Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion.

Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.

The evolving landscape of salivary gland tumors.

Salivary gland cancers are a rare, histologically diverse group of tumors. They range from indolent to aggressive and can cause significant morbidity and mortality. Surgical resection remains the mainstay of treatment, but radiation and systemic therapy are also critical parts of the care paradigm. Given the rarity and heterogeneity of these cancers, they are best managed in a multidisciplinary program. In this review, the authors highlight standards of care as well as exciting new research for salivary gland cancers that will strive for better patient outcomes.

Salivary gland function, development, and regeneration.

Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

Organogenetic transcriptomes of the Drosophila embryo at single cell resolution.

To gain insight into the transcription programs activated during the formation of Drosophila larval structures, we carried out single cell RNA sequencing during two periods of Drosophila embryogenesis: stages 10-12, when most organs are first specified and initiate morphological and physiological specialization; and stages 13-16, when organs achieve their final mature architectures and begin to function. Our data confirm previous findings with regards to functional specialization of some organs - the salivary gland and trachea - and clarify the embryonic functions of another - the plasmatocytes. We also identify two early developmental trajectories in germ cells and uncover a potential role for proteolysis during germline stem cell specialization. We identify the likely cell type of origin for key components of the Drosophila matrisome and several commonly used Drosophila embryonic cell culture lines. Finally, we compare our findings with other recent related studies and with other modalities for identifying tissue-specific gene expression patterns. These data provide a useful community resource for identifying many new players in tissue-specific morphogenesis and functional specialization of developing organs.

Geospatial joint modeling of vector and parasite serology to microstratify malaria transmission.

The World Health Organization identifies a strong surveillance system for malaria and its mosquito vector as an essential pillar of the malaria elimination agenda. Anopheles salivary antibodies are emerging biomarkers of exposure to mosquito bites that potentially overcome sensitivity and logistical constraints of traditional entomological surveys. Using samples collected by a village health volunteer network in 104 villages in Southeast Myanmar during routine surveillance, the present study employs a Bayesian geostatistical modeling framework, incorporating climatic and environmental variables together with Anopheles salivary antigen serology, to generate spatially continuous predictive maps of Anopheles biting exposure. Our maps quantify fine-scale spatial and temporal heterogeneity in Anopheles salivary antibody seroprevalence (ranging from 9 to 99%) that serves as a proxy of exposure to Anopheles bites and advances current static maps of only Anopheles occurrence. We also developed an innovative framework to perform surveillance of malaria transmission. By incorporating antibodies against the vector and the transmissible form of malaria (sporozoite) in a joint Bayesian geostatistical model, we predict several foci of ongoing transmission. In our study, we demonstrate that antibodies specific for Anopheles salivary and sporozoite antigens are a logistically feasible metric with which to quantify and characterize heterogeneity in exposure to vector bites and malaria transmission. These approaches could readily be scaled up into existing village health volunteer surveillance networks to identify foci of residual malaria transmission, which could be targeted with supplementary interventions to accelerate progress toward elimination.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

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.

FGF signaling regulates salivary gland branching morphogenesis by modulating cell adhesion.

Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and found that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1 and Fgfr2 (Fgfr1/2) knock-in alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF-dependent mechanisms play a role in salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cell-basement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild-type or signaling alleles that are incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through cell-adhesion processes.

Malaria: influence of Anopheles mosquito saliva on Plasmodium infection.

Malaria is caused by Plasmodium protozoa that are transmitted by anopheline mosquitoes. Plasmodium sporozoites are released with saliva when an infected female mosquito takes a blood meal on a vertebrate host. Sporozoites deposited into the skin must enter a blood vessel to start their journey towards the liver. After migration out of the mosquito, sporozoites are associated with, or in proximity to, many components of vector saliva in the skin. Recent work has elucidated how Anopheles saliva, and components of saliva, can influence host-pathogen interactions during the early stage of Plasmodium infection in the skin. Here, we discuss how components of Anopheles saliva can modulate local host responses and affect Plasmodium infectivity. We hypothesize that therapeutic strategies targeting mosquito salivary proteins can play a role in controlling malaria and other vector-borne diseases.

Nkx2.3 transcription factor is a key regulator of mucous cell identity in salivary glands.

