Overview of human salivary glands: highlights of

Overview of human salivary glands: highlights of morphology and developing processes Fernanda de Paula1, Tathyane Harumi Nakajima Teshima2, Ricardo Hsieh2, Milena Monteiro Souza1, Marcello Menta Simonsen Nico1, Silvia Vanessa Lourenco2 1 2

Department of Dermatology, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil Department of Stomatology, School of Dentistry, University of Sao Paulo, Sao Paulo, Brazil Corresponding author: Tathyane Harumi Nakajima Teshima Department of Stomatology School of Dentistry University of Sao Paulo Av Prof Lineu Prestes, 2227 Cidade Universitaria Sao Paulo, SP – Brazil 05508-000 E-mail: [email protected] [email protected] Telephone number: +551126488219

Running title: Overview of human salivary gland development Grant sponsor: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) – Grant 2011/18865-2; 2015/02824-6.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ar.23569 This article is protected by copyright. All rights reserved.

The Anatomical Record

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ABSTRACT Salivary glands are essential organs that produce and secrete saliva to the oral cavity. During gland morphogenesis, many developmental processes involve a series of coordinated movements and reciprocal interactions between the epithelium and mesenchyme that generate the ductal system and the secretory units. Recent studies have shown new findings about salivary gland development, particularly regarding lumen formation and expansion, with the involvement of apoptosis and cell polarization, respectively. Moreover, it has been observed that human minor salivary glands start forming earlier than previously published and that distinct apoptotic mediators can trigger duct lumen opening in humans. This review summarizes updated morphological and cellular features of human salivary glands and also explores new aspects of the human developmental process.

Keywords: human salivary glands; morphogenesis; morphology; lumen formation.

John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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INTRODUCTION Human salivary glands (SG) are fundamental for the maintenance of the oral cavity homeostasis. They synthesize and secrete saliva, a multi-functional fluid, which provides mucosal lubrication, salivary electrolytes, antibacterial compounds and various enzymes to protect the oral mucosa and teeth surface (Feller et al., 2013; Carpenter, 2013). Salivary secretory function is therefore critical for oral health maintenance and its straight correlation with oral function and quality of life has been recently demonstrated (Furuta & Yamashita, 2013). Partial or total reduction of saliva due to gland loss or degeneration occurs for distinct reasons and the increasing comprehension of the biogenesis of SG development as well as their morphology may provide new methods to interpret glandular health and disease (Holmberg & Hoffman, 2014; Pringle et al., 2013; Nelson et al., 2013a; Vissink et al., 2010).

Defining the morphology of adult human salivary glands Human major salivary glands consist of three pairs of glands known as parotid, submandibular and sublingual glands; together they are responsible for 90% of the total saliva. Minor salivary glands in turn, comprise approximately 600 to 1000 glands distributed throughout the oral cavity. Although they secrete less than 10% of the total secretion, this secretion serves as the main lubricant saliva due to its protective and mucous components (Edgar, 1990; Amano et al., 2012; Holmberg & Hoffman, 2014). Adult salivary glands consist of specialized cells derived from the epithelium with two morphologically well-defined functional segments. They are predominantly composed of acinar cells and a complex ductal system associated with contractile myoepithelial cells, which



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contribute to salivary secretion into the ducts (Figure 1) (see Humphrey & Williamson, 2001 for review; Dodds et al., 2005) The ductal network is formed of different types of ducts (intercalated, striated and excretory ducts). Intercalated ducts are the first structures to receive the initial secretion, which is then conveyed to the striated ducts, where several electrolytic changes occur between the salivary fluid and the extracellular matrix via plasma membrane. As the saliva turns more hypotonic, wide extralobular excretory ducts collect all fluid from the glandular lobules, and secrete it to the oral cavity (Varga, 2012; Edgar, 1992)

Major salivary glands Parotid glands Human parotid glands are the largest major glands (average weight: 25-30g) disposed on each side of the head, behind the external auditory canal of the mandible and the skull base (Berkovitz et al., 1992; Ferraris & Muñoz, 2006). The development of human parotid glands starts from the 5th and 6th week of the intrauterine stage (Ellis & Auclair, 2008). The main excretory duct of the parotid glands is composed of Stensens duct, running through the anterolateral edge of the gland over the masseter muscle, culminating at the buccal mucosa in the upper molar region (Carlson, 2000; Ferraris & Muñoz, 2006; Katchburian & Arana, 2012; Holmberg & Hoffman, 2014). Parotid glands are exclusively formed by serous acini, which secrete aqueous saliva, rich in amylase, sulphomucins and sialomucins (Ferraris & Muñoz, 2006). The salivary secretion of the parotid glands is stimulated by sympathetic nerves and the auriculotemporal plexus (Katchburian & Arana, 2012).


