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Development and secretions of salivary glands using mouse models

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Development and secretions of salivary glands using mouse models

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original o r copy submitted. Thus, some thesis and dissertation copies are in typewriter free, while others may be from any type o f computer printer. The quality of this reproduction is dependent upon th e quality of the copy subm itted. Broken o r indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. 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DEVELOPMENT AND SECRETIONS OF SALIVARY GLANDS USING MOUSE MODELS by Jeffrey Michael Burstein A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment o f the Requirements for the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR BIOLOGY) August 1997 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UMI Number: 1387814 Copyright 1998 by Burstein, Jeffrey Michael All rights reserved. i i UMI Microform 1387814 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UNIVERSITY O F SOUTHERN CALIFORNIA T H E GRADUATE SCHOOL. UNIVERSITY PARK LOS A N G ELES. CALIFORNIA 9 0 0 0 7 This thesis, written by Jeffrey Michael Burstein__________ under the direction of hi§. Thesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of Master of Science THESIS COMMITTEE/^ Cksi rmmn R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TABLE OF CONTENTS A bstract » i Introduction 1 M aterials and M ethods 22 Results 39 Discussion 44 The Future 48 Figures 51 Bibliography 73 f i » t I I \ f « 1 I L i i R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Jeffrey Bnrstein Student Tnltan Tokes Department Chairman DEVELOPMENT AND SECRETIONS OF SALIVARY GLANDS PS1NG MOUSE MODELS Abstract: The von Ebner’s gland (VEG), a minor salivary gland that directly bathes the taste buds, secretes proteins believed to mediate taste transduction. This thesis provides evidence by Northern analysis that transcripts encoding a VEG protein (T61) are initially detected at day 6 in mice, and expression increases as the animal matures. This is consistent with the timing of development of other taste machinery and is further evidence that this protein is involved in taste reception. The transcription factor Msx2’s role in submandibular gland development was examined by comparing glands from normal mice expressing Msx2 endogenously to transgenic mice (CMVMsx2) overexpressing the Msx2 gene. No differences were observed in lobule size or number between the two animals. Northern blot analysis failed to detect Msx2 mRNA in the submandibular glands suggesting low levels of Msx2 expression. No differences were seen between the transgenic and normal mice suggesting that Msx2 may not play a critical role in submandibular gland development. i i i R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. INTRODUCTION von Ebner’s Gland Protein Involved In Taste Transduction Considering the fundamental value of taste to human health and preventative medicine, it is quite surprising that, until recently, little research has been done on the physiology of taste and the mechanisms of taste reception. The recent surge in research concerning the gustatory sense has produced a great deal of knowledge about the . processes involved in taste, the function and mechanisms of the von Ebner’s gland (VEG), and the proteins which it secretes. This recent surge has been catalyzed by developmental anomalies in taste machinery, drugs which effect our taste reception, and the decrease in stimulus intensity in aging patients. The factors responsible for these ( • S clinical phenomena cannot be understood until the structure and mechanism by which the § taste system operates is better defined. { S von Ebner’ s Gland Secretes a Unique Protein Designated T61 I i Saliva is the first digestive fluid secreted by the gastrointestinal pathway and u about 90% is produced and secreted into the oral cavity by the parotid, sublingual, and ; submandibular glands. The other 10% is produced by minor salivary glands such as the I von Ebner’s gland which is located inferior to the circumvallate papillae in the posterior of the tongue. The von Ebner’s gland secretes lipases and other proteins, including a 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. unique von Ebner’s gland protein which Snead and colleagues (unpublished) have cloned and localized to the secretory ducts of the gland. This protein will be referred to as VEG protein of T61 or just T61, relating to the cDNA clone isolated. Comparisons of the amino acid sequence of this VEG protein with a protein superfamily of lipophilic molecule carriers which bind and transport small hydrophobic molecules reveals a high homology (Buck and Axel, 1991). The amino acid sequence specifically shows over 32% homology with a rat olfactory receptor protein and weak homology with the Bombex mori pheromone binding protein as well (Figure 1). The homology between the translated sequence of this VEG protein and lipophilic molecule carriers reveals that the VEG protein may have a role in binding and transporting lipophilic gustatory molecules to taste receptors in the tongue. On the other hand, this protein may take on the opposite role of transporting lipophilic tastants out of the circumvallate and foliate papillae so that receptors are ready to accept new taste stimuli. In order to review our current understanding of how this VEG protein may regulate taste stimuli, it is critical to understand how the von Ebner’s gland anatomy, structure and secretions play an integral role in the overall mechanism involved in taste reception. Mechanisms & Principles Involved in Taste Reception The primary function of the sense of taste is to evaluate food for nutritional or deleterious content. The taste system also plays a crucial role in salivation and R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. swallowing, reflexes such as ingestion and egestion, as well as mediating conscious taste sensations. These taste sensations are the result of four different stimuli triggering specific sensory taste centers on the tongue (Frank and Hettinger, 1992). The four taste stimuli are sweet, sour, salty, and bitter, and each is putatively mediated by a distinct receptor (Kinnamon, 1988). However, if each of these four taste stimuli have their own receptor, does each taste bud express one or multiple receptors? The ordering of receptors on the epithelium might determine patterning information during development and regulate taste receptor signals. A better understanding of the gustatory epithelium which detects these taste stimulants is essential. The gustatory epithelium lines both the circumvallate papillae in the posterior of the tongue as well as the taste buds within these papillae (Frank and Hettinger, 1992). I First discovered in the oral cavity of fish (Leydig, 1851), taste buds of varied shapes were later associated with the foliate and vallate papillae of different mammalian species (Schwalbe, 1867). Taste buds, which contain taste receptors inside, are clustered into four distinct sensory fields in the oral cavity. In the anterior lingual field, although not ! ; heavily concentrated, taste buds are present at the tip of the tongue (Frank and Hettinger, 1992). In the palatal field containing the stratified squamous and columnar epithelia lining the soft palate, the few taste buds present are not associated with any papillae (Frank and Hettinger, 1992). Although the physiologic roles and structures of these taste buds have not been extensively studied, they represent a unique opportunity to study the function of chemical receptor cells in the oral cavity. While isolated from the von 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Ebner’s gland, the palatal and anterior taste buds merit study to compare their sensory detection of taste with taste buds of the foliate and circumvallate papillae, the third and fourth taste sensory fields, respectively. A more detailed morphological analysis of taste buds in the papillae of the spherical organ that is structurally similar in all vertebrates (Frank and Hettinger, 1992). These spherical organs are comprised of dark cells containing secretory granules as well as less numerous light cells thought to be involved in the direct transduction of taste stimuli (Kinnamon, 1987). At the base of the taste buds are basal stem cells, which are a supposed source for the continuous generation of taste bud cells which have a maximum these cells which form the taste bud, extends beyond the surface of the epithelium to form the taste pore, a structure largely comprised of mucus (Frank and Hettinger, 1992). These taste pores represent the initial site of tastant entry and the first site of sensory transduction of taste stimuli. There are probably many mechanisms for the transduction of taste stimuli (Figure 2). One theory divides specific chemical stimuli into ionic and nonionic signals. Ionic taste stimuli (salty and sour stimulants) are believed to transduce direcdy through ion channels, while nonionic taste stimuli (sweet and bitter stimulants) are believed to bind to protein receptors or the cell membranes of receptor cells (Teeter and Brand, 1987; to one hundred cells forming a \ life of ten days (Beidler and Smallman, 1965; Delay et al., 1986). In addition, the apex of 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Kinnamon, 1988). Once the stimulus has penetrated the taste bud, primary afferent (sensory) neurons utilize cranial nerves to project these stimuli into the brain stem (Kinnamon, 1988). For example, the glassopharyngeal nerve, which is discussed later, is responsible for transmitting nerve signals from the posterior region of the tongue (i.e., von Ebner’s gland). How are these primary taste stimulants removed so that the limited number of receptors are free to receive new tastants? Cellular Anatomy o f the von Ebner’ s Gland One predominant theory is that the von Ebner’s gland secretions play a critical role in “washing out taste substances from the trough and readying the taste receptors in 1 the walls of the papilla for new stimuli” (Hand, 1970). This tubulo-acinar serous gland, I ! located directly beneath the circumvallate and foliate papillae of the tongue, was first described by von Ebner (1873). More recendy, scanning and transmission electron r ' I microscopy was used to characterize the predominant secretory and ductal cells within the gland as well as their subcellular components. The secretory cells of the von Ebner’s j gland surround a central lumen, which is part of a long canal or groove extending between the cells of the gland (Figure 3 A). These secretory cells which line the acini k (pyramidal cells) or tubuli (cuboidal cells), contain thin lateral folds which help them to interlock with neighboring cells. On a subcellular level, the Golgi apparatus contains stacks of saccules, vesicles, tubules, and condensing vacuoles (Riva et al., 198S). The t rough endoplasmic redculum is well organized at the base of the cell while randomly R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. arranged at the apex (Riva et al., 1985). Also located predominantly near the apex of these cells are secretory granules which undergo exocytosis by fusing with the plasma membrane (Riva et al., 1985). The complex structure of the golgi, endoplasmic reticulum, and secretory granules are crucial not only for understanding the anatomy of the secretory cells, but suggest the presence of complex protein making machinery, which support the initial observations by Hamosh and Bums (1977) that the von Ebner’s gland was involved in protein secretion. The second major group of cells within the von Ebner’s gland are ductal in nature and line the ducts which open into the trough at the base of the circumvallate papillae (Figure 3 A). Morphologically tall, cuboidal or columnar, ductal cells contain desmosomes on both their lateral and basal surfaces helping to connect them to other ductal cells as well as to myoepithelial elements (Hand, 1970). Characterized by a well developed Golgi apparatus, ductal cells are rich in mitochondria and contain only a small amount of secretory granules scattered throughout their apex (Riva et al., 1985). Although these cells contain a large amount of mitochondria in addition to a high degree of lateral infolding, they differ from the major salivary glands in lacking striations (Riva et al., 1985). Striated ducts, responsible for adding a secretory product from the major salivary glands to the final saliva mixture, are believed to be necessary to contribute to the saliva modification process (Riva et al., 1985). This therefore leaves unanswered the mechanism by which the von Ebner’s gland contributes components to the saliva. Perhaps, myoepithelial cells surrounding the complex, and the well developed secretory 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. and ductal cells, work together to secrete a product which is added to the final saliva mixture and contributes to taste reception as well as the enzymatic breakdown of food products. Understanding the role of the von Ebner’s gland in taste reception will give us a clearer picture of its mechanism of salivary secretion. VEG Protein’ s Role in Taste Reception The role the von Ebner’s gland plays in taste reception has been studied in rats and mice. In these mammals, the gland appears as a triangular mass in the posterior of the tongue with the circumvallate papillae located at the apex of the triangle and the foliate papillae at the ends of the base. As the majority of the lingual taste buds are located in the circumvallate and foliate papillae (Miller, 1977), there is no doubt that the von Ebner’s gland secretions which drain into the troughs of these papillae have a role in the transduction of taste stimuli to taste receptor cells. Gurkan and Bradley (1988) suggest that the role the salivary secretions of the von Ebner’s gland play in taste reception closely parallels the function of the nasal mucus of the Bowman’s gland. To examine the extent the von Ebner’s gland mediates taste reception, Gurkan and Bradley (1988) measured the effects of von Ebner gland secretions on taste reception in rats. In the first experiment, chemical stimuli (Ammonium Chloride, citric acid, Potassium Chloride and Sodium Chloride) were applied to the circumvallate papillae of rats while access to the foliate papilla was blocked with cryanoacrylate cement painted into the clefts of the papilla. Glossopharyngeal nerve stimulation, which corresponds to R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. taste stimulus response in the posterior region of the tongue (Gurkan and Bradley, 1988), was electrically monitored as the normal responses to these four chemical stimuli were measured. The mice were then injected with the parasympathetic antagonist atropine which blocks salivary secretions. The results demonstrated that normal salivary secretion are able to reduce the taste response and prepare the taste buds for the next stimulus. In analyzing the data, Gurkan and Bradley (1988) were quick to point out that salivary secretions effected different stimuli by different magnitudes. They concluded (Gurkan and Bradley, 1988) that the different magnitudes of stimuli reduction signified that the chemical stimuli were not just diluted by the salivary secretions of the von Ebner’s gland, but rather specific factors released from the von Ebner’s gland were controlling the transduction of chemical taste stimuli before the stimulants activated receptor ceils. The unique von Ebner’s gland protein (T61) isolated by Snead and colleagues may possibly play a role in mediating taste reception. It’s homology to sequences of other lipophilic binding proteins, as well as immunohistochemical and in situ experiments revealing its expression in the secretory granules of the gland, have led to further investigation into the possibility that this protein may play an integral role in taste reception. In addition, northern blot analysis reveals (to be discussed below) that the first detectable mRNA transcripts coding for the von Ebner gland protein are detectable in 6 day post natal mice with levels increasing into adulthood. This is consistent with the time course of the development of taste adnexa necessary to assess the nutritional or 8 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. deleterious content of food and can be considered further evidence that this protein is likely involved in taste reception. As a model for understanding the development of this minor salivary gland, the next phase of my thesis project involved the development and regulation of the submandibular gland by the transcription factor Msx2. Since this major salivary gland has been well characterized, it posed as a model for studying how increased expression of this transcription factor would alter development Submandibular Gland Development In attempting to determine how the transcription factor Msx2 regulates submandibular gland morphogenesis, an understanding of the embryological development and histology of the salivary glands as well as the functions of the products which it secretes is required. Only after realizing how Msx2 may regulate salivary gland development can we begin to look at its clinical value in promoting or preventing developmental anomalies. Morphogenesis of the submandibular gland involves a series of epithelial mesenchymal interactions (Screebny, 1987) which are defined by the following stages: first the initiation, then the branching, and then the lumenization of the epithelium 9 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. followed by the cytodifferentiation of cells within the salivary gland. In mice, differentiation extends into post-natal life. (Initiation - Day 12) Initiation of salivary gland morphogenesis, which normally occurs at the end of 12 days post-conception, is characterized by a focal thickening of the primitive oral epithelium as it migrates into adjacent mesenchymal tissue (Figure 4A). As the cluster of epithelial cells is enveloped, the surrounding mesenchymal cells, while rapidly proliferating, begin to condense around the migrating epithelium. This is soon followed by the rapidly dividing epithelial cells pushing further into the mesenchyme, forming a terminal bulb and a trailing cord consisting of a solid web of epithelial cells (Figure 4B; Screebny, 1987). Soon thereafter, the spherical terminal bulb takes shape during the branching phase. C Branching - Day 13) Branching morphogenesis of the submandibular gland direcdy follows initiation in properly developing submandibular glands, and is characterized by the formation of clefts in the originally spherical terminal lobule (Figure 4Q. Multiple bulbs, which develop from the original terminal bulb, migrate further into the surrounding mesenchyme while connected to an epithelial cord still attached to the original stalk (Screebny, 1987). Each new terminal bulb continues to proliferate, giving rise to 10 perm ission of the copyright owner. Further reproduction prohibited without perm ission. successive generations of terminal bulbs and cords forming a solid epithelial web held together through tight junctions, desmosomes, and microfilaments (Screebny, 1987). Initiation and branching morphogenesis, leading to the eventual development of functional salivary glands, would not be possible without the intercellular signaling between the expanding epithelium and the surrounding mesenchyme. In fact, without mesenchyme, the epithelium would merely spread out and undergo small amounts of aberrant forms of branching (Fleming, 1992). Furthermore, tissue recombination experiments have demonstrated that when submandibular gland mesenchyme is mixed with mammary and lung epithelium, the mesenchyme is able to induce submandibular gland like branching patterns (Spooner and Wessels, 1970). These experiments demonstrated that the mesenchyme was exerting a strong influence over the epithelium during the initiation and branching stages of salivary gland morphogenesis. It is also quite possible that Bone Morphogenic Protein (BMP) is inducing tissue specific expression of the Msx transcription factors involved in development, as is the case in tooth morphogenesis. (Jowett et al., 1993). (Lumenization - Day 13) Following the branching of the lobules, apoptosis, or programmed cell death of the interior epithelial cells occurs resulting in an internal lumen in the main stalk and eventually throughout the entire salivary gland (Figure 4D). The formation of the lumen in the terminal bulbs is accompanied by a deepening of the peripheral clefts to form more 11 perm ission of the copyright owner. Further reproduction prohibited without perm ission. defined spherical terminal lobules (Figure 4E). Initiation, branching, and lumenization of the stalk and bulbs, signify the first step in the differentiation of the primitive salivary rudiments into a functional gland. The cytodifferentiation of the undifferentiated cells in the terminal bulbs and cord is the next step in the development of the submandibular glands. (Cytodifferentiation - Day 13 - Post Natal Life) The differentiation of the lobular epithelial cells into secretory acinar cells and the epithelial cord cells into ductal cells occur during cytodifferentiation (Figure 4F). In the terminal bulbs, the cells lining the lumen differentiate into tubuloacinar cells while cells in the periphery (on the exterior of the lobules) differentiate into myoepithelial cells. The formation of acinar cells is characterized by a strong increase in the rough endoplasmic reticulum and the production and synthesis of secretory granules from the Golgi apparatus (Screebny, 1987). Simultaneously, the epithelial cells in the stalk elongate to form tall columnar cells lined with numerous mitochondria that together form the excretory, striated, and intercalated ducts (Figure 5). Submandibular Gland Anatomy/Histology These salivary glands appear as lobules separated from each other by mesenchymally derived connective tissue. The acini are located in the terminal lobules at the tips of the intercalated ducts. After passing through the intercalated ducts, salivary 12 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. secretions pass through the striated ducts and into the excretory ducts from where they join a large tubule which empties into the oral cavity. Acini Acini are comprised o f pyramidal cells clustered around a small central lumen established during development These acini can be distinguished as serous or mucous depending on the excretory products they synthesize and secrete(Rankow and Polayes, 1976). While serous cells are predominantly found in the parotid and von Ebner’s glands, mucous cells are found in the three major salivary glands. In addition, these acini are separated from the surrounding connective tissue by a basal lamina as well as by myoepithelial cells on the periphery of the gland. Cholinergic and adrenergic nerve terminals enervating the acinar cells have been found between the secretory (acinar) cells and the outer myoepithelial cells (Rankow and Polayes, 1976). Ducts Secretions from acinar cells first pass through the intercalated ducts (Figure S) formed by a single layer of cuboidal cells. These ducts are not believed to play a major role in modifying the saliva as it passes into the striated duct. These ducts, composed of a single layer of columnar cells, are important in removing Sodium (Na*) and Chloride (Cf) ions as well as water from the salivary secretions while concurrently adding components such as Potassium (K*)(Figure 6). The secretions then enter into the 13 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. excretory ducts consisting of several layers of cuboidal and squamous epithelial ceils. These ducts also have a role in absorbing Na+ ions while adding K+ cations to the salivary secretions in order to help convert saliva to its hypotonic end product (Kinnamon, 1976) Function o f Submandibular Gland Secretions In order to understand why congenital defects in salivary development cause various oral diseases, the normal secretion and composition of saliva as well as the role it plays in maintaining a healthy oral environment must be understood. Saliva Production and Composition Saliva production is thought to occur by a two step process. In the first step, an isotonic electrolyte solution is produced by the acinar cells, while in the second step, ions such as Na+ and Cl' are absorbed while passing through the ductal system leaving a hypotonic end product (Screebny, 1987) (Fig 6). This hypotonic endproduct consists of a mixture of both inorganic and organic substances consisting of electrolytes (Na+ and Cl*), enzymes (e.g., salivary amylase), and other glycoproteins (mucins) and vitamins. In addition, there are a number of factors one must consider when trying to evaluate the various products released in saliva. Factors such as diet, circadian rhythms, age, sex, flow rate and the size of the salivary gland can all effect the composition of salivary secretions. For this reason, when using immunohistochemisty to measure protein 14 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. secretions from salivary glands in mice, it is essential that pregnant mothers are bred under as regimented and controlled o f a schedule as possible. Function o f Saliva The enzyme components of salivary secretion play an integral role in aiding the digestion of food products in addition to acting as an anti-microbial agent to maintain a strong healthy bacteria free oral cavity. Enzymes such as salivary amylase, which breakdown starch into the oligosacharide molecules, are secreted from the serous acinar cells in the lobules of the submandibular gland (Berne & Levy, 1996). Another primary function of saliva involves maintaining the integrity of the mucous membrane in both oral and oral pharyngeal regions as well as maintaining the health of the teeth and tongue. An I integral component to this function is the glycoprotein mucin secreted from mucous f | acinar cells. Mucin plays a major role in lubricating food particles to aid in mastication | and swallowing as well as in protecting the oral epithelium from irritants such as the | hydrolytic and proteolytic enzymes produced in plaque, carcinogens (from smoking by ’ products) and breathing (Rankow & Polayes, 1976). Mucin glycoproteins also help r i prevent tooth destruction by coating the teeth to prevent constant abrasion during mastication (Rankow & Polayes, 1976). The constant flow of saliva aids in removing food and bacterial debris for elimination by the digestive tract. Finally, one of the most crucial components of the submandibular gland secretion are the antibacterial components IgA and lysozyme. Immunoglobulin A is secreted into submandibular gland 15 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. saliva via sinusoidal capillaries innervating the connective tissue surrounding the ducts (Mason & Chisholm, 1975). This antibody, directed at specific bacteria present throughout the mucus coating of the oral tissue, will agglutinate the invading bacteria. Lysozyme, another antibacterial agent and being non-specific (Rankow and Polayes, 1976), is secreted into the striated ducts. This enzyme is able to hydrolyze glycopeptides containing muramic acid which are present in the bacterial cell walls (Rankow and Polayes, 1976). Together, IgA and lysozyme play major roles in defending against bacterial infection and caries. Developmental Anomalies leading to Salivary Gland Disease Understanding the mechanisms by which salivary glands mature is essential to understanding which factors play a role in developmental irregularities. Some congenital defects of salivary glands include: the agenesis of the glands, the absence of ducts, congenital fistula and the presence of salivary tissue in abnormal areas. Agenesis of the major salivary glands involves the congenital absence of either one or both of the symmetrical glands (Mason & Chisholm, 1975) leading to xerostomia which in turn leads to caries formation. In salivary gland atresia, which occurs most often in the submandibular and sublingual gland (Mason and Chisholm, 1977), congenital occlusion or absence of at least one major salivary gland duct occurs. Clinically, this leads to severe xerostomia and possibly to the retention of cyst formation (Mason and Chisholm, 1977). Congenital fistula formation of the ductal system involves the abnormal formation 16 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. of branchial clefts (Mason & Chisholm, 1977), while aberrancey involves the presence of salivary rudiments at abnormal sites, such as within the body of the mandible (Mason and Chisholm, 1975). Although all are quite rare, these congenital anomalies effecting salivary gland development may be due to errors in the signaling cascade responsible for epithelial mesenchymal tissue development. The transcription factor Msx2 could interact in the signaling cascade to play a regulatory role in salivary gland development by a similar mechanism by which it regulates development of craniofacial structures such as the sutures (Jabs et al, 1993). It has recently been shown that premature closure of the sutures and ectopic bone growth occurs in mice with a mutation in the homeodomain of Msx2 (Liu et al., 1995). The mutation in the functional homeodomain of Msx2 demonstrates its importance in development and brought us one step closer to ? understanding the molecular processes that Msx2 regulates. The structure, patterns of expression and possible functions of Msx2 are discussed in greater detail below. I i I s MSX2 REGULATION OF SUBMANDIBULAR GLAND DEVELOPMENT ! A combination of growth factors, their receptors and transcriptional regulators are expressed in tissues undergoing morphogenesis via epithelial mesenchymal interactions. i The homeodomain containing transcription factor Msx2, is an example of one of the 17 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. more evolutionary conserved DNA binding proteins which regulates the expression of genes involved in development. Utilizing the CMV promoter to non-specifically increase Msx2 levels in mice, we can attempt to characterize the regulatory role of Msx2 in major salivary glands. In order to characterize the function of Msx2 in submandibular gland development, it is first necessary to understand the structure, patterns of expression, and probable function of the transcription factor Msx2. Structure o f Msx2 The isolation of Msx2 from the homeobox containing Msh (muscle segmenting homeobox gene) gene family signified the potential discovery of yet another gene thought F to play a key role in the developmental process. Goned out of a mouse genomic library using degenerate primers for the conserved homeobox domain found in the Msh gene i superfamily, Msx2 is comprised of 2 exons separated by a 3.5 kb intron (Bell et al., 1993) ; (Figure 7). Exons 1 and 2 are 600 bp and 691 bp long respectively, with the 180 bp (60 ! | Amino Acid) homeodomain present in exon 2 (Bell et al., 1993). \ Homeodomain f I Transcription factors regulate cellular development, differentiation, and growth by interacting with targeted DNA binding sites of a gene promoter thus regulating or \ | influencing gene expression. There are many structures within these DNA binding ! proteins which allow transcription factors to bind and regulate expression. Transcription 18 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. factors are often characterized by binding structures such as zinc fingers and leucine zippers; the transcription factor Msx2 is characterized by its homeodomain. The homeodomain is based on the helix-tum-helix (HTH) motif, part of many prokaryotic DNA-binding proteins. In addition to the helix-tum-helix motif, the 60 amino acid homeodomain contains a third helix perpendicular to the first two which provides intrinsic stability to the homeodomain. One of the better analyzed homeodomain complexes has been observed through the crystal structure of the complex from Drosophilia (Figure 8). The homeodomain structure involves helix I and 2 in antiparallel arrangement, perpendicular to the homeodomain containing 3rd helix which packs its hydrophobic face against the first two helices, forming the interior foundation of the protein. The homeodomain contacts the DNA primarily through amino acids in helix 3, which recognize the common recognition element 5’TAAT 3* in the major groove of the DNA. There are still many unanswered questions concerning accessory proteins which might play a role in recognizing and regulating the binding of transcription factors to DNA, and without the crystal structure of the Msx2 homeodomain, the best manner of characterizing Msx2 is perhaps by examining its patterns of expression. Patterns ofMsx2 Gene Expression Perhaps the most convincing evidence that Msx2 plays a role in regulating epithelial mesenchymal tissue interactions is by examining its patterns of expression during embryogenesis. In mice, it is found to be heavily expressed in the premigratory 19 perm ission of the copyright owner. Further reproduction prohibited without perm ission. neural crest, neural crest derived structures of the skull and face (Liu et al., 1995; Jabs et al., 1993), limbs, in epithelial mesenchymal interactions during tooth induction (Jowett (1993), and most recently in both the epithelium and mesenchyme of the submandibular gland (Malcolm Snead and colleagues, unpublished data). Defining the Function ofM sx2 There are many different ways to examine the function of a gene after determining its expression pattern. One possible model involves eliminating a functional domain of a gene and reintroducing it to the genome through homologous recombination. A second method, which we utilized here, involves overexpressing the gene in order to identify how excessive amounts of the protein alter development. This alteration can be used as a first step in determining the path by which a transcription factor such as Msx2 regulates transcriptional mechanisms. To examine how Msx2 regulates development, submandibular glands from normal mice were compared with those containing CMVMsx2 (Figure 9A) randomly inserted into the genome (Figure 9B). The CMV promoter, from a fragment of the early CMV promoter, will continually express the gene to which it is ligated. The constituitively high expression of Msx2 will increase levels of Msx2 expression such as it does in the sutures (Liu et al, 1995) and skin (Kundru, unpublished data). Therefore, the possible role Msx2 is playing in development could be ascertained by comparing the phenotype of mice overexpressing the gene to normal mice. 20 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The focus of my research was to characterize the expression of Msx2 in the developing submandibular glands in order to better understand its role in development. Understanding the molecular processes by which salivary glands develop will not only aid our knowledge and appreciation for the basis of human congenital malformation, but may also play a role in a greater understanding of other biological processes such as oncogenesis and tissue repair. Ascertaining the role transcription factors such as Msx2 play in the developmental process could also eventually help devise treatment protocols for various diseases. 21 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. MATERIALS AND METHODS Tissue Dissection for RNA Extraction: (von Elmer gland experiment) To determine the postnatal day upon which the message for the von Ebner gland protein is initially expressed, the von Ebner* s gland inferior to the circumvallate papillae in the posterior region o f the tongue was microscopically dissected. To correctly establish the time onset of expression, von Ebner gland tissue from 2-, 4>, 6-, 8-, 10-, 12- and 14-day post natal mice were dissected and frozen immediately in liquid nitrogen and < ■ k \ then stored at -70 °C until needed. Dissections of all tissue for RNA extraction was 1 always performed using RNAse-free, sterile instruments, and upon removal the tissue was immediately placed in RNAzol (Tel-Test, Inc., Friendswood, TX) for RNA extraction or \ frozen at -70 °C until needed. ! i (Msx2 experiment) | Extensive analysis of the embryonic expression patterns of Msx2 has revealed f that the developing cranium and underlying neural tissue express high levels of Msx2 f I transcripts (Liu et al., 1995). Therefore the superior half of the cranium from normal Swiss-Webster embryonic mice E16 (E0 signifying day of conception) through gestation ^ were used as positive controls for the detection o f Msx2 mRNA in the submandibular ( gland through Northern blot analysis. 22 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . To look for the endogenous expression o f Msx2 in the submandibular glands, these salivary glands were dissected from normal Swiss-Webster E16.5 mice. Microsdissection o f the mouse’s craniofacial cavity, with the removal of surrounding tissue from the submandibular glands, was required to microscopically dissect these salivary glands. In order to examine how Msx2 is regulating developmental processes in the submandibular gland, a mouse line bearing a copy o f the gene CMVMsx2 (which overexpresses Msx2) was used. To compare the phenotypic expression of Msx2 in wild type mice with those expressing the transgene, the submandibular glands of CMVMsx2 | transgenic mice (see determination of transgenic mice in Materials & Methods) were f c microdissected from E14,E15,16 and E17 mice, placed into an eppendorf tube, and r: [ stored in 4% paraformaldehyde until their transgenic status had been ascertained through tail blot analysis. Once the transgene status had been determined, the number of lobules as well as the area of the cross sectional area of these lobules, was determined and | compared. ? I RNA Extraction: | RNA extraction for all tissue was performed under similar conditions. After the | tissue was placed in RNAzol (2mL RNAzol/100 mg tissue) it was homogenized (Biospecs products, Inc. homogenizer) and 0.2 volumes of chloroform was added to every 1 volume of homogenate. After vigorous shaking o f the mixture for 15 seconds and 23 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . chilling on ice for 5 minutes, the samples were centrifuged at 12,000g for 15 minutes at 4 °C. The upper aqueous layer containing the RNA from the tissue was then removed, careful to leave behind the DNA and proteins present in the interphase and organic phase. To precipitate the RNA, an equal volume o f isopropanol was then added to this aqueous phase, and the mixture was stored for 15 minutes on ice and then centrifuged at 12,000g (4 °C) to pellet the RNA. The supernatant was removed and the pellet was washed with 70 % ethanol and then recentrifuged for 5 minutes at 7,500g. At this point the pellet was either stored at -70 °C under the 70 % ethanol used to wash it, or the Ethanol was removed allowing the pellet to dry. Once dry, the pellet was resuspended in RNAse free DEPC (Diethyl Pyrocarbonate) treated water (water treated with 0.1% DEPC). (von Ebner’ s Gland experiment) RNA was extracted from 2,4,6,8,10,12,14 days post natal mice in order to determine at what stage of development the messenger RNA for the von Ebner gland protein was being expressed. In addition, RNA from a gland which was from a 33 day post natal mouse was used as a positive control for the VEG message, and RNA from the brain and intestines were used as negative controls. (Submandibular Gland Experiment) RNA was extracted from 2,5,10 and 20 glands of normal E16.5 Swiss-Webster mice in order to determine the basal levels needed to detect endogenous Msx2 expression. The minimum amount o f submandibular glands needed to detect endogenous Msx2 24 p erm issio n o f th e cop yright ow ner. Further reproduction prohibited w ithout p erm issio n . 