Embryonic Submandibular Gland Research Articles

A novel approach to describing the pancreas and submandibular gland: Can they be classified as primary and secondary tissue organs?

Our aim is to examine the relationship between the submandibular gland (SMG) and the pancreas by discussing similarities and differences in their embryology, histology, physiology, and pathology, and to introduce the question of classifying them as primary or secondary organs. From an embryonic standpoint, SMG and pancreas originate from different germ layers, yet they share a variety of similar elements in their morphogenesis, branching process, and mesenchymal molecular interaction. The histological and anatomical comparison between these two organs reveals parallels in the basic function of their exocrine physiology. With both the SMG and pancreas playing a significant role in digestive processes, there are also common aspects in their exocrine function. Furthermore, recent research has unraveled an intricate novel system of hormonal interaction between the salivary glands and pancreas which regulates pancreatic cellular differentiation and injury repair mechanism. Lastly, there are analogous features in the pathological mechanisms and inflammatory processes in the course of chronic disease in both organs. By defining this close relationship between the SMG and the pancreas, we aim to provide alternative insights for scholars and physicians in the shared characteristics of basic function of these organs, and possible pathological consequences of their dysregulation.

Adamts18 Deficiency Causes Spontaneous SMG Fibrogenesis in Adult Mice

Chronic sclerosing sialadenitis of the submandibular gland (also known as Küttner tumor) is characterized by concomitant swelling of the submandibular glands secondary to strong lymphocytic infiltration and fibrosis. The pathogenesis of this disease has been unclear, but it is associated with immune disorders. ADAMTS18 is a member of the ADAMTS superfamily of extracellular proteinases. In this study, we showed that Adamts18 is highly expressed in submandibular salivary gland (SMG) during embryonic development and decreases but is retained in adult SMG tissue in mice. Adamts18 deficiency led to reduced cleft formation and epithelial branching in embryonic SMG before embryonic day 15.5 in mice. No significant histologic changes in the later stages of branching or the morphology of SMG were detected in Adamts18−/− mice. However, Adamts18 deficiency causes spontaneous SMG fibrogenesis and fibrosis in adult mice. At 8 wk of age, Adamts18−/− mice began to manifest the first signs of pathologic changes of mild fibrosis and CD11b+ cell infiltration in SMG tissues. At ≥8 mo, all male and female Adamts18−/− mice developed unilateral or bilateral SMG scleroma that is similar to patients with chronic sclerosing sialadenitis of the submandibular gland. Adamts18−/− mice also showed secretory dysfunction and severe dental caries. Histologically, SMG scleroma is characterized by progressive periductal fibrosis, acinar atrophy, irregular duct ectasis, and dense infiltration of IgG-positive plasma cells. A significant infiltration of CD4+ T lymphocytes and CD11b+ monocytes and macrophages was also detected in the SMG scleroma of Adamts18−/− mice. The levels of TGF-β1, IL-6, and IL-33 were significantly increased in Adamts18−/− SMGs, which induces chronic inflammation and myofibroblast activation, ultimately leading to fibrosis. This study indicates that Adamts18 regulates the early branching morphogenesis of embryonic SMG and plays a role in protecting from spontaneous SMG fibrogenesis via modulating local inflammation, autoimmune reaction, and myofibroblast activation in adult mice.

Efficient Surface Immobilization of Chemically Modified Hyaluronans for Enhanced Bioactivity and Survival of In Vitro-Cultured Embryonic Salivary Gland Mesenchymal Cells.

Embryonic salivary gland mesenchyme (eSGM) secretes various growth factors (bioactives) that support the proper growth and differentiation of salivary gland epithelium. Therefore, eSGM cells can be used as feeder cells for in vitro-cultured artificial salivary gland if their survival and bioactivity are properly maintained. As eSGM is encapsulated in a hyaluronan (HA)-rich developmental milieu, we hypothesized that mimicking this environment in vitro via surface immobilization of HA might enhance survival and bioactivity of eSGM. In this study, various HA derivatives, conjugated with catechol (HA–CA), thiol (HA–SH), or amine (HA–EDA) moieties, respectively, were screened for their efficacy of culturing eSGM-derived feeder cells in vitro. Among these HA derivatives, HA–CA showed the highest surface coating efficiency and growth enhancement effect on the embryonic submandibular gland. In addition, the HA–CA coating enhanced the production of growth factors EGF and FGF7, but not FGF10. These effects were maintained when eSGM cells isolated from the embryonic salivary gland were re-seeded to develop the feeder layer cells. CD44s (a major HA receptor) in eSGM cells were clustered at the cell membrane, and enhanced EGF expression was detected only in CD44 cluster-positive cells, suggesting that membrane clustering of CD44 is the key mechanism for the increased expression of EGF.

A mechanogenetic role for the actomyosin complex in branching morphogenesis of epithelial organs.

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the crucial genes required for the distribution of epithelial progenitor cells were regulated by YAP and TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.

Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5.

Branching morphogenesis is a crucial developmental mechanism for the formation of the typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we have investigated whether cross-talk may exist between the Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. An embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles and possible interactions of the Wnt/beta-catenin signaling pathway, FGF signaling, and lama5 in the branching process were investigated by morphogenesis assays and gene expression patterns. Here, we show that the E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. Our branching morphogenesis assay revealed that the epithelial branching process can be promoted when the canonical Wnt pathway is inhibited and significantly suppressed when the wnt pathway is over activated. Further experiments indicated that FGF signaling most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. Finally, we show that Wnt/beta-catenin signaling regulates branching morphogenesis through Lama5. We conclude that the Wnt/beta-catenin signaling pathway acting downstream of FGF signaling can serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

Characteristics of aquaporin 1, 3, and 5 expression during early murine salivary gland development.

Aquaporins (AQPs) are essential to coordinate the transit of water and ions through the cell membrane. In salivary glands (SGs), AQPs have been associated with saliva formation, facilitating water absorption through the epithelium during the formation of hypotonic saliva, which is then secreted into the oral cavity. Different members of the AQP family have been suggested to play distinct roles during embryonic development, highlighted by their specific expression patterns. Here, we have investigated the expression patterns of AQP-1, AQP-3 and AQP-5 by immunofluorescence at key stages of salivary gland development, utilising cultured mouse embryonic submandibular (SMG) and sublingual (SLG) glands. The expression of AQPs was compared to a mitotic marker, phospho-histone 3 (PH3), a myoepithelial marker, smooth muscle actin (SMA), and a vascular marker, CD31. Qualitative analysis revealed that AQP-1 and AQP-3 were primarily expressed during the earlier phases of SG morphogenesis and were associated with cells undergoing mitotic processes (PH3-positive). AQP-5, in contrast, was not associated to mitotic figures, but was predominantly expressed during late stages of SG morphogenesis. Our results highlight that AQPs are expressed from early stages of SG morphogenesis and exhibit complimentary expression patterns that may contribute to the morphogenesis of salivary glands.

Developmental role of hyaluronic acid and its application in salivary gland tissue engineering

Dry mouth, or xerostomia, caused by salivary gland dysfunction significantly impacts oral/systemic health and quality of life. Although in vitro-generated artificial salivary glands have been considered as the fundamental solution, its structural complexity is difficult to reproduce using current biomaterials. Therefore, understanding and recapitulating the roles of biomacromolecules in salivary gland organogenesis is needed to solve these problems. Hyaluronic acid (HA) is a macromolecule abundant during salivary gland organogenesis, but its role remains unknown. Here, we verify the effects of HA on salivary gland organogenesis and artificial organ germ formation in solubilized and substrate-immobilized forms. In embryonic submandibular glands (eSMG), we found dense HA layers encapsulating proliferative c-Kit+ progenitor cells that were expressing CD44, an HA receptor. The blockage of HA synthesis, or degradation of HA, impaired eSMG growth by ablating the c-Kit+ progenitor cell population. We also found that high-molecular-weight (HMW) HA has a significant role in eSMG growth. Based on these findings, we discovered that HA is also crucial for in vitro formation of salivary gland organ germs, one of the most promising candidates for salivary gland tissue regeneration. We significantly enhanced salivary gland organ germ formation by supplementing HMW HA in solution; this effect was further increased when the HMW HA was immobilized on the substrate by polydopamine/HA co-immobilization. Our study suggests that the current use of HA in salivary gland tissue engineering can be further optimized.

NiCHE Platform: Nature-Inspired Catechol-Conjugated Hyaluronic Acid Environment Platform for Salivary Gland Tissue Engineering.

Recently, there has been growing interest in replacing severely damaged salivary glands with artificial salivary gland functional units created in vitro by tissue engineering approaches. Although various materials such as poly(lactic-co-glycolic acid), polylactic acid, poly(glycolic acid), and polyethylene glycol hydrogels have been used as scaffolds for salivary gland tissue engineering, none of them is effective enough to closely recapitulate the branched structural complexity and heterogeneous cell population of native salivary glands. Instead of discovering new biomaterial candidates, we synthesized hyaluronic acid-catechol (HACA) conjugates to establish a versatile hyaluronic acid coating platform named "NiCHE (nature-inspired catechol-conjugated hyaluronic acid environment)" for boosting the salivary gland tissue engineering efficacy of the previously reported biomaterials. By mimicking hyaluronic acid-rich niche in the mesenchyme of embryonic submandibular glands (eSMGs) with NiCHE coating on substrates including polycarbonate membrane, stiff agarose hydrogel, and polycaprolactone scaffold, we observed significantly enhanced cell adhesion, vascular endothelial and progenitor cell proliferation, and branching of in vitro-cultured eSMGs. High mechanical stiffness of the substrate is known to inhibit eSMG growth, but the NiCHE coating significantly reduced such stiffness-induced negative effects, leading to successful differentiation of progenitor cells to functional acinar and myoepithelial cells. These enhancement effects of the NiCHE coating were due to the increased proliferation of vascular endothelial cells via interaction between CD44 and surface-immobilized HAs. As such, our NiCHE coating platform renders any kind of material highly effective for salivary gland tissue culture by mimicking in vivo embryonic mesenchymal HA. Based on our results, we expect the NiCHE coating to expand the range of biomaterial candidates for salivary glands and other branching epithelial organs.

Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2’-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

Generating Embryonic Salivary Gland Organoids.

Organoids are important research tools for studying organ morphogenesis and differentiation because they recapitulate ex vivo the native 3D organization of cells that is essential for proper cell and organ function. The composition of organoids can be manipulated to incorporate specific cell types to facilitate molecular interrogation of cell-cell interactions during organoid formation. A method for generating organoids derived from both embryonic salivary gland epithelial progenitor cells and mesenchymal support cells is described. Methods for isolating enriched populations of the epithelial cells as clusters and the mesenchyme cells as single cells from mouse embryonic submandibular salivary glands are also provided. Separating the epithelial and mesenchymal cell populations allows for independent molecular manipulation of each cell type. In addition, methods for lentiviral transduction of the mesenchyme cells and quantitative image analysis of organoids are provided. The methods described here are useful for exploring mechanisms driving organ formation. © 2018 by John Wiley & Sons, Inc.

TGF-β1 attenuated branching morphogenesis of embryonic murine submandibular gland through Smad3 activation.

Transforming growth factor-β1 (TGF-β1) plays several crucial regulatory roles in multiple physiological and pathological processes. The aim of this work was to investigate the role of TGF-β1 in branching morphogenesis of salivary gland. We harvested and cultured submandibular salivary glands (SMGs) from murine embryos, which were then treated with exogenous TGF-β1, or its neutralized antibody, Smad3 inhibitor, or Smad3 small interfering RNA (siRNA). Our results suggested that TGF-β1 attenuated branching morphogenesis of embryonic murine SMG via Smad3 activation, thus playing a negative regulatory role in salivary gland development.

MSCs feeder layers induce SMG self-organization and branching morphogenesis.