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Submandibular glands Human submandibular gland development starts from the completion of the sixth week of intrauterine life (Ellis & Auclair, 2008). Representing the second largest pair of human salivary glands, submandibular glands range in weight from 7g to 15g and are located in the submandibular triangle behind the free insertion of the mylohyoid muscle. Submandibular ducts open at each side of the lingual frenulum through Wharton’s duct (Berkovitz et al., 2004; Carlson, 2000; Ferraris & Muñoz, 2006; Ellis & Auclair, 2008; Holmberg & Hoffman, 2014). The intercalated ducts of the submandibular parenchyma are shorter compared to the parotid gland, whereas striated ducts are more branched and extensive (Ferraris & Muñoz, 2006; Katchburian & Arana, 2012). Regarding the salivary secretion, submandibular glands are composed of mixed acini with mucous and serous components. In general, the serous acini are predominant, although the proportion may vary among lobes. (Ferraris & Muñoz, 2006; Nanci, 2013). Submandibular glands contribute substantially to the amount of secreted saliva within the oral cavity, secreting a viscous saliva composed mainly of glycoproteins sulfated cystatins and neuronal and epidermal growth factors, promoting lubrication and protection of the oral mucosa (Ferraris & Muñoz, 2006). The neural component which stimulates the submandibular gland occurs via facial and lingual branches of the mandibular nerve and the sympathetic trunk, arising from the submandibular ganglion (Katchburian & Arana, 2012). In rodent submandibular glands, only serous acinar cells are identified. In this gland type, the granular ducts or granular convoluted



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tubules composed by high-columnar secretory cells with secretory granules are detected between intercalated duct and striated duct (Amano et al., 2012).

Sublingual glands Human sublingual glands are the last pair of major glands to form. They start to form between the 7th and 8th week of embryonic life (Ellis & Auclair, 2008). With an average weight of approximately 3g, the sublingual glands represent the smallest group of the major salivary glands. They are deeply located between the connective tissue of the floor of the mouth and the mylohyoid muscle (Ferraris & Muñoz, 2006; Katchburian & Arana, 2012). The sublingual glands present predominantly mucous acinar cells and serous acini are rarely found (Ferraris & Muñoz, 2006; Nanci, 2013; Katchburian & Arana, 2012). Its final secretion is sent to the Bartholin’s excretory duct, which opens at the sublingual caruncle, near to the Wharton's duct of the submandibular gland. In addition, several accessory excretory ducts of smaller glandular lobes are found nearby the lingual frenulum (Ferraris & Muñoz, 2006; Holmberg & Hoffman, 2014).

Minor salivary glands It has been reported that human minor salivary gland development starts at the third month of pregnancy (Ellis & Auclair, 2008), although data from our research group already reveal fully developed minor glands at this stage.


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Minor salivary glands are located throughout oral submucosa, except in the gum and hard palate. They are predominantly formed by mucous acini that are surrounded by loose connective tissue that defines a more irregular mesenchymal capsule (Ferraris & Muñoz, 2006; Katchburian & Arana, 2012). Von Ebner glands are other minor glands found at the base of the circumvallate papilla of the tongue, however these glands only secrete a serous sort of saliva, which is essential for breast milk lipid digestion (Ferraris & Muñoz, 2006; Nanci, 2013; Katchburian & Arana, 2012). Minor salivary glands also display a complex ductal system similar to those of the major glands, although with shorter tracts, which make the distinction between both gland types more difficult. Importantly, the salivary secretion from minor glands usually occurs homogeneously through several small ducts spread over the oral mucosal surface, instead of being collected by a single large duct, which contributes to an efficient lubrication of the oral cavity (Ferraris & Muñoz, 2006; Nanci, 2013). Paradoxically, minor salivary glands are considered the most important for the mucosal protective and lubricant functions due to their saliva composition (Edgar, 1990). Representing 6 to 10% of the daily secretion of saliva, minor salivary glands play a crucial role in the protective mechanisms of oral mucosa and enamel surface, where saliva forms the dental biofilm. They produce about 70% of the salivary mucins and significant quantities of immunoglobulins (mainly IgA), salivary acid phosphatase and lysozymes, preventing colonization of microorganisms on the teeth surface and the occurrence of infections. The structure and function of major and minor salivary glands are potentially affected by the use of alcohol and drugs, as well as by poor nutrition, aging and head and neck radiotherapy (Ferraris & Muñoz, 2006; Nanci, 2013).



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Histological characterization of human salivary glands Acini The oral cavity saliva is mainly composed of water, ions and proteins; this complex fluid is secreted by the salivary gland end-pieces – the acinar lobules, which are composed by acinar cells. Acinar cells are classified according to the type of secretion as serous or mucous acini, presenting a distinct cellular architecture and intracellular components. Differences of secretion are related to the diet type and may vary among the different species (Berkovitz et al., 1992; Tandler & Phillips, 1998). The acinar serous secretory portion consists of 8 to 12 pyramid-shaped cells, occupying the broadest portion adjacent to the stroma at the basal side. Nuclei are round and pronounced in the middle of the cytoplasm. These cells are arranged in a spherical structure forming a narrow central lumen apex. A significant amount of molecular components of serous saliva is found within the secretory granules located in the apical cytoplasm (Berkovitz et al., 1992; Nanci, 2013). Different from serous cells, mucous acini are formed by tubule-shaped secretory structures, in which secretory cells present flat nuclei dislocated towards the basal cell surface due to large numbers of mucin granules accumulated in the apical cytoplasm. The main function of this mucous saliva is to provide oral lubrication and form the protective barrier surface (Munger, 1964; Nanci, 2013). Serous acini have only low expression of glycoconjugates, which are thought to have antimicrobial and enzymatic activity, binding to calcium and a large amount of ions and water; their main secretion is marked by high levels of amylase (Nanci, 2013; Ligtenberg et al., 2015).


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Salivary gland secretory acini of major glands are generally divided into several lobes and lobules surrounded by a mesenchymal capsule with fat cells between the lobular divisions; increased numbers of adipocytes that may replace the functional parenchymal tissue due to aging and potentially cause symptoms of ‘dry mouth’ syndrome. To date there is no evidence of how this process is regulated and the reason for this replacement; further studies are needed to prevent such inconvenience (Ferraris & Muñoz, 2006; Katchburian & Arana, 2012). Mucous acinar cells are also pyramidal-shaped with wide cytoplasm, where the intense accumulation of secretory granules defines the flat-shaped nuclei located in the basal portion. The whole acinar strucuture is usually tubular and these volumous acini produce large amount of mucins, which provide a viscous character to the saliva and acts as essential lubricant in the oral cavity (Ferraris & Muñoz, 2006; Nanci, 2013; Katchburian & Arana, 2012)

Ducts The ductal complex participates actively in modifying the primary hypotonic saliva into an isotonic fluid by mediating several ionic changes between saliva and ducal cells. This unique function requires specific ductal structures and the ductal network, is therefore formed by three different types of ducts known as intercalated, striated and excretory ducts (Nanci, 2013). Connected directly to the acini, the intercalated ducts are the first to receive the primary saliva, while remaining hypotonic, as the lumen of the secretory acini is contiguous with the lumen of the intercalated duct. This type of duct is formed by cells of simple cuboidal epithelium and is partially covered by contractile myoepithelial cells that contribute to the salivary flow. Intercalated ductal cells present central nuclei and a small amount of cytoplasm with small



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secretory granules in the apical region, which has microvilli projections into the lumen space. These granules contain components such as lysozyme and lactoferrin that are secreted in the saliva (Berkovitz et al., 1992; Nanci, 2013). Intercalated ducts are considered important for salivary gland homeostasis and replenishment, as many authors consider that this structure harbors salivary gland pluripotent/stem cells that give rise to acinar, myoepithelial and ductal cells (Ellis & Auclair, 2008). Our group demonstrated the presence of beta-1 integrin positive cells in the intercalated ducts of developing human salivary glands, suggesting that these might be progenitor cells at early stages of salivary gland morphogenesis (Lourenço et al., 2007). This hypothesis is yet to be confirmed. Striated ducts comprise the majority of the ductal system in the major salivary glands, being also considered as intralobular ducts, as they permeate the glandular lobes. They are specialized in the regulation of secretion and reabsorption of electrolytes through a bi-directional transport between the lumen and the extracellular space, promoting the essential salivary modification from isotonic to hypotonic saliva. This process requires cell energy and striated ductal cells therefore present a large amount of mitochondria along their basolateral membrane, characterizing the striated aspect by their multiple folding. Striated duct cells are particularly columnar and the nuclei are central within the cytoplasm (Berkovitz et al., 1992; Nanci, 2013). The final collecting ducts, interlobular excretory ducts, are generally found between the glandular lobules. They are responsible for continuing the reabsorption of sodium and potassium secretion, and subsequently conducting the final saliva to the oral cavity. Excretory ducts are formed by pseudostratified epithelium, which transforms into stratified structures when


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approaching the oral mucosa. Moreover, various types of columnar epithelial cells present luminal microvilli that may be involved in the sensitivity, reabsorption or secretion of the salivary flow (Berkovitz et al., 1992; Nanci, 2013).

Myoepithelial cells Associated with the glandular parenchyma, the contractile myoepithelial cells play an essential role in acinar salivary secretion. Their dual epithelial and contractile properties are supported by immunohistochemical findings, showing the expression of keratin filaments and also a massive expression of contractile proteins such as actin, caldesmon, calponin and smooth muscle actin (Redman, 1994; Ianez et al., 2010; Chitturi et al., 2015). It has been extensively described that myoepithelial cells wrap the secretory units between the acinar basement membrane and mesenchyme in many mammalian exocrine glands, such as salivary, mammary, sweat and tear glands. Its distribution varies considerably between types of glands and even during the course of glandular development. Tamarin, in 1966, described such glands as "like an octopus sitting on a rock." In the terminal portion of the salivary glands, myoepithelial cells "embrace" the acini. There is, however, no agreement about the specific location of myoepithelial cells in the literature yet. In a qualitative analysis of salivary glands, myoepithelial cells were identified around mucous and serous acini and also in different regions of the ductal system (Redman, 1994; Hardy & Kramer, 1998; Ogawa, 2003; Chitturi et al., 2015). Myoepithelial cells have also been associated with functions other than their primary contractile role. The production of the basement membrane, which contributes to tumor



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suppression and transport of metabolites has also been reported. Furthermore, it has been suggested that myoepithelial cells play a role in propagation neural stimuli. Others have described that they are important during gland development and that they represent a potential stem/progenitor cell source in salivary glands, however further investigations need to be performed (Caselitz et al, 1986; Redman., 1994; Ogawa, 2003; Ianez et al., 2010; Chitturi et al., 2015).

Salivary gland neural supply The composition and volume of secreted saliva depends on neural stimulation, and the normal secretion is associated with the autonomic nerve supply. In this scenario the parasympathetic nerve is of significant importance. The parasympathetic nervous system, which is intricately associated with the development of the salivary gland from early stages, is essential for branching morphogenesis and initiation of lumens. The sympathetic nervous system, in contrast, does not become associated with the gland until later stages, i.e., once a branched structure has formed. This timepoint coincides with the expansion of the luminal space and differentiation of the acini and ductal structures. The role of the sympathetic nervous system during salivary gland development however, has not been studied. Neural growth factor (NGF) has been shown to be a key player in sympathetic neuron regulation in other systems and this pathway is expressed in human salivary glands (Ghasemlou et al., 2004; Proctor & Carpenter, 2007; Knox et al., 2010; Datta et al., 1991; Naesse et al., 2013; Nedvetsky et al., 2014).


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Proctor & Carpenter (2007) and Proctor (2016) reported that reflex of saliva secretion is mediated by autonomic nerves in adult salivary glands. The parasympathetic stimulation causes an increase in the volume of saliva while the sympathetic stimulation interferes in a salivary secretion rich in proteins. The muscarinic receptors M1 and M3 interact with acetylcholine in the parasympathetic nerves to stimulate fluid secretion, while the sympathetic nerves are activated by noradrenaline and β1 adrenoceptors to promote protein secretion (Asking & Gjörstrup, 1987; Proctor, 2016). Almost all cell types of salivary glands appear to be innervated by both sympathetic and parasympathetic nerves. The cholinergic innervation in the vasculature occurs in all salivary glands. In the glandular blood vessels, the sympathetic innervation causes vasoconstriction, but this has no effect in the salivary reflex (Rossoni et al., 1981; Proctor & Carpenter, 2007). Some neural receptors like mechanoreceptors, olfactory receptors, gustatory receptors and nociceptors induce saliva secretion through the stimulated reflex, such as mastication, taste and other stimuli. The activation of some different mechanoreceptors located in the periodontal ligament and mucosae, where taste buds are found over the epithelium of the dorsal surface of the tongue, evokes the salivary secretion in the major salivary glands and this increased flux may occur in the minor salivary glands, also in response to taste stimulation (Shannon et al., 1969; Speirs, 1984; Hector & Linden, 1999).

Blood supply The external carotid artery provides the blood supply to the major glands, as there is access to the arterial vascularization via small branches. Capillaries and arterioles extend around



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secretory terminal units and striated ducts. These arterioles are present due to one or more arteries that penetrate the gland and divide into smaller arterioles, also found close to excretory ducts. The blood flow is able to increase up to 15 times during the period of maximum salivary secretion (Nanci, 2013; Katchburian & Arana, 2014). An essential role of endothelial-epithelial interactions during organogenesis has already been reported in liver development as early endothelial cells surround newly specified endoderm, driving the liver bud growth within the mesenchyme. In salivary glands, the role of the vasculature during branching morphogenesis remains unclear and whether the vascular glandular supply is critical for the maintenance of salivary secretion requires further investigation (Matsumoto et al., 2001; Patel et al., 2006).

Overview of salivary gland morphogenesis (Figure 2) During salivary gland development important interactions between epithelial and mesenchymal tissues occur, which induces and controls salivary gland morphogenesis and cytodifferentiation. The patterns of salivary gland development and branching morphogenesis are essentially controlled by the mesenchymal capsule and the epithelial rudiments (Bernfield et al., 1972; Cutler & Gremski, 1991). Organogenesis is an essential developmental process to form all human organs that will generate specific morphology of each structure (Otani et al., 2016). Overall, the development of secretory organs such as kidneys, lungs, salivary and mammary glands undergoes similar embryonic process and depends on a specific mechanism named branching morphogenesis (Denny et al., 1997; Carlson, 2000; Lourenço et al., 2007, 2008; Varner & Nelson, 2014).


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Interactions between epithelium and mesenchyme provide the main signals that orchestrate branching morphogenesis. It involves highly coordinated mechanisms that include gene expression and regulation of cell shape changes through growth, proliferation, differentiation, migration, apoptosis and epithelial mesenchymal interaction, endothelial and neuronal communication. (Cutler, 1990; Kashimata & Gresik, 1996; Patel et al., 2006; Nelson et al., 2013b). There is no direct lineage tracing evidence revealing the ectodermal origin of major salivary glands; however, Rhotova et al. (2012) reported no endodermal contribution to mouse salivary gland formation in their Sox17-2A-iCre/Rosa26 lineage tracing studies. Overall, bud-like structures from the invagination of the oral epithelium also known as stomodeum migrate towards the underlying mesenchyme, which will progressively proliferate, branch and canalize to form the salivary glands (Cutler, 1990; Kashimata & Gresik, 1996; Carlson, 2000; Ellis & Auclair, 2008; Teshima et al, 2011). All salivary gland developmental stages in both human and mouse models have recently been illustrated and compared (Teshima et al., 2016b), showing a similar developmental pattern. The first stage of salivary gland morphogenesis shows only the initial thickening of the oral epithelium characterizing the prebud stage, which occurs at embryonic day 11.5 in mouse (E11.5) (Jaskoll & Melnick, 1999; Melnick & Jaskoll, 2000). Next, at the initial bud stage, a single bud-like structure is connected to the original oral epithelium by a solid stalk, surrounded by a highly condensed mesenchyme that contains the main signaling regulators that control the gland development (Figure 2, A1 to A3). Neural crest-derived precursors wrap around the epithelial stalks, which then coalesce to form the submandibular parasympathetic ganglia. The signals that initiate this neural-epithelial interaction have not been fully described yet (Jaskoll & Melnick, 1999; Jaskoll et al., 2002; Knox & Hoffman, 2008; Patel & Hoffman, 2014).



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Further at the pseudoglandular stage (Figure 2, B1 and B2), there are multiple epithelial buds (approximately 4 to 5 buds in the submandibular gland) associated with more developed stalks that start branching at the distal portions. Recently, it has been reported that lumen formation already starts at this stage by removing the epithelial cells from the centre of the solid stalks through programmed cell death apoptosis (Jaskoll & Melnick, 1999; Melnick & Jaskoll, 2000; Teshima et al., 2016a). In addition, other studies have shown the requirement of cell polarization for lumen formation at later stages and also that lumen expansion is mediated by electrolytic flow within developing ducts (Nedvetesky et al., 2014). Branching morphogenesis then progresses with the formation of new and extensive duct and bud structures (presumptive acini), characterizing the canalicular stage (Figure 2, C1 and C2). At this point, the majority of the presumptive ducts will develop the lumen (Jaskoll & Melnick, 1999; Melnick & Jaskoll, 2000). By the terminal bud stage (Figure 2, D1 and D2) the glands are extensively branched, showing differentiated terminal end buds and a presumptive ductal system with respective well-developed lumens (Melnick & Jaskoll, 2000). Myoepithelial cells are present.

Duct lumen formation is driven by apoptosis at early stages and is expanded by cell polarization – lessons from mouse research and human perspectives Mouse duct canalization and lumen expansion are remarkable processes at the pseudoglandular and canalicular stage (Tucker, 2007; Teshima et al., 2016a; Nedvetesky et al., 2014). It has been recently reported that early lumen formation is regulated by apoptosis of central cells within the solid epithelial stalk from E12.5, earlier than previously shown. After the initial lumen onset, the expression of apoptotic cells is substantially reduced at the same regions


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at E14.5, and tissue cultures have also shown no difference in duct formation in absence of apoptosis during this developmental stage (Teshima et al., 2016a). Cell polarization has also been reported to be required during lumen formation, and later lumen expansion was correlated with the presence of electrolytic fluid within the developing ducts (Nedvetesky et al., 2014). Although recent studies have produced growing data on the molecular mechanisms regulating the lumen formation and expansion of salivary glands, it is still intriguing whether mouse models can completely mimic human morphogenesis. This is based on previous findings where there were differences in apoptotic expression during lumen formation of human salivary glands, where the main executioner regulator seems to be caspase-7 instead of caspase-3, which is normally seen in mouse development. As these two regulators share the same function, the higher expression of caspase-7 and the absence of caspase-3 in the same specimens suggest more activity of caspase-7 in humans instead (Teshima et al, 2016b). Similarly, other evidence in human developing glands has shown that lumen space expansion and maintenance seems to be regulated by early secretion of glycoproteins Muc1 and Muc4 within the lumen throughout human development (Teshima et al., 2011). However, no specific correlation of mucin expression during mouse salivary gland development has yet been reported. Further studies are therefore required to investigate the particular differences between human and animal salivary gland development, aiming to understand whether they would be important to subsequent investigations.



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ACKNOWLEDGEMENTS The authors declare no potential conflicts of interest with respect to authorship and/or publication of this article. de Paula, F was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) from Brazil, and Teshima THN by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) – Grant 2011/18865-2; 2015/02824-6. This work has the support of the NAP-Saliva Research Group (School of Dentistry of University of Sao Paulo) coordinated by Dr Lourenço SV. We would like to acknowledge Fabio Andriolo for schematic figure of human salivary gland morphogenesis and Andrew Foye for help with revising the final manuscript.


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Katchburian E, Arana V. 2012. Histologia e Embriologia Oral. Texto, Atlas e Correlações Clinicas. 3° ed. rev. atual. Rio de Janeiro: Guanabara Koogan. Knox SM, Hoffman MP. 2008. Salivary gland development. Ames, IA: Blackwell Publication Knox SM, Lombaert IM, Reed X, Vitale-Cross L, Gutkind JS, Hoffman MP. 2010. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science. Sep 24;329(5999):1645-7. Ligtenberg AJ, Brand HS, van den Keijbus PA, Veerman EC. 2015. The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme. Arch Oral Biol. Nov;60(11):1639-44. Lourenço SV, Coutinho-Camillo CM, Buim ME, Uyekita SH, Soares FA. 2007. Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers. Histochem Cell Biol. Oct;128(4):361-9. Lourenço SV, Uyekita SH, Lima DM, Soares FA. 2008. Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation. Virchows Arch. Apr;452(4):427-34. Matsumoto K, Yoshitomi H, Rossant J, Zaret KS. 2001. Liver Organogenesis Promoted by Endothelial Cells Prior to Vascular Function. Science. Oct 19; 294(5542):559-63. Melnick M, Jaskoll T. 2000. Mouse submandibular gland morphogenesis: a paradigm for embryonic signal processing. Crit Rev Oral Biol Med. 11(2):199-215. Munger BL. 1964. Histochemical studies on seromucous- and mucous-secreting cells of human salivary glands. Am J Anat. Nov;115:411-29. Naesse EP, Schreurs O, Messelt E, Hayashi K, Schenck K. 2013. Distribution of nerve growth factor, pro-nerve growth factor, and their receptors in human salivary glands. Eur J Oral Sci. Feb;121(1):13-20. Nanci A. 2013. Ten Cate histologia oral: desenvolvimento, estrutura e função. 8. ed. Rio de Janeiro: Elsevier. Nedvetsky PI, Emmerson E, Finley JK, Ettinger A, Cruz-Pacheco N, Prochazka J, Haddox CL, Northrup E, Hodges C, Mostov KE, Hoffman MP, Knox SM. 2014. Parasympathetic innervation regulates tubulogenesis in the developing salivary gland. Dev Cell. Aug 25;30(4):449-62. Nelson, J., Manzella, K. & Baker, O.J., 2013a. Current cell models for bioengineering a salivary gland: A mini-review of emerging technologies. Oral Diseases, 19, pp.236–244. Nelson DA, Manhardt C, Kamath V, Sui Y, Santamaria-Pang A, Can A, Bello M, Corwin A, Dinn SR, Lazare M, Gervais EM, Sequeira SJ, Peters SB, Fiona Ginty F, Gerdes MJ, Larsen M. 2013b. Quantitative single cell analysis of cell population dynamics during



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FIGURE LEGENDS

Figure 1: Schematic figure of fully developed salivary gland showing detailed parenchymal cells and their respective transversal section Representative scheme illustrates a mixed secretory salivary gland composed of mucous and serous acini. Mucous acinar cells are highlighted as more elongated structures featuring peripheral compressed nuclei at the basal region due to high mucous production within the cytoplasm. Alternatively, serous acini are composed by triangular-shaped cells and round nuclei component responsible for secreting a more aqueous secretion. The detailed ductal network structure can also be observed where the proximal intercalated ducts are directly connected to the secretory units, showing a simpler wall structure as a single cuboidal epithelial layer. These are followed by striated ducts, which are functionally important to the gland for performing most of



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the ion exchange between the initial salivary fluid and the extracellular matrix through several membrane folding (striations) on their basal side. Finally, a calibrated and stratified epithelial collecting duct is connected to all parts of the gland, which is responsible for carrying the final secretion to the oral cavity. Myoepithelial cells are also represented in this scheme where their cellular extensions are wrapping around the end bud structures and striated ducts, contributing to expelling the salivary content to the ductal network.

Figure 2: schematic pattern of human salivary gland developmental stages and their respective histological examples A1 to A3: initial bud stage Solid proliferation of a single bud-like structure connected to the original epithelium by a solid stalk, invaginating towards a condensed mesenchyme (A1). In A2, note a similar histological aspect of a solid proliferation of basaloid cells invaginating into the mesenchyme from the oral epithelium; in A3, solid buds and the initial ductal lumen space can be observed at the final stages of the bud phase (H&E, original magnification X600 and X600). B1 and B2: pseudoglandular stage Schematic representation of epithelial buds connected to the main stalk, which starts cavitating (B1). In B2, note the extended solid cord of epithelial cells associated with bud proliferation and initial lumen formation (H&E, original magnification X400). C1 and C2: canalicular stage


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Schematic representation of branching morphogenesis progression with a developed luminal system and the presence of myoepithelial cells surrounding the end buds (C1). In C2, histological aspect of a developing human salivary gland with a well-established ductal system and end-buds. Heterogeneous developments can be observed at this stage, where some end buds are still solid and show a basaloid aspect while others already present initial acinar cell differentiation (clearcells). (H&E, original magnification X600). D1 and D2: terminal bud stage D1: Schematic representation of a well-developed salivary gland. Note the presence of acinar buds, myoepithelial cells, and distinct ductal segments (intercalated, striated and excretory ducts). In D2, histological sample of human salivary gland at the terminal bud stage, where the glands are already extensively branched and show differentiated terminal end buds (acinar units) (H&E, original magnification X600).



Figure 1: Schematic figure of fully developed salivary gland showing detailed parenchymal cells and their respective transversal section Representative scheme illustrates a mixed secretory salivary gland composed of mucous and serous acini. Mucous acinar cells are highlighted as more elongated structures featuring peripheral compressed nuclei at the basal region due to high mucous production within the cytoplasm. Alternatively, serous acini are composed by triangular-shaped cells and round nuclei component responsible for secreting a more aqueous secretion. Detailed ductal network structure can also be observed where the proximal intercalated ducts are directly connected to the secretory units, showing a simpler wall structure as a single cuboidal epithelial layer. These are followed by striated ducts, which are functionally important to the gland for performing most of ion exchange between the initial salivary fluid and the extracellular matrix through several membrane folding (striations) on their basal side. Finally, a calibrated and stratified epithelial collecting duct is connected to all parts of the gland, which will be responsible for carrying the final secretion to the oral cavity. Myoepithelial cells are also represented in this scheme where their cellular extensions are wrapping around the end bud structures and striated ducts, contributing to expel the salivary content to the ductal network. 105x86mm (600 x 600 DPI)


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Figure 2: schematic pattern of human salivary gland developmental stages and their respective histological examples A1 to A3: initial bud stage Solid proliferation of a single bud-like structure connected to the original epithelium by a solid stalk, invaginating towards a condensed mesenchyme (A1). In A2, note a similar histological aspect of a solid proliferation of basaloid cells invaginating into the mesenchyme from the oral epithelium; in A3, solid buds and initial ductal lumen space can be observed at the final stages of the bud phase (H&E, original magnification X600 and X600). B1 and B2: pseudoglandular stage Schematic representation of epithelial buds connected to the main stalk, which starts cavitating (B1). In B2, observe the extended solid cord of epithelial cells associated with bud proliferation and initial lumen formation (H&E, original magnification X400). C1 and C2: canalicular stage



Schematic representation of branching morphogenesis progression with a developed luminal system and the presence of myoepithelial cells surrounding the end buds (C1). In C2, histological aspect of a developing human salivary gland with a well-established ductal system and end-buds. Heterogeneous developments can be observed at this stage, where some end buds are still solid and show a basaloid aspect while others already present initial acinar cell differentiation (clear-cells). (H&E, original magnification X600). D1 and D2: terminal bud stage D1: Schematic representation of a well-developed salivary gland. Note the presence of acinar buds, myoepithelial cells, and distinct ductal segments (intercalated, striated and excretory ducts). In D2, histological sample of human salivary gland at terminal bud stage, where the glands are already extensively branched and show differentiated terminal end buds (acinar units) (H&E, original magnification X600). 250x312mm (300 x 300 DPI)


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Suggested cover image. Histological image of human minor salivary gland at late stage of development showing fully differentiated secetory structures associated with proximal ducts. 194x146mm (300 x 300 DPI)
