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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.
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Regeneración , Glándulas Salivales , Linaje de la Célula , Humanos , Salud Bucal , Regeneración/fisiología , Glándulas Salivales/fisiología , Transducción de SeñalRESUMEN
Functional salivary glands (SG) are essential for maintaining oral health, and salivary dysfunction is a persistent major clinical challenge. Several cancer therapies also have off-target effects leading to SG dysfunction. Recent advances highlight the role of SG immune populations in homeostasis, dysfunction and gland regeneration. Here, we review what is known about SG immune populations during development and postnatal homeostasis. We summarize recent findings of immune cell involvement in SG dysfunction following cancer treatments such as irradiation (IR) for head and neck cancers, immune transplant leading to graft-versus-host-disease (GVHD) and immune checkpoint inhibitor (ICI) treatment. The role of immune cells in SG in both homeostasis and disease, is an emerging field of research that may provide important clues to organ dysfunction and lead to novel therapeutic targets.
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Epithelial-mesenchymal interactions are required to coordinate cell proliferation, patterning, and functional differentiation of multiple cell types in a developing organ. This exquisite coordination is dependent on various secreted molecules that provide developmental signals to mediate these tissue interactions. Recently, it was reported that mature mesenchymal-derived microRNAs (miRNAs) in the fetal mouse salivary gland are loaded into exosomes, and transported to the epithelium where they influence progenitor cell proliferation. The exosomal miRNAs regulated epithelial expression of genes involved in DNA methylation in progenitor cells to influence morphogenesis. Thus, exosomal miRNAs are mobile genetic signals that cross tissue boundaries within an organ. These findings raise many questions about how miRNA signals are initiated to coordinate organogenesis and whether they are master regulators of epithelial-mesenchymal interactions. The development of therapeutic applications using exosomal miRNAs for the regeneration of damaged adult organs is a promising area of research.
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Exosomas/metabolismo , MicroARNs/genética , Organogénesis , Transducción de Señal , Animales , Transporte Biológico , Células Epiteliales/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Humanos , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , MicroARNs/uso terapéutico , Técnicas de Diagnóstico Molecular , Glándulas Salivales/embriología , Glándulas Salivales/metabolismoRESUMEN
The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review will focus on research on mouse submandibular gland development and the translation of this basic research toward therapy for patients suffering from salivary hypofunction. Here we review the most recent literature that has enabled a better understanding of the mechanisms of salivary gland development. Additionally, we discuss approaches proposed to restore salivary function using gene and cell-based therapy. Increasing our understanding of the developmental mechanisms involved during development is critical to design effective therapies for regeneration and repair of damaged glands.
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Glándulas Salivales/crecimiento & desarrollo , Animales , Procesos de Crecimiento Celular/fisiología , Humanos , Ratones , Morfogénesis , Regeneración/fisiología , Glándulas Salivales/citología , Glándulas Salivales/embriología , Células Madre/citologíaRESUMEN
The multiscale attributes of mammalian muscle confer significant challenges for structural imaging in vivo. To achieve this, we employed a magnetic resonance method, termed "generalized Q-space imaging", that considers the effect of spatially distributed diffusion-weighted magnetic field gradients and diffusion sensitivities on the morphology of Q-space. This approach results in a subvoxel scaled probability distribution function whose shape correlates with local fiber orientation. The principal fiber populations identified within these probability distribution functions can then be associated by streamline methods to create multivoxel tractlike constructs that depict the macroscale orientation of myofiber arrays. We performed a simulation of Q-space input parameters, including magnetic field gradient strength and direction, diffusion sensitivity, and diffusional sampling to determine the optimal achievable fiber angle separation in the minimum scan time. We applied this approach to resolve intravoxel crossing myofiber arrays in the setting of the human tongue, an organ with anatomic complexity based on the presence of hierarchical arrays of intersecting myocytes. Using parameters defined by simulation, we imaged at 3T the fanlike configuration of the human genioglossus and the laterally positioned merging fibers of the styloglossus, inferior longitudinalis, chondroglossus, and verticalis. Comparative scans of the excised mouse tongue at 7T demonstrated similar midline and lateral crossing fiber patterns, whereas histological analysis confirmed the presence and distribution of these myofiber arrays at the microscopic scale. Our results demonstrate a magnetic resonance method for acquiring and displaying diffusional data that defines highly ordered myofiber patterns in architecturally complex tissue. Such patterns suggest inherent multiscale fiber organization and provide a basis for structure-function analyses in vivo and in model tissues.