Saliva is vital to oral health, fulfilling multiple functions in the oral cavity. Three pairs of major salivary glands and hundreds of minor salivary glands contribute to saliva production. The secretory acinar cells within these glands include two distinct populations. Serous acinar cells secrete a watery saliva containing enzymes, while mucous acinar cells secrete a more viscous fluid containing highly glycosylated mucins. Despite their shared developmental origins, the parotid gland (PG) is comprised of only serous acinar cells, while the sublingual gland (SLG) contains predominantly mucous acinar cells. The instructive signals that govern the identity of serous versus mucous acinar cell phenotypes are not yet known. The homeobox transcription factor Nkx2.3 is uniquely expressed in the SLG. Disruption of the Nkx2.3 gene was reported to delay the maturation of SLG mucous acinar cells. To examine whether Nkx2.3 plays a role in directing the mucous cell phenotype, we analyzed SLG from Nkx2.3-/- mice using RNAseq, immunostaining and proteomic analysis of saliva. Our results indicate that Nkx2.3, most likely in concert with other transcription factors uniquely expressed in the SLG, is a key regulator of the molecular program that specifies the identity of mucous acinar cells.

Swip-1 promotes exocytosis of glue granules in the exocrine Drosophila salivary gland.

Exocytosis is a fundamental cellular process by which cells secrete cargos from their apical membrane into the extracellular lumen. Cargo release proceeds in sequential steps that depend on coordinated assembly and organization of an actin cytoskeletal network. Here, we identified the conserved actin-crosslinking protein Swip-1 as a novel regulator controlling exocytosis of glue granules in the Drosophila salivary gland. Real-time imaging revealed that Swip-1 is simultaneously recruited with F-actin onto secreting granules in proximity to the apical membrane. We observed that Swip-1 is rapidly cleared at the point of secretory vesicle fusion and colocalizes with actomyosin network around the fused vesicles. Loss of Swip-1 function impairs secretory cargo expulsion, resulting in strongly delayed secretion. Thus, our results uncover a novel role of Swip-1 in secretory vesicle compression and expulsion of cargo during regulated exocytosis. Remarkably, this function neither requires Ca2+ binding nor dimerization of Swip-1. Our data rather suggest that Swip-1 regulates actomyosin activity upstream of Rho-GTPase signaling to drive proper vesicle membrane crumpling and expulsion of cargo.

Multifunctional role of GPCR signaling in epithelial tube formation.

Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G-protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the medioapical region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data support a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

Single-cell RNA sequencing reveals PDGFRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFß1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFß1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Exocrine gland structure-function relationships.

Fluid secretion by exocrine glandular organs is essential to the survival of mammals. Each glandular unit within the body is uniquely organized to carry out its own specific functions, with failure to establish these specialized structures resulting in impaired organ function. Here, we review glandular organs in terms of shared and divergent architecture. We first describe the structural organization of the diverse glandular secretory units (the end-pieces) and their fluid transporting systems (the ducts) within the mammalian system, focusing on how tissue architecture corresponds to functional output. We then highlight how defects in development of end-piece and ductal architecture impacts secretory function. Finally, we discuss how knowledge of exocrine gland structure-function relationships can be applied to the development of new diagnostics, regenerative approaches and tissue regeneration.

Transforming Growth Factor-ß Signaling Guides the Differentiation of Innate Lymphoid Cells in Salivary Glands.

The signals guiding differentiation of innate lymphoid cells (ILCs) within tissues are not well understood. Salivary gland (SG) ILCs as well as liver and intestinal intraepithelial ILC1 have markers that denote tissue residency and transforming growth factor-ß (TGF-ß) imprinting. We deleted Tgfbr2 in cells expressing the ILC and NK marker NKp46 and found that SG ILCs were reduced in number. They lost distinct tissue markers, such as CD49a, and the effector molecules TRAIL and CD73. Expression of the transcription factor Eomes, which promotes NK cell differentiation, was elevated. Conversely, Eomes deletion in NKp46(+) cells enhanced TGF-ß-imprinting of SG ILCs. Thus, TGF-ß induces SG ILC differentiation by suppressing Eomes. TGF-ß acted through a JNK-dependent, Smad4-independent pathway. Transcriptome analysis demonstrated that SG ILCs had characteristic of both NK cells and ILC1. Finally, TGF-ß imprinting of SG ILCs was synchronized with SG development, highlighting the impact of tissue microenvironment on ILC development.