1 ^1 ) 1 ■ ■ * > ■ » KiiiP , - ■ , ■ expression were then used to compare Msx2 expression from wild type E l6.5 mice with the same number o f submandibular glands from CMVMsx2 transgenic E l6.5 mice. DNA Extraction (to determine whether the transgene CMVMsx2 was incorporated into the embryo’s genome) The cytomegalovirus promoter (CMV) is constitutively active as a promoter in viral DNA sequences. This promoter was inserted 5’ to the Msx2 gene and the construct (Figure 9A) was inserted into the pronuclei of fertilized mouse eggs (figure 9B) where it randomly incorporated into the mouse’s genome (Liu et al, 1995). These mouse eggs were then implanted into the uterus o f a pseudo-pregnant female which was mated with a sterile male. Offspring which caned this exogenous copy of Msx2, continually expressed by the CMV promoter and showed overexpression of Msx2 activity in tissue which endogenously express Msx2 (Liu et al, 1995); Ramen, unpublished data). Therefore using the CMV promoter to drive Msx2 expression, we could examine what developmental factors are effected by overexpressing Msx2. My experiments involved the offspring o f heterozygous males many generations removed from the original female in which the transgene was introduced. The transgene status of the offspring was determined by taking a fragment of the embryo’s tail and incubating it overnight at 55 °C in digestion solution (50 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS + Proteinase K (10mg/ml)). In the morning, the DNA was extracted by adding an equal volume of phenol to the digested tail solution, shaking gently for 3 25 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . minutes and centrifuging for 3 minutes at 14,000 rpm. The supernatant was transported to a fresh tube and an equal volume o f phenolrchloroform (1:1 mix) was added. The mixture was again shaken and centrifuged. The supernatant was removed and placed in a new tube, to which an equal volume o f ethanol was added to precipitate the DNA. After centrifugation for 5 minutes at 14,000 rpm, the ethanol was removed and the pellet was washed in 70% ethanol, then air dried and resuspended in TE buffer (10 mM Tris-Cl (pH 7.4), I mM EDTA (pH 8)). To determine whether CMVMsx2 was incorporated into the genome, EcoRl was used to cut the genomic DNA, so that a probe for CMV could detect the presence of the CMV promoter within the animal’s genome. f j Spectrophotomic Determination of Nucleic Acid Concentration: | To determine the quantity o f RNA recovered from tissue, spectrophotomic measurements (Beckman DU 640 Spectrophotometer) were taken from small volumes of nucleic acid sample. Cuvettes were progressively filled with increasing amounts of nucleic acid volumes (5ul, lOul, 15ul) as spectrophotomic optical density (OD) measurements (ultraviolet wavelengths o f260 nm and 280 am) were recorded. The OD reading at 260 nm allows for the calculation o f the concentration o f nucleic acids in the sample. A measurement of 1 OD corresponds to around 40 ug/ml for single stranded RNA and DNA, and 50 ug/ml for double stranded DNA. The ratio of OD260/OD280 should be in the range between 1.8-2.0 if there is no contamination from protein or phenol used during extraction o f the nucleic acid. 26 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . Formaldehvde-Agarose Gel Electrophoresis: Extracted RNA samples from both submandibular glands and the von Ebner glands were size separated on formaldehyde-agarose gels (1% agarose, 3.5% formaldehyde, 1 X formaldehyde running buffer (0.1 M 3-(N-morpholino) propanesulfonic acid (pH 7), 40 mM sodium acetate, 5 mM EDTA (pH 8.0)), 5 ul ethydium bromide). Prior to loading the gel, RNA samples were mixed with an equal volume o f 2 X RNA loading buffer (stock:7.2ml formamide, 1.6 m l 10X MOPS, 2.6 ml formaldehyde, 1.8 ml H2O) and incubated at 65 °C. After 15 minutes the samples were chilled on ice and rapidly centrifuged, before adding 2 pi of formaldehyde gel loading buffer (50% glycerol, 1 mM EDTA (pH 8.0), 0.25 % bromophenol blue, 0.25 % xylene cyanol). Samples were loaded in to the gel submerged in 1 X formaldehyde running buffer, and run at approximately 75 V until desired separation of the 18S and 28S bands could be visualized after which the gel was photographed. The separated nucleic acids were then transferred to nitrocellulose in order to detect messenger RNA from the donor tissue. Capillary Transfer of Nucleic Acids to Nvlon Filters: Briefly, in capillary transfer, nucleic acid fragments migrate from the gel and are deposited on the surface of the nylon solid support. Fluid is drawn from both the gel and the surrounding 20 X SSC (Sodium Chloride/Sodium Citrate (Sambrook et al) reservoir, by capillary action maintained by a stack of dry paper towels on top of the filter, with the 27 p erm issio n o f th e cop yright ow n er. Further reproduction prohibited w ithout p erm issio n . rate o f transfer being dependent on the size of the nucleic acid fragments and the concentration o f agarose in the gel. (Capillary Transfer o f RNA) Following two 20 minute washes in 20 X SSC and 2 rinses in DEPC treated water to remove the formaldehyde from the gel, the RNA was efficiently transferred to a nylon membrane. The gel was inverted and placed on top o f a piece o f Whatman 3 MM paper, which extended over the plexiglass supporting the gel, and into the surrounding baking dish containing 20 X SSC (3 M sodium chloride, 0.3 M sodium citrate, (pH 7.0)). A nylon filter (Schleicher & Schuell, 0.45 pm pore), with the same length and width as the gel was then immersed in water, followed by 20 X SSC for 5 minutes, before being placed on top o f the gel. Two additional pieces o f Whatman 3MM paper immersed in 2 X SSC buffer were laid on top o f the nitrocellulose, followed by an evenly layered stack of towels to allow for complete capillary transfer o f the RNA from the gel on to the nitrocellulose. Following an overnight transfer, the nitrocellulose is rinsed briefly in 6 X SSC to remove any agarose adhering to its surface and the filter is observed under UV to I examine the efficiency o f transfer. The filter is then dried briefly and crosslinked (UV Stratalinker 1800), which crosslinks the immobilized RNA to the surface of the nitrocellulose. The filter is now ready for hybridization with a radiolabeled probe. 2 8 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . (Capillary Transfer o f DNA) During the capillary transfer, larger DNA molecules do not transfer with high efficiency. Therefore, DNA in the gel is exposed first to a weak acid (1.5 M NaCl, 0.5 N NaOH) for 45 minutes to fractionate larger DNA, followed by a strong base (1 M Tris (pH 7.4,1.5 M NaCl) which allows the DNA to be transferred rapidly from the gel with higher efficiency. Following the acid-base hydrolysis of the DNA, it is ready to be transferred to a nylon membrane as done with RNA above. Msx2 cDNA Template for Probe: (Msx2 cDNA template) The mouse Msx2 cDNA (1 kb) was used to make a probe complementary to the single stranded Msx2 mRNA immobilized on the nitrocellulose filter. This plasmid (pSKcMsx2) was given to me as an insert ligated into the parental vector pBluescript II SK-(2.9 kb) by Dr. Yi Liu (Figure 11). This plasmid DNA was then used in bacterial transformation (the genetic change o f bacterium after exposure to and recombination with the plasmid). This was done by mixing the plasmid pSKcMsx2 with chemically competent E. coli DH5-alpha cells (have hole in cell wall for plasmid to get inserted into bacterial cell). The sample was put at 42 °C for 2 minutes, quickly cooled on ice for two minutes, and then plated on agar containing ampicillin. Since the parental Bluescript r | vector contains the gene for ampicillin resistance, all bacterial cells which incorporate the plasmid will be resistant to the toxic ampicillin. After incubating the bacterial cells for 16 2 9 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . yp-?ir: ™ viri y hours at 37 °C, colonies appeared on the agar. One colony was removed and cultured overnight at 37 °C in LB media containing ampicillin. In the morning the mixture was centrifuged at 12,000 g for 30 seconds and the medium was easily removed. The pellet was then washed with STE (0.1 M NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH8.0)) in order to avoid problems caused by some strains of bacteria which shed their cell wall components into the medium, which can inhibit the action o f restriction enzymes used later to cleave out the insert from the plasmid. Upon removal of the STE, the pellet was resuspended in 100 ul of an alkali lysing solution (50 mM glucose, 25 mM Tris-Cl (pH8.0), 10 mM EDTA (pH 8.0) and vortexed thoroughly. A second lysing solution (0.2 • N NaOH, 1% SDS) is then added to the sample, and the contents are inverted five times. # 1 A third lysing solution (5 M potassium acetate, glacial acetic acid) is then added to the [ e lysate and the tube is vortexed and centrifuged for 5 minutes. After transferring the supernatant to a fresh tube, 2 volumes o f ethanol are added to it to precipitate the double stranded DNA, and the tube is then centrifuge at 12,000g for 5 minutes to pellet the DNA. The pellet is then rinsed with 70% ethanol and finally air dried before redissolving the nucleic acids in water. To cut the insert out of the precipitated DNA plasmids, I used the enzymes BamHl and EcoRl (Figures 11 and 12). lOpl of DNA (34 pg), was then mixed with 2 pi EcoRl (20units/itl), 1 pi BamHl (40 units/(il), 6 til buffer B and 41 til water. It was necessary to use an enzyme volume less than 1/10 the total volume so that glycerol in the enzyme buffer does not inhibit the restriction enzymes ability to cleave out the insert. 30 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . Since 1 unit of enzyme digests 1 fig in 1 hr, the reaction was complete after one hour. The DNA was then loaded in a 1% low melting agarose gel along side a Stratagene kilobase marker. After the insert (1 kb) had size separated from the plasmid (2.9 kb remaining) and I verified its size with the 1 kb ladder, I cut the insert out o f the gel and put it in an eppendorf with the enzyme Beta-agarase (l|xl Beta agarase enzyme for every 200 mg agarose). Following an overnight digestion at 42 °C, the mixture was put on ice and centrifuged, so that the supernatant could be removed from any remaining agarose. To precipitate the Msx2 cDNA insert, sodium acetate (to a final concentration of 0.3 m) and 2.5 volumes o f ethanol were added. The tube was put on dry ice for fifteen minutes and then spun for 20 minutes. The remaining pellet was washed with 70 % ethanol, dried, and resuspended in TE (Tris-EDTA). To achieve the optimal concentration o f Msx2 cDNA, the DNA was run next to the Stratagene marker once again until the concentration of the insert appeared to be around 16 ng/|il. The template was then ready to be randomly primed to create a radiolabeled probe that could bind to the Msx2 mRNA immobilized of the nitrocellulose filter. (von Ebner Gland cDNA probe) The 900 base pair cDNA (T61) sequence for the unique von Ebner’s gland protein we are characterizing was used to make a radiolabeled template probe that recognized the 1680 nucleotide mRNA sequence in von Ebner’s gland tissue samples. The concentration of the T61 cDNA was 12 ng/pl. 31 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . (CMVMsxl Probe) The 600 base pair CMV promoter, was used as a template to make a radiolabeled probe which would recognize the presence o f CMVMsx2 incorporated into a mouse’s genome. The concentration o f the CMV template was 12 ng/pl. Initiating a cDNA P32 Radiolabeled Probe from a cDNA Template Utilizing the method o f “random primed” DNA labeling (Feinberg and Vogelstein, from random primed labeling kit protocol) a complementary DNA strand is synthesized from the 3’OH termini of the random hexanucleotide primer using Klenow enzyme. The reaction proceeds by adding 2 pi (25 ng) of template DNA to 7.5 ul o f water and then boiling the mixture for 10 minutes. After boiling the template to separated the double stranded DNA, the template is quickly cooled on ice so that the DNA does not reanneal. 3 pi of nucleotides (a 1:1:1 mix of dATP, dGTP and dTTP), 2 pi of hexanucleotide primers, 5 pi o f alpha-3 2 P dCTP (50 pCi at 3000 Ci/mmol), and 1 pi of Klenow enzyme are added and the sample is then incubated at 37 °C for 1 hour. During the incubation the deoxy-nucleotides, including the alpha P labeled dCTP are incorporated into the newly synthesized complementary DNA strands. Following a half hour incubation, the mixture is heated to 65 °C to stop the reaction and the percent incorporation of the dCTP into the complementary strands is measured with a G-25 cephalose column. The amount of newly synthesized DNA is calculated with the following formula: 32 p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . iiCi dNTP x 13.2 x % incorporation = ng of DNA *use 2 ng/ml in hyb. buffer specific activity dNTP (Ci/mmol) The amount o f incorporated radioactivity in dpm is: (iCi dNTP x (2.2X104) x % incorporation = incorporated radioactivity (dpm) The specific activity o f the probe in dpm/ug: incorporated radioactivity x 103 __________ = dpm/flg (good probe = 109 dpm/pg) input DNA(ng) + newly synthesize DNA (ng) (Troubleshooting with Probe) | For a long period of time I was unable to get a good signal when hybridizing the Msx2 cDNA probe to RNA from the sutures and underlying neural tissue. To visually asses the actual amount of full length complementary sequence that was being synthesized from the Msx2 cDNA template I used 3 MM paper and a capillary reaction technique (Figure 13). After the probe was synthesized, one drop was placed on the bottom of a piece o f 3MM paper (Dot A). The probe was then precipitated (10 |xg tRNA, 7.5 M ammonium acetate, and 2.5 volumes of ethanol), chilled on dry ice for 20 minutes, and spun at 14,000 rpm for 20 min. The supernatant was then removed and one drop 33 R ep ro d u ced with p erm issio n o f th e copyrigh t ow ner. Further reproduction prohibited w ithout p erm issio n . * ► V I f (Dot B) was placed next to drop A on the 3 MM paper. The DNA pellet was then washed with 70 % EtOH, dried, and resuspended in TE buffer. One drop o f the resuspended probe (Dot Q was then added to the filter. The filter was then placed in a beaker containing about 20 ml of Buffer (0.7S M KH2 P 04, pH 3.5). Through capillary reaction the buffer, and the unincorporated nucleotides from the probe, migrated up the filter paper. The filter was then placed on x-Ray film, which was developed after five minutes. The migration o f the three dots were then analyzed to see how much o f the nucleotides had incorporated into the full length complementary probe. Hybridization and Film Development: i (RNA) i i f I Prior to hybridizing a radiolabeled cDNA probe to RNA immobilized on a nitrocellulose filter, the filter is incubated in prehybridization solution (6XSSC, 5X Denhardt’s reagent, 0.5% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 50 % formamide) at a temperature 12 to 20°C below the calculated Tm . The Tm for DNA hybridized to RNA is as follows: Tm = 79.8°C + 18.5(logio<Na+ >) + 0.58(fraction G + C) + 11.8(fraction G + C)2 - i 0.50(% formamide) - (820/1) Tr a = 79.8 + 18.5(logio<l.l M>) + 0.58(1/2) + 11.8(.5)2 - 0.5(50) - (820/1000) =79.8 + 18.5(0.04) + .3 + 2.95 - 25 - .82 = 57.95°C hybridization temp = 38 - 46 °C (I used 42°Q 3 4 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . After 2 hours in prehybridization solution at 42°C, the radiolabeled probe with no less than 40 % incorporation was added to the hybridization, for overnight incubation at 42 °C. In the morning, the filter was washed for IS minutes at room temperature with a solution of 2 X SSC, 0.1 % SDS, and then for another 30 minutes at 68°C in 0.1 X SSC and 0.1% SDS. If all of the non specifically bound probe had not been removed after these two washes, a third wash using 0.1 X SSC, 0.1 % SDS was done. The filter was then briefly dried and exposed to X-ray film (Kodak, XAR-Scientific Imaging Film) overnight at -70°C, and developed in the morning. (DNA) When hybridizing a radiolabeled probe to DNA immobilized on a nitrocellulose filter, the filter must be incubated in prehybridization solution (6XSSC, 5X Denhardt’s reagent, 0.5% SDS, 100 |xg/ml denatured, fragmented Salmon Sperm DNA) at a temperature 12-20°C below the calculated Tm . The Tm for DNA hybridized to DNA is as follows: Tm = 79.8 degrees Celsius + 18.5 f l o g i o < N a + > ) + o.58 (fraction G + C) + 11. 