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Imagen por Resonancia Magnética , Músculos/citología , Animales , Difusión , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , LenguaRESUMEN
The regulation of epithelial proliferation during organ morphogenesis is crucial for normal development, as dysregulation is associated with tumor formation. Non-coding microRNAs (miRNAs), such as miR-200c, are post-transcriptional regulators of genes involved in cancer. However, the role of miR-200c during normal development is unknown. We screened miRNAs expressed in the mouse developing submandibular gland (SMG) and found that miR-200c accumulates in the epithelial end buds. Using both loss- and gain-of-function, we demonstrated that miR-200c reduces epithelial proliferation during SMG morphogenesis. To identify the mechanism, we predicted miR-200c target genes and confirmed their expression during SMG development. We discovered that miR-200c targets the very low density lipoprotein receptor (Vldlr) and its ligand reelin, which unexpectedly regulate FGFR-dependent epithelial proliferation. Thus, we demonstrate that miR-200c influences FGFR-mediated epithelial proliferation during branching morphogenesis via a Vldlr-dependent mechanism. miR-200c and Vldlr may be novel targets for controlling epithelial morphogenesis during glandular repair or regeneration.
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Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , MicroARNs/metabolismo , Morfogénesis/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de LDL/metabolismo , Glándula Submandibular/embriología , Análisis de Varianza , Animales , Western Blotting , Proliferación Celular , Biología Computacional , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Reelina , TransfecciónRESUMEN
The lacrimal gland (LG) develops through branching morphogenesis and produces secretions, including tears, that lubricate and protect the ocular surface. Despite the prevalence of LG disorders such as dry eye, relatively little is known about the regulation of LG development. In this study, we show that the homeobox transcription factor Barx2 is highly expressed in conjunctival epithelium, eyelids and ocular [lacrimal, harderian (HG), and meibomian (MG)] glands and is necessary for normal ocular gland and eyelid development. Barx2(-/-) mice show defective LG morphogenesis, absence of the HG, and defects in MG and eyelid fusion. Ex vivo antisense assays confirm the requirement for Barx2 in LG bud elongation and branching. Gene expression profiles reveal decreased expression of several adhesion and matrix remodeling molecules in Barx2(-/-) LGs. In culture, Barx2 regulates expression of matrix metalloproteinases (MMPs) and epithelial cell migration through the extracellular matrix. Fibroblast growth factors are crucial regulators of LG development and we show that Barx2 is required for Fgf10-induced LG bud elongation and that both Barx2 and Fgf10 cooperate in the regulation of MMPs. Together, these data suggest a mechanism for the effects of loss of Barx2 on ocular gland development. Intriguingly, salivary glands that also express a high level of Barx2 develop normally in Barx2(-/-) mice and do not show altered levels of MMPs. Thus, the function of Barx2 is specific to ocular gland development. Based on our data, we propose a functional network involving Barx2, Fgf10 and MMPs that plays an essential role in regulating branching morphogenesis of the ocular glands.
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Matriz Extracelular/metabolismo , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Aparato Lagrimal/anatomía & histología , Aparato Lagrimal/embriología , Aparato Lagrimal/crecimiento & desarrollo , Morfogénesis/fisiología , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Ojo/anatomía & histología , Ojo/crecimiento & desarrollo , Factor 10 de Crecimiento de Fibroblastos/genética , Proteínas de Homeodominio/genética , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.
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Heparan sulfate (HS) regulation of FGFR function, which is essential for salivary gland (SG) development, is determined by the immense structural diversity of sulfated HS domains. 3-O-sulfotransferases generate highly 3-O-sulfated HS domains (3-O-HS), and Hs3st3a1 and Hs3st3b1 are enriched in myoepithelial cells (MECs) that produce basement membrane (BM) and are a growth factor signaling hub. Hs3st3a1;Hs3st3b1 double-knockout (DKO) mice generated to investigate 3-O-HS regulation of MEC function and growth factor signaling show loss of specific highly 3-O-HS and increased FGF/FGFR complex binding to HS. During development, this increases FGFR-, BM- and MEC-related gene expression, while in adult, it reduces MECs, increases BM and disrupts acinar polarity, resulting in salivary hypofunction. Defined 3-O-HS added to FGFR pulldown assays and primary organ cultures modulates FGFR signaling to regulate MEC BM synthesis, which is critical for secretory unit homeostasis and acinar function. Understanding how sulfated HS regulates development will inform the use of HS mimetics in organ regeneration.
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Membrana Basal , Diferenciación Celular , Células Epiteliales , Heparitina Sulfato , Ratones Noqueados , Glándulas Salivales , Transducción de Señal , Sulfotransferasas , Animales , Heparitina Sulfato/metabolismo , Membrana Basal/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Sulfotransferasas/metabolismo , Sulfotransferasas/genética , Ratones , Células Epiteliales/metabolismo , Células Epiteliales/citología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Masculino , Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Understanding the transcriptional landscape that results in chronic salivary hypofunction after irradiation will help identify injury mechanisms and develop regenerative therapies. We present scRNA-seq analysis from control and irradiated murine parotid glands collected 10 months after irradiation. We identify a population of secretory cells defined by specific expression of Etv1, which may be an acinar cell precursor. Acinar and Etv1+ secretory express Ntrk2 and Erbb3, respectively while the ligands for these receptors are expressed in myoepithelial and stromal cells. Furthermore, our data suggests that secretory cells and CD4+CD8+T-cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement. Lastly, evaluation of cell-cell communication networks predicts that neurotrophin, neuregulin, ECM, and immune signaling are dysregulated after irradiation, and thus may play a role in the lack of repair. This resource will be helpful to understand cell-specific pathways that may be targeted to repair chronic damage in irradiated glands.