TGF-ß3 from fibroblasts promotes necrotising sialometaplasia by suppressing salivary gland cell proliferation and inducing squamous metaplasia.

Necrotising sialometaplasia (NSM) is a non-neoplastic lesion mainly arising in the minor salivary glands of the oral cavity. In the clinical features, NSM shows swelling with or without ulceration, and can mimic a malignant disease such as squamous cell carcinoma. Histopathologically, NSM usually shows the lobular architecture that is observed in the salivary glands. Additionally, acinar infarction and squamous metaplasia of salivary ducts and acini are observable. The aetiology of this lesion remains unknown, although it has a characteristic feature that sometimes requires clinical and histopathological differentiation from malignancy. In this study, we investigated upregulated genes in NSM compared with normal salivary glands, and focused on the TGF-ß3 (TGFB3) gene. The results of the histopathological studies clarified that fibroblasts surrounding the lesion express TGF-ß3. Moreover, in vitro studies using mouse salivary gland organoids revealed that TGF-ß3 suppressed salivary gland cell proliferation and induced squamous metaplasia. We demonstrated a possible aetiology of NSM by concluding that increased TGF-ß3 expression during wound healing or tissue regeneration played a critical role in cell proliferation and metaplasia. © 2024 The Pathological Society of Great Britain and Ireland.

Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB::NFIB or MYBL1::NFIB translocations. We have developed the conditions for the generation of both 2D and 3D tumour organoid patient-derived ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies uncover a novel mechanistic effect of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cellular and molecular diversity in Sjogren's syndrome salivary glands: Towards a better definition of disease subsets.

Primary Sjögren's syndrome (pSS) is a highly heterogeneous disease in terms of clinical presentation ranging from a mild disease localised to the salivary and lacrimal glands, to multiorgan complications of various degrees of severity, finishing with the evolution, in around 5% of pSS patients, to B cell lymphomas most commonly arising in the inflamed salivary glands. Currently, there are poor positive or negative predictors of disease evolution able to guide patient management and treatment at early stages of the diseases. Recent understanding of the pathogenic mechanisms driving immunopathology in pSS, particularly through histological and transcriptomic analysis of minor and parotid salivary gland (SG) biopsies, has highlighted a high degree of cellular and molecular heterogeneity of the inflammatory lesions but also allowed the identification of clusters of patients with similar underlying SG immunopathology. In particular, patients presenting with high degrees of B/T cell infiltration and the formation of ectopic lymphoid structures (ELS) in the SG have been associated, albeit with conflicting results, with higher degree of disease severity and enhanced risk of lymphoma evolution, suggesting that a dysregulated adaptive immune response plays a key role in driving disease manifestations in pSS. Recent data from randomised clinical trials with novel biological therapies in pSS have also highlighted the potential role of SG immunopathology and molecular pathology in stratifying patients for trial inclusion as well as assessing proof of mechanisms in longitudinal SG biopsies before and after treatment. Although significant progress has been made in the understanding of disease pathogenesis and heterogeneity through cellular and molecular SG pathology, further work is needed to validate their clinical utility in routine clinical settings and in randomised clinical trials.

An inhibitor of leukotriene-A4 hydrolase from bat salivary glands facilitates virus infection.

SignificanceAn immunosuppressant protein (MTX), which facilitates virus infection by inhibiting leukotriene A4 hydrolase (LTA4H) to produce the lipid chemoattractant leukotriene B4 (LTB4), was identified and characterized from the submandibular salivary glands of the bat Myotis pilosus. To the best of our knowledge, this is a report of an endogenous LTA4H inhibitor in animals. MTX was highly concentrated in the bat salivary glands, suggesting a mechanism for the generation of immunological privilege and immune tolerance and providing evidence of viral shedding through oral secretions. Moreover, given that the immunosuppressant MTX selectively inhibited the proinflammatory activity of LTA4H, without affecting its antiinflammatory activity, MTX might be a potential candidate for the development of antiinflammatory drugs by targeting the LTA4-LTA4H-LTB4 inflammatory axis.