results demonstrate that LSD-1 is involved in cell cycle and cell death programs in the salivary gland, providing novel insight into the effects of LSD-1 in regulating salivary gland development and the interaction between LSD-1 and Myc.

ß-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, ß-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of ß-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither ß-H-Spectrin nor Big bang require microtubules for their localisation. ß-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of ß-H-Spectrin (ß-H-33) displaces endogenous ß-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.

Salivary gland developmental mechanics.

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

Salivary Gland Tissue Recombination Can Modify Cell Fate.

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

Developmental events in the mandibular salivary gland of Indian sheep (Ovis aries): Morphological and histochemical studies.

At 22nd day of fetal development, the primordial anlage of mandibular gland was first noticed as a solid epithelial bud from oral epithelium. The terminal buds were arranged in the form of clusters with undifferentiated epithelial cells and terminated in a bulb like structure in 28-day-old fetus. The lumenization and branching of the main cord was noticed at 35th day. The primary septa, which divide the glandular mass into lobes was observed from 53rd day onwards which resulted in the formation of distinct lobulation at 58th day. At 61st day, the capsule formation was initiated by the aggregation of mesenchymal tissue. The terminal tubules differentiated to form the secretory end pieces and the tubular portion leads to the formation of intercalated and striated ducts at 98th day. Predominantly mucous types of acinar cells were seen from 108th day onwards. The number of lobules increased with steep increase in parenchyma from 128th day onwards. Micrometrical studies revealed that the mean diameter of acinar cells and all ducts was increased with the advancement of age and the significant differences were observed between groups. Localization of acidic and neutral mucopolysaccharides was observed in mucous cells and goblet cells. RESEARCH HIGHLIGHTS: The primordial anlage of mandibular salivary gland was seen at 22nd day. Lobulation of gland was appeared at 53rd day of development, however; it was completed at 58th day. At 98th day, the terminal tubules differentiated to form the secretory end pieces. The parenchyma of the gland showed predominantly mucous type of cells from 108th day onwards. Myoepithelial cells were first appeared as flattened basal cells initially around the developing acinar cells at 132nd day. Localization of acidic as well as neutral mucopolysaccharides was observed in mucous cells and goblet cells. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue, however; phospholipids were observed in the cell membrane of secretory cells and ducts.

Bioengineered Salivary Gland Microtissues─A Review of 3D Cellular Models and their Applications.

Salivary glands (SGs) play a vital role in maintaining oral health through the production and release of saliva. Injury to SGs can lead to gland hypofunction and a decrease in saliva secretion manifesting as xerostomia. While symptomatic treatments for xerostomia exist, effective permanent solutions are still lacking, emphasizing the need for innovative approaches. Significant progress has been made in the field of three-dimensional (3D) SG bioengineering for applications in gland regeneration. This has been achieved through a major focus on cell culture techniques, including soluble cues and biomaterial components of the 3D niche. Cells derived from both adult and embryonic SGs have highlighted key in vitro characteristics of SG 3D models. While still in its first decade of exploration, SG spheroids and organoids have so far served as crucial tools to study SG pathophysiology. This review, based on a literature search over the past decade, covers the importance of SG cell types in the realm of their isolation, sourcing, and culture conditions that modulate the 3D microenvironment. We discuss different biomaterials employed for SG culture and the current advances made in bioengineering SG models using them. The success of these 3D cellular models are further evaluated in the context of their applications in organ transplantation and in vitro disease modeling.

Exploring the Cocktail Factor Approach to Generate Salivary Gland Progenitors through Co-Culture Techniques.