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Exocrine secretory acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific epithelial SG populations later in development and during acinar differentiation are unknown. Here, we predicted FGFR dependence in specific populations using scRNAseq data and conditional mouse models to delete FGFRs in vivo. We identifed essential roles for FGFRs in craniofacial and early SG development, as well as progenitor function during duct homeostasis. Importantly, we discovered that FGFR2b was critical for seromucous and serous acinar cell differentiation and secretory gene expression (Bpifa2 and Lpo) via MAPK signaling, while FGFR1b was dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activated the FGFR2b-dependent seromucous transcriptional program. We propose a model where MEC-derived FGF7 drives seromucous acinar differentiaton, providing a rationale for targeting FGFR2b signaling in regenerative therapies to restore acinar function.
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Exocrine acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific populations later in development and during acinar differentiation are unknown. Here, we use scRNAseq and conditional deletion of murine FGFRs in vivo to identify essential roles for FGFRs in craniofacial, early SG development and progenitor function during duct homeostasis. Importantly, we also discover that FGFR2 via MAPK signaling is critical for seromucous acinar differentiation and secretory gene expression, while FGFR1 is dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activates the FGFR2-dependent seromucous transcriptional program. Here, we propose a model where MEC-derived FGF7 drives seromucous acinar differentiation, providing a rationale for targeting FGFR2 signaling in regenerative therapies to restore acinar function.
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Orosomucoide , Transducción de Señal , Animales , Ratones , Diferenciación Celular/genética , Homeostasis , Glándulas SalivalesRESUMEN
The mechanisms that prevent regeneration of irradiated (IR) salivary glands remain elusive. Bulk RNAseq of IR versus non-IR human salivary glands showed that neurotrophin signaling is highly disrupted post-radiation. Neurotrophin receptors (NTRs) were significantly upregulated in myoepithelial cells (MECs) post-IR, and single cell RNAseq revealed that MECs pericytes, and duct cells are the main sources of neurotrophin ligands. Using two ex vivo models, we show that nerve growth factor (NGF) induces expression of MEC genes during development, and upregulation of NTRs in adult MECs is associated with stress-induced plasticity and morphological abnormalities in IR human glands. As MECs are epithelial progenitors after gland damage and are required for proper acinar cell contraction and secretion, we propose that MEC-specific upregulation of NTRs post-IR disrupts MEC differentiation and potentially impedes the ability of the gland to regenerate.
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HS3ST1 is a genetic risk gene associated with Alzheimer's disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.
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Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/genética , Cromatografía Liquida , Sulfatos , Espectrometría de Masas en Tándem , Heparitina Sulfato , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Ratones Noqueados , Encéfalo/metabolismoRESUMEN
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.
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Factor 10 de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Glándulas Salivales , Animales , Células Epiteliales/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ratones , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Transducción de SeñalRESUMEN
Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1-/- and Hs3st3b1-/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1-/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1-/- HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.
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Heparitina Sulfato , Sulfotransferasas , Animales , Factores de Crecimiento de Fibroblastos , Ratones , Morfogénesis , Glándulas Salivales , Sulfotransferasas/genéticaRESUMEN
Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.
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Células Ciliadas Auditivas/metabolismo , Audición/fisiología , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Vertebrados/metabolismo , Proteínas de Pez Cebra/metabolismo , Adenoviridae/genética , Animales , Animales Recién Nacidos , Anticuerpos/inmunología , Oído Interno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Canales Iónicos/inmunología , Ratones , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Rana catesbeiana , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio , Pez Cebra/metabolismo , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/inmunologíaRESUMEN
Understanding the dynamic transcriptional landscape throughout organ development will provide a template for regenerative therapies. Here, we generated a single-cell RNA sequencing atlas of murine submandibular glands identifying transcriptional profiles that revealed cellular heterogeneity during landmark developmental events: end bud formation, branching morphogenesis, cytodifferentiation, maturation, and homeostasis. Trajectory inference analysis suggests plasticity among acinar and duct populations. We identify transcription factors correlated with acinar differentiation including Spdef, Etv1, and Xbp1, and loss of Ybx1, Eno1, Sox11, and Atf4. Furthermore, we characterize two intercalated duct populations defined by either Gfra3 and Kit, or Gstt1. This atlas can be used to investigate specific cell functions and comparative studies predicting common mechanisms involved in development of branching organs.
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Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis.
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Epithelial-mesenchymal interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR.