Apoptosis in Early Salivary Gland Duct Morphogenesis and Lumen Formation.

Salivary glands are essential for the maintenance of oral health by providing lubrication and antimicrobial protection to the mucosal and tooth surfaces. Saliva is modified and delivered to the oral cavity by a complex multifunctional ductal system. During development, these ducts form as solid tubes, which undergo cavitation to create lumens. Apoptosis has been suggested to play a role in this cavitation process along with changes in cell polarity. Here, we show that apoptosis occurs from the very earliest stages of mouse salivary gland development, much earlier than previously reported. Apoptotic cells were observed in the center of the first epithelial stalk at early-stage embryonic day 12.5 (E12.5) according to both TUNEL staining and cleaved caspase 3 immunofluorescence. The presumptive lumen space was highlighted by the colocalization of a predictive lumen marker, cytokeratin 7. At E14.5, as lumens start to form throughout the glands, apoptotic expression decreased while cytokeratin 7 remained positive. In vitro inhibition of all caspases in E12.5 and E13.5 salivary glands resulted in wider ducts, as compared with the controls, and a defect in lumen formation. In contrast, no such defect in lumen formation was observed at E14.5. Our data indicate that apoptosis is involved during early stages of gland formation (E12.5 onward) and appears important for shaping the forming ducts.

NFIB regulates embryonic development of submandibular glands.

NFIB (nuclear factor I B) is a NFI transcription factor family member, which is essential for the development of a variety of organ systems. Salivary gland development occurs through several stages, including prebud, bud, pseudoglandular, canalicular, and terminal. Although many studies have been done to understand mouse submandibular gland (SMG) branching morphogenesis, little is known about SMG cell differentiation during the terminal stages. The goal of this study was to determine the role of NFIB during SMG development. We analyzed SMGs from wild-type and Nfib-deficient mice (Nfib (-/-)). At embryonic (E) day 18.5, SMGs from wild-type mice showed duct branching morphogenesis and differentiation of tubule ductal cells into tubule secretory cells. In contrast, SMGs from Nfib (-/-) mice at E18.5 failed to differentiate into tubule secretory cells while branching morphogenesis was unaffected. SMGs from wild-type mice at E16.5 displayed well-organized cuboidal inner terminal tubule cells. However, SMGs from Nfib (-/-) at E16.5 displayed disorganized inner terminal tubule cells. SMGs from wild-type mice at E18.5 became fully differentiated, as indicated by a high degree of apicobasal polarization (i.e., presence of apical ZO-1 and basolateral E-cadherin) and columnar shape. Furthermore, SMGs from wild-type mice at E18.5 expressed the protein SMGC, a marker for tubule secretory cells. However, SMGs from Nfib (-/-) mice at E18.5 showed apicobasal polarity, but they were disorganized and lost the ability to secrete SMGC. These findings indicate that the transcription factor NFIB is not required for branching morphogenesis but plays a key role in tubule cell differentiation during mouse SMG development.

VIP pipes up: neuronal signals direct tubulogenesis.

Biological tubes serve as the body's plumbing system, transporting fluids and gases throughout secretory, circulatory, and respiratory organs. In this issue of Developmental Cell, Nedvetsky et al. (2014) find that vasoactive intestinal peptide (VIP), secreted by parasympathetic nerves, is a surprising player in directing epithelial tubulogenesis in salivary glands.

Parasympathetic innervation regulates tubulogenesis in the developing salivary gland.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

Morphological study of the fetal parotid duct and buccinator muscle and the relationship to salivary secretion.

The parotid glands secrete about 25% of all saliva produced. In the presence of a stimulus, the amount of saliva secreted from the parotid gland increases to 50%. A decrease in the amount of produced saliva due to aging and parotiditis results in a dry mouth. Therefore, the parotid duct is important to maintaining a healthy oral cavity. In human adults, the parotid duct, approximately 6-8-cm long, travels over the masseter muscle and penetrates the buccinator muscle to enter the oral cavity. Although there have been various studies regarding the parotid gland, only few suggest a functional role of the parotid duct, especially its area of penetration of the buccinator muscle. In this study, 34 fetal specimens ranging from 4 to 10 months of age at death were dissected for anatomical and histological examinations. The area of the parotid duct penetrating the buccinator muscle was fully formed in 5-month-old fetuses. This study found buccinator muscle fibers invading the parotid duct wall near its opening in 6-month-old fetuses and older. Our results support the claim that the buccinator muscle may act as a sphincter, playing a role in regulating and possibly preventing the reflux of salivary secretions into the parotid duct.

Lumen formation in salivary gland development.

During salivary gland morphogenesis, the developing ducts and acini must hollow out to form lumina which will eventually allow the free passage and modification of saliva on its journey from acini to oral cavity. The molecular mechanisms that participate in the creation of this tubular structure are of great research interest. Histological studies show that lumen formation begins during the mid stages of branching morphogenesis. At this stage, apoptotic cells are detectable in the developing salivary ducts at sites where lumina are forming, suggesting that programmed cell death is instrumental in clearing the luminal space. The formation of cell-cell junctions between the epithelial cells lining the space is also an integral part of lumen formation, since these junctions form a barrier around the lumen and allow the surfaces of the lumen-lining cells to become specialized. This chapter will discuss the mechanisms involved in salivary gland lumen formation during development, and draw on the most recent research in this interesting field.

Organogenesis of the juxta-oral organ in mice.

The juxta-oral organ is a bilateral organ in the mammalian bucca. It consists of epithelial cords with surrounding mesenchyme. It develops from embryonic oral epithelium, but its macroscopic morphology in mice is less studied and seems to be very different from that of humans. The juxta-oral organ in mice extends more widely from the subcutaneous tissue of the mandible near the lateral fascia of the masseter to the submucosa of the soft palate. In this paper, we report that the mutant mouse allele Bmp7(lacZ) presented intense lacZ expression in the epithelial component of the juxta-oral organ in its homo- and heterozygous states. The main aims of this study were to show that this mutant mouse allele is suitable for observing macroscopic structure of the juxta-oral organ and to describe the development of this organ during embryonic and postnatal stages. Whole-mount beta-gal staining of this strain of mouse showed that the juxta-oral organ in mice appeared at E12.0 from oral epithelium and lost connection with it before E12.5. Then, the juxta-oral organ extended anteriorly to the lateral fascia of the masseter and posteriorly to the submucosal layer of the soft palate via the orbit. The mature juxta-oral organ had no connection to other epithelia such as those of the bucca and parotid duct. It persisted until adulthood and there seemed to be no tendency to regress. Transmission electron microscopy showed that each part of the juxta-oral organ was an epithelial cord surrounded by a basement membrane and mesenchymal tissue.

Diverse roles of E-cadherin in the morphogenesis of the submandibular gland: insights into the formation of acinar and ductal structures.

The formation of acinar and ductal structures during epithelial tissue branching morphogenesis is not well understood. We report that in the mouse submandibular gland (SMG), acinar and ductal cell fates are determined early in embryonic morphogenesis with E-cadherin playing pivotal roles in development. We identified two morphologically distinct cell populations at the single bud stage, destined for different functions. The outer layer of columnar cells with organized E-cadherin junctions expressed the neonatal acinar marker B1 by E13.5, demonstrating their acinar fate. The interior cells initially lacked distinct E-cadherin junctions, but with morphogenesis formed cytokeratin 7 (K7) -positive ductal structures with organized E-cadherin junctions and F-actin filaments. Inhibition of E-cadherin function with either siRNA or function blocking antibody caused extensive apoptosis of ductal cells and aberrantly dilated lumens, providing the first evidence that E-cadherin regulates ductal lumen formation during branching morphogenesis of the salivary gland.

Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands.

Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

Grainyhead-related transcription factor is required for duct maturation in the salivary gland and the kidney of the mouse.

Duct epithelial structure is an essential feature of many internal organs, including exocrine glands and the kidney. The ducts not only mediate fluid transfer but also help to maintain homeostasis. For instance, fluids and solutes are resorbed from or secreted into the primary fluid flowing through the lumen of the ducts in the exocrine glands and kidneys. The molecular mechanism underlying the functional maturation of these ducts remains largely unknown. Here, we show that a grainyhead-related transcription factor, CP2-like 1 (CP2L1), is required for the maturation of the ducts of the salivary gland and kidney. In the mouse, Cp2l1 is specifically expressed in the developing ducts of a number of exocrine glands, including the salivary gland, as well as in those of the kidney. In Cp2l1-deficient mice, the expression of genes directly involved in functional maturation of the ducts was specifically reduced in both the salivary gland and kidney, indicating that Cp2l1 is required for the differentiation of duct cells. Furthermore, the composition of saliva and urine was abnormal in these mice. These results indicate that Cp2l1 expression is required for normal duct development in both the salivary gland and kidney.

Immunoexpression of extracellular matrix proteins in human salivary gland development.

Immunoexpression of the extracellular matrix (ECM) proteins laminin, fibronectin, tenascin and types I, III and IV collagen was analyzed in the major and minor salivary glands of seven human fetuses at different gestational ages. The results showed the presence and localization of laminin, collagen IV and fibronectin around glandular structures at all stages of development. Tenascin was only detectable around excretory ducts. In the earliest stages of development, type I and type III collagen were presented as fine fibers delineating the glandular structures and delimiting the extension of the future lobule. As glandular development proceeded, the lobule was gradually filled with collagens and glandular tissue.

Prenatal development of the palatine gland of rats.

The present study aimed to elucidate the prenatal development of the rat palatine gland. Parasagittal 5 microm thick serial sections made from Wistar rats at embryonic days (E) 17 to 22 were stained with haematoxylin-eosin (HE), Alcian blue-Kernechtrot or immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) as a marker of proliferating cells. Additionally, three-dimensional images of developing glandular parenchyma were reconstructed from serial HE sections with a personal computer. At E 17, several thickenings of the palatal epithelium had appeared which thereafter became the epithelial cords. Branching and lumenization commenced at E 20, and immature acini were observed at E 21. Three-dimensional reconstruction showed that the proximal part of the epithelial cord differentiated into the duct, and the distal part of the epithelial cord differentiated into the acinus. In immunohistochemical staining, there were many BrdU-positive cells in the epithelial cords including thickenings of the palatal epithelium, ducts, and acini. The BrdU labeling index of the cells of the epithelial cord was the highest (statistically significant) of the three in the primitive palatine gland. In conclusion, during the development of the rat palatine gland, epithelial cords with very high proliferative activity arise from the palatal epithelium, and then the proximal part of the epithelial cord differentiates into the duct, and the distal part of the epithelial cord differentiates into the acinus. Proliferation of these glandular parenchyma contributes to the growth of the developing palatine gland.

Specification of cell fates within the salivary gland primordium.

The Drosophila salivary gland is a simple tubular organ derived from a contiguous epithelial primordium, which is established by the activities of the homeodomain-containing proteins Sex combs reduced (SCR), Extradenticle (EXD), and Homothorax (HTH). EGF signaling along the ventral midline specifies the salivary duct fate for cells in the center of the primordium, while cells farther away from the source of EGF signal adopt a secretory cell fate. EGF signaling works, at least in part, by repressing expression of secretory cell genes in the duct primordium, including fork head (fkh), which encodes a winged-helix transcription factor. FKH, in turn, represses trachealess (trh), a duct-specific gene initially expressed throughout the salivary gland primordium. trh encodes a basic helix-loop-helix PAS-domain containing transcription factor that has been proposed to specify the salivary duct fate. In conflict with this model, we find that three genes, dead ringer (dri), Serrate (Ser), and trh itself, are expressed in the duct independently of trh. Expression of all three duct genes is repressed in the secretory cells by FKH. We also show that SER in the duct cells signals to the adjacent secretory cells to specify a third cell type, the imaginal ring cells. Thus, localized EGF- and Notch-signaling transform a uniform epithelial sheet into three distinct cell types. In addition, Ser directs formation of actin rings in the salivary duct.

Expression of cytoskeletal proteins in developing human minor salivary glands.

The presence of an epithelium at different stages of proliferation and differentiation raises interesting questions concerning the histogenesis, cell turnover and differentiation of normal salivary glands. In order to expand knowledge of these aspects, we investigated the expression of cytokeratins (CKs) 7,8,10,13,14,16,18 and 19, vimentin (VIM), and smooth muscle actin (SMA) in developing human minor salivary glands using monoclonal antibodies. Labial, buccal, palatine, and lingual salivary glands and those from the floor of the mouth were obtained from human fetuses (forensic postmortem) ranging in age from gestational weeks 10 to 29. Serial sections, 3 microm thick, were immunostained using a strepto-avidin-biotin technique. Reactivity for all antibodies was negative in the salivary gland epithelium during the developmental stages of bud formation, cord growth, and branching of cord. During canalization and cytodifferentiation, the glandular epithelial cells showed a positive reaction to some CKs and SMA. Cytokeratins 7, 8, 18, and 19 showed strong labeling in luminal duct cells that exhibited some degree of morphological differentiation. Myoepithelial cellc were recognized by antibodies to SMA. Cytoskeletal protein expression changes according to the cell type, degree of differentiation, and stage of morphological development of the glandular structure. These changes occur independently of the localization of the gland.

An alternatively spliced Muc10 glycoprotein ligand for putative L-selectin binding during mouse embryonic submandibular gland morphogenesis.

Late-gestation (embryonic day 18; E18) mouse submandibular glands (SMG) comprise a network of large and small ducts that terminate in lumen-containing, presumptive acini (terminal buds) expressing unique, cell membrane-associated embryonic mucin. The objective here was to clone and sequence embryonic low molecular-weight SMG mucin, predict its secondary structure, and begin to investigate its possible role in SMG development. Evidence was found that: (1) embryonic low molecular-weight mucin is an alternatively spliced Muc10 gene product, 220 amino acids in size (approximately 25 kDa), rich in potential O-glycosylation sites, and variably glycosylated (approximately 40 and 68 kDa); (2) consensus secondary-structure prediction for embryonic low molecular-weight mucin is consistent with a molecule that is anchored to the plasma membrane, directly or indirectly (via a glycolipid), and has a protein core that serves as a scaffold for carbohydrate presentation; (3) embryonic L-selectin is immunolocalized to the plasma membrane region of terminal-bud epithelial cells in a pattern similar to that seen for embryonic mucin; (4) embryonic, but not adult, mucin is able to bind L-selectin and does so endogenously in E18 SMG. As the primary role of L-selectin is to mediate cell adhesion and its ligands are mucin-like glycoproteins, it is suggested that this embryonic low molecular-weight mucin be termed MucCAM.

Spatial and temporal localisation of bone morphogenetic protein-3 (osteogenin) in the developing rat submandibular gland.

Submandibular gland morphogenesis is a highly regulated process modulated by epithelio-mesenchymal interactions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are known to be soluble mediators of epithelio-mesenchymal interactions during the development of certain organs. The aim of this study was to localise bone morphogenetic protein-3 (BMP-3 or osteogenin), spatially and temporally in the developing rat submandibular gland. Immunocytochemistry was performed on sections of the developing submandibular gland (gestation ages E14.5-E19.5). BMP-3 was localised in the extra-cellular matrix of the mesenchyme of the gland in all the stages examined. Intense staining of BMP-3 was observed in the epithelial cells of the developing end-buds between stages E14.5 and E16.5. As cytodifferentiation progressed (stage E17.5 onwards) the number of epithelial cells in the developing acini in which BMP-3 was present became markedly reduced. Similarly, the number of cells containing BMP-3 in the developing ducts decreased as duct development progressed. By stage E19.5, BMP-3 was located mainly in the immature ducts. While the effects of BMP-3 on matrix macromolecules could not be deduced from this study, its spatial and temporal location within the developing glands may indicate a role in the co-ordinated regulation of branching morphogenesis.

Early genes required for salivary gland fate determination and morphogenesis in Drosophila melanogaster.

Studies of Drosophila salivary gland formation have elucidated the regulatory pathway by which the salivary gland fate is determined and the morphogenetic processes by which the primordial cells are internalized to form the tubular glands. Both the position of the salivary primordia and the number of cells recruited to a salivary gland fate are established through a combination of the localized expression of the transcription factors SEX COMBS REDUCED (SCR), TEASHIRT (TSH) and ABDOMINAL-B (ABD-B), and localized DPP-signaling. Similarly, the distinction between the two major cell types, duct and secretory, is determined by spatially limited EGF-signaling. Salivary gland formation also requires the function of two transcription factors expressed in nearly all cells of the developing embryo, EXTRADENTICLE (EXD) and HOMOTHORAX (HTH). Once the salivary gland fate is determined, cells of the secretory primordia are internalized by an apical constriction mode of invagination. We have characterized three genes encoding transcription factors, trachealess (trh), hückebein (hkb), and fork head (fkh), that are downstream targets of the salivary gland regulators. Mutations in these transcription factors profoundly affect salivary gland morphogenesis. trh is required for the formation of the salivary duct tubes. hkb determines the order of secretory cell invagination, a regulated process critical for determining the final shape of the salivary gland. fkh has two early roles in salivary gland formation. fkh both promotes secretory cell survival and facilitates secretory cell internalization. trh, hkb, and fkh are involved in the formation of not only the salivary duct and secretory tubes, but also of other tubular structures, such as the trachea and the gut endoderm. We propose that trh, hkb, and fkh may serve as "morphogenetic cassettes" responsible for forming tubular structures in a variety of tissues.

The Drosophila Pax gene eye gone is required for embryonic salivary duct development.

What are the developmental mechanisms required for conversion of an undifferentiated, two-dimensional field of cells into a patterned, tubular organ? In this report, we describe the contribution of the Drosophila Pax gene eye gone to the development of the embryonic salivary glands and ducts. eye gone expression in salivary tissues is controlled by several known regulators of salivary fate. After the initial establishment of the salivary primordium by Sex combs reduced, fork head excludes eye gone expression from the pregland cells so that its salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eye gone expression in the posterior preduct cells. We have previously described the process by which fork head and the EGF receptor pathway define the border between the gland and duct primordia. Here we show that eye gone is required for the subdivision of the duct primordium itself into the posterior individual duct and the anterior common duct domains. In the absence of eye gone, individual ducts as well as the precursor of the adult salivary glands, the imaginal ring, are absent. We took advantage of this ductless phenotype to show that Drosophila larvae do not have an obligate requirement for salivary glands and ducts. In addition to its role in the salivary duct, eye gone is required in the embryo for the development of the eye-antennal imaginal disc and the chemosensory antennal organ.

Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis.

The Drosophila Single-minded and Tango basic-helix-loop-helix-PAS protein heterodimer controls transcription and embryonic development of the CNS midline cells, while the Trachealess and Tango heterodimer controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess is highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins is investigated because of their similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicate that they accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells where it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Conversely, Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since they are cytoplasmic in the absence of Tango. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription. The lineage-specific action of Single-minded and Trachealess derives from transcriptional activation of their genes in their respective lineages, not from extracellular signaling.