BACKGROUND: The derivation of salivary gland (SG) progenitors from pluripotent stem cells (PSCs) presents significant potential for developmental biology and regenerative medicine. However, the existing protocols for inducing SG include limited factors, making it challenging to mimic the in vivo microenvironment of embryonic SGs. METHODS: We reported a cocktail factor approach to promote the differentiation of mouse embryonic stem cell (mESC)-derived oral epithelium (OE) into SG progenitors through a three-dimensional co-culture method. Upon confirming that the embryonic SG can promote the differentiation of mESC-derived OE, we performed RNA sequence analysis to identify factors involved in the differentiation of SG progenitors. RESULTS: Our findings highlight several efficient pathways related to SG development, with frequent appearances of four factors: IFN-γ, TGF-ß2, EGF, and IGF-1. The combined treatment using these cocktail factors increased the expression of key SG progenitor markers, including Sox9, Sox10, Krt5, and Krt14. However, absence of any one of these cocktail factors did not facilitate differentiation. Notably, aggregates treated with the cocktail factor formed SG epithelial-like structures and pre-bud-like structures on the surface. CONCLUSION: In conclusion, this study offers a novel approach to developing a differentiation protocol that closely mimics the in vivo microenvironment of embryonic SGs. This provides a foundation for generating PSC-derived organoids with near-physiological cell behaviors and structures.

Specific localization of fibroblasts at the intercalated duct in the major salivary glands of rats.

OBJECTIVES: Immunohistochemical methods were employed to investigate the morphological heterogeneity and localization of fibroblasts associated with the function of major salivary glands in rats. METHODS: Histochemical and electron microscopic observations were made in rat parotid, submandibular, and sublingual glands and pancreas. Fibroblasts were immunostained using their specific marker, 47 kDa heat shock protein (Hsp47). RESULTS: Hsp47-immunopositive fibroblasts within the intralobular connective tissue exhibited a notably smaller size compared with the interlobular connective tissue. They were loosely distributed throughout the connective tissue. However, fibroblasts with elongated long processes were explicitly identified at the intercalated ducts in parotid, sublingual, and submandibular glands. Fibroblastic bodies and processes were tightly approximated with the basement membrane of the duct. Electron microscopy confirmed these findings, revealing a thin layer consisting of collagen fibers was found between the fibroblasts and the basement membrane. Double staining of Hsp47 and α-smooth muscle actin (αSMA) in parotid glands indicating that Hsp47-positive fibroblasts enveloped both the duct and αSMA-positive myoepithelial cells. Additionally, They projected long and thin processes longitudinally at the straight portion or circularly at the bifurcated portion of the duct. The three-dimensional reconstruction showed a frame-like structure of fibroblasts surrounding the intercalated duct with longitudinal myoepithelial cells. However, such specific localization of fibroblasts was not detected in the exocrine pancreas lacking myoepithelium. CONCLUSIONS: Small fibroblasts with long processes connecting or overwrapping each other and thin collagen layers surround the intercalated ducts in rat major salivary glands, presumably contributing to protecting the ducts from salivary flow and myoepithelial contraction.

The Usefulness of Salivary Gland Organoids for Evaluation of the Potency of Botulinum Neurotoxin.

BACKGROUND AND OBJECTIVES: Botulinum neurotoxin (BoNT) is a substance used to treat chronic sialorrhea, muscle dystonia, and is used in cosmetic applications. Measuring the potency of BoNT is crucial because it acts even with a small amount. However, the current methods for measuring the potency of BoNT involve using two-dimensional neuroblastoma cell line-based methods. In this study, we aimed to develop a new method to measure the potency of BoNT using a three-dimensional organoid culture system. MATERIALS AND METHOD: We established the optimal conditions for coculturing N2a neuronal cells with murine salivary gland organoids (SGOs). After determining the appropriate chemical concentrations, we treated the SGOs cocultured with N2a cells with BoNT type A (BoNT/A). We confirmed the expression of salivary gland-related genes and proteins using real-time polymerase chain reaction (PCR) and immunofluorescence staining. RESULTS: The SGOs cocultured with N2a cells showed that the dendrites or axons of neuronal cells were in contact with the outermost layer of the SGOs. When we applied acetylcholine and neostigmine to the coculture systems, the mRNA expression of Aqp5 and Bhlha15, associated with salivary gland secretory cells, increased. However, this effect was reversed when BoNT/A was applied, as confirmed through real-time PCR. CONCLUSION: We found that the coculture system of SGOs and N2a neuronal cells can potentially serve as a potency testing platform for BoNT. LEVEL OF EVIDENCE: NA Laryngoscope, 134:2697-2704, 2024.

Matrix Degradability Contributes to the Development of Salivary Gland Progenitor Cells with Secretory Functions.

Synthetic matrices that are cytocompatible, cell adhesive, and cell responsive are needed for the engineering of implantable, secretory salivary gland constructs to treat radiation induced xerostomia or dry mouth. Here, taking advantage of the bioorthogonality of the Michael-type addition reaction, hydrogels with comparable stiffness but varying degrees of degradability (100% degradable, 100DEG; 50% degradable, 50DEG; and nondegradable, 0DEG) by cell-secreted matrix metalloproteases (MMPs) were synthesized using thiolated HA (HA-SH), maleimide (MI)-conjugated integrin-binding peptide (RGD-MI), and MI-functionalized peptide cross-linkers that are protease degradable (GIW-bisMI) or nondegradable (GIQ-bisMI). Organized multicellular structures developed readily in all hydrogels from dispersed primary human salivary gland stem cells (hS/PCs). As the matrix became progressively degradable, cells proliferated more readily, and the multicellular structures became larger, less spherical, and more lobular. Immunocytochemical analysis showed positive staining for stem/progenitor cell markers CD44 and keratin 5 (K5) in all three types of cultures and positive staining for the acinar marker α-amylase under 50DEG and 100DEG conditions. Quantitatively at the mRNA level, the expression levels of key stem/progenitor markers KIT, KRT5, and ETV4/5 were significantly increased in the degradable gels as compared to the nondegradable counterparts. Western blot analyses revealed that imparting matrix degradation led to >3.8-fold increase in KIT expression by day 15. The MMP-degradable hydrogels also promoted the development of a secretary phenotype, as evidenced by the upregulation of acinar markers α-amylase (AMY), aquaporin-5 (AQP5), and sodium-potassium chloride cotransporter 1 (SLC12A2). Collectively, we show that cell-mediated matrix remodeling is necessary for the development of regenerative pro-acinar progenitor cells from hS/PCs.

Dental pulp stem cell-derived exosomes revitalize salivary gland epithelial cell function in NOD mice via the GPER-mediated cAMP/PKA/CREB signaling pathway.

BACKGROUND: Restoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet. METHODS: DPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis. RESULTS: DPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER. CONCLUSIONS: Our results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.

A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands.

Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse, we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos population express the hallmarks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2bpos ductal and myoepithelial cells.

Does HTLV-1 Infection Show Phenotypes Found in Sjögren's Syndrome?

Viruses are a possible cause for Sjögren's syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

Correct regionalization of a tissue primordium is essential for coordinated morphogenesis.

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviors of cell wedging, tilting, and cell intercalation that are key to the initial stages of tube formation. Here, we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signaling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behavior from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviors that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.

ß-Adrenergic signaling induces Notch-mediated salivary gland progenitor cell control.

ß-Adrenergic signaling blockade is a mainstay of hypertension management. One percent of patients taking ß-blockers develop reduced salivary gland (SG) function. Here we investigate the role of SG progenitor cells in ß-blocker-induced hyposalivation, using human SG organoid cultures (SGOs). Compared with control SGs, initial low SG progenitor cell yield from patients taking ß-blockers was observed. When passaged, these SGOs recovered self-renewal and upregulated Notch pathway expression. Notch signaling was downregulated in situ in ß-adrenergic receptor-expressing luminal intercalated duct (ID) cells of patients taking ß-blockers. Control SGOs treated with ß-adrenergic agonist isoproterenol demonstrated increased proportion of luminal ID SGO cells with active Notch signaling. Control SGOs exposed to isoproterenol differentiated into more mature SGOs (mSGOs) expressing markers of acinar cells. We propose that ß-blocker-induced Notch signaling reduction in luminal ID cells hampers their ability to proliferate and differentiate into acinar cells, inducing a persistent hyposalivation in some patients taking ß-blocking medication.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.