Dysfunction of salivary glands leads to several oral health problems, including dental caries, mastication and swallowing dysfunctions and multiple oral infections. Conventional treatments for such condition fell short of providing satisfying therapeutic results. Recent advances in organ regeneration therapy which utilize tissue stem cells to fabricate bioengineered 3D organ buds, have introduced a promising therapeutic tool for full functional organ regeneration. However, finding a sustainable and easily accessible cell source for such approaches is still challenging, especially in case of severely atrophied tissues such as irradiated salivary glands. In response to this, we hypothesized that bone marrow derived mesenchymal stem cells (MSCs) could be used as feeder cells to induce salivary epithelial tissues/cells branching. Indeed, in 2D cultures, MSCs supported branching of embryonic submandibular salivary gland (SMG) epithelium. Interestingly, this enhancing effect was dependent on the initial number of MSC feeder cells. In addition, MSCs supported the self-assembly of SMG epithelial progenitor cells into well-patterned and branched 3D salivary organoids. Therefore, these findings propose MSCs as a valuable candidate cell source for induced SMG epithelial branching, which can potentially be applied in future methods for SMG regeneration approaches.

Endothelial cell regulation of salivary gland epithelial patterning.

Perfusion-independent regulation of epithelial pattern formation by the vasculature during organ development and regeneration is of considerable interest for application in restoring organ function. During murine submandibular salivary gland development, the vasculature co-develops with the epithelium during branching morphogenesis; however, it is not known whether the vasculature has instructive effects on the epithelium. Using pharmacological inhibitors and siRNA knockdown in embryonic organ explants, we determined that VEGFR2-dependent signaling is required for salivary gland epithelial patterning. To test directly for a requirement for endothelial cells in instructive epithelial patterning, we developed a novel ex vivo cell fractionation/reconstitution assay. Immuno-depletion of CD31+ endothelial cells in this assay confirmed a requirement for endothelial cells in epithelial patterning of the gland. Depletion of endothelial cells or inhibition of VEGFR2 signaling in organ explants caused an aberrant increase in cells expressing the ductal proteins K19 and K7, with a reduction in Kit+ progenitor cells in the endbuds of reconstituted glands. Addition of exogenous endothelial cells to reconstituted glands restored epithelial patterning, as did supplementation with the endothelial cell-regulated mesenchymal factors IGFBP2 and IGFBP3. Our results demonstrate that endothelial cells promote expansion of Kit+ progenitor cells and suppress premature ductal differentiation in early developing embryonic submandibular salivary gland buds.

Data supporting chitosan facilitates structure formation of the salivary gland by regulating the basement membrane components.

To investigate the role of basement membrane (BM) in chitosan-mediated morphogenesis of the salivary glands, the embryonic submandibular gland (SMG) experimental model was used. Chitosan promotes branching at distinct stages in SMG morphogenesis. When enzymes such as type IV collagenase, dispase, and cathepsin B were used to digest the BM components, the morphogenetic effect mediated by chitosan disappeared. Immunofluorescence revealed that the corresponding receptors for BM components, including CD49c, CD49f, CD29, and dystroglycan, were locally enriched at the epithelial–mesenchymal junction around BM areas. The functional roles of laminin α1 and α5 in SMG branching were explored via siRNA knockdown, and suppression was confirmed at both the RNA and protein levels (Yang and Hsiao, Biomaterials, http://dx.doi.org/10.1016/j.biomaterials.2015.06.028, 2015). This data article demonstrates the experimental approaches to investigate the role of basement membrane in the structure formation of the salivary gland engineered by biomaterials.

Melatonin inhibits embryonic salivary gland branching morphogenesis by regulating both epithelial cell adhesion and morphology.

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.

Regenerating Salivary Glands in the Microenvironment of Induced Pluripotent Stem Cells.

This report describes our initial attempt to regenerate salivary glands using induced pluripotent stem (iPS) cells in vivo and in vitro. Glandular tissues that were similar to the adult submandibular glands (SMGs) and sublingual glands could be partially produced by the transplantation of iPS cells into mouse salivary glands. However, the tumorigenicity of iPS cells has not been resolved yet. It is well known that stem cells affect their microenvironment, known as a stem cell niche. We focused on the niche and the interaction between iPS cells and salivary gland cells in our study on salivary gland regeneration. Coculture of embryonic SMG cells and iPS cells have better-developed epithelial structures and fewer undifferentiated specific markers than monoculture of embryonic SMG cells in vitro. These results suggest that iPS cells have a potential ability to accelerate differentiation for salivary gland development and regeneration.

LIM kinase regulation of cytoskeletal dynamics is required for salivary gland branching morphogenesis.

Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein-dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation--cleft initiation, stabilization, and progression--via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis.

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.