8(fraction G + Q 2 - (820/1). Tm = 79.8 + 18.5(logio<l.l M>) + 0.58(1/2) + ll.8(.5)2 - (820/1000) 35 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . =79.8 + 18.5(0.04) + .3 + 2.95 - .82 = 82.85 degrees Celsius hybridization temp = 63-70 degrees Celsius (I used 68 degrees Celsius) After 2 hours in prehybridization solution at 68°C, the radiolabeled probe with no less than 40 % incorporation was added to the hybridization for overnight incubation at 68°C. The following morning, the filter with immobilized DNA, was washed and put on film in the same manner as with the filter containing immobilized RNA. Morphological Comparison of Submandibular Glands (Normal E14.5 vs. CMVMsx2 E14.5): To examine whether the transcription factor Msx2 regulates submandibular gland development, the number of lobules in a gland and the area o f cross section from lobules within wild type and transgenic (CMVMsx2) E14.5 mice were compared. Initially, I had the intention of studying the development of lobules from embryos of different ages: E14, E15, E16, and E17. However, after analysis o f submandibular glands from these embryonic mice, it was evident that after E 14.5, the increase in branching and lobule number made it virtually impossible to asses the area o f individual lobules (see Results). After the transgenic status of the embryos had been determined, the glands were preserved in 4% paraformaldehyde (2g paraformaldehyde, 1 X PBS) until photographed. Using an obliquely lit microscope (Edge R400, Edge Scientific Instruments) with a 10 X magnification submergible lens, I took color slides o f both normal E14.5 submandibular 36 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . glands and glands from E14.5 transgenic mice (CMVMsx2). The slides were then placed in a viewing projector, which projected the image o f the gland onto a box like screen. Transparencies were then placed over the image on the screen and the circumference all distinctly visible lobules was traced (Figure I4A). Due to the large quantity o f both normal and transgenic submandibular glands, I made relative measurements to calculate their area. This was accomplished by using a template with increasingly larger circles, to draw as many small circles as possible within the recently sketched submandibular glands. I then drew a large circle around the entire gland (Figure 14B). I then calculated the difference in area between the large circle and the smaller circles (Large area-Small area total; Figure 14Q. After estimating the % of gland (eg., 20% ,40%,60%,80%) that was outside o f the smaller circles and inside of the larger circle, I used the following formula to calculate the total area of each lobule (Figure 14DJE): Small area to ta l + % (Large area-Small areatotai)= total area of lobule To account for any error that may have been made in estimating the percent of lobule present in (Large area-Small area), I increased and decreased each of my estimated decimal percentages (0.2,0.4,0.6,0.8) by .1 and .2, for my positive control wild type animals. This allowed me to examine whether or not an error in estimating the percent of lobule present in (Large area-Small Area) would have caused a big discrepancy in comparing the average sizes of submandibular glands from E14.5 non-transgenic and transgenic mice. 37 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . The one way Anova test was then utilized to determine whether the embryos from different litters were all at the same developmental stage. The Anova test compared the averages and variability of lobule area from nontransgenic animals from liter to litter. If there was a statistically significant difference in the averages and variability o f the areas used to calculated those averages, then the P value would be less than 0.05. If on the other hand, the areas from lobules of nontransgenic animals from litter 1 had the same degree of variance as those from litter 2,3,4,5 then the P value would be greater than 0.05. This would signify that there was no statistical significance between the areas from the lobules o f non-transgenic animals from different litters. The purpose of this test, was to discover whether it was possible to compile all the transgenic and nontransgenic areas into 2 big groups representing all 5 litters. This way I would get a more realistic average to use in comparing how similar or dissimilar the nontransgenic and transgenic lobules were. Counting the number of lobules present within each gland was the second method I used to try and identify how the overexpression o f Msx2 was affecting submandibular gland development. I compared the average, range and standard deviation of the number of lobules in transgenic mice and nontransgenic mice from the same litter to examine any possible difference between normally developing animals and those overexpressing the gene Msx2. 3 8 p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . RESULTS Characterizing a Novel von Ebner Gland Protein Characterizing the von Ebner Gland protein that Malcolm Snead & colleagues had recently isolated revealed that it displays sequence homology with both pheromone and odorant binding proteins (Figure 1) which ate involved in binding and transporting olfactory stimuli. Attempting to determine whether this protein plays a role in taste transduction, we first investigated the levels of expression and localization of its mRNA. In situ hybridization results from our group revealed that an 3 5 S labeled antisense t strand synthesize from our cDNA template anneals to VEG mRNA in the cytoplasm of the serous acinar cells. There is no mRNA expression in the ductal cells, suggesting that ' the protein is primarily secreted from the acinar cells before heading through the ducts and into the oral cavity. j * ■ Immunohistochemistry was performed by our group using an antibody specific for I this VEG protein. The antibody was obtained by transforming cDNA (T61)into E.coli, * I extracting the purified VEG protein from the bacteria, injecting it into a chicken, and collecting antibody present in the yolk of the chicken’s egg. The primary antibody was combined with sections cut from the tongue of 6 day post natal mice, reacted with a f j : secondary antibody (anti-chick antibody conjugated with peroxidase) and then reacted s t with chromogen (aminoethyl carbazole) which changes tissue containing VEG protein red. Immunostaining revealed that the VEG protein is localized to the cytoplasm of 39 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . secretory acinar cells, as seen in the figure (Figure 3B). In addition, we found that the protein is expressed in small amounts in the lumen of the von Ebner’s gland. Northern blot analysis o f von Ebner Gland tissue from adult mice, displayed prominent expression of the VEG protein mRNA with an intense band (1700 bp) slightly smaller in size than the 18S rRNA (around 1.9 kilobase pairs). RNA from the adult gland could then be used as a positive control to look at transcript expression in glands from 2 day, 4 day, 6 day, 10 day, and 12 day post natal mice. Northern analysis revealed that the message encoding the von Ebner Gland protein is initially expressed in 6 day post natal mice, with expression increasing into adulthood (Figure 15 A 3)- I failed to detect mRNA expression in any of the negative controls examined, including brain, intestines and water. Msx2 Regulating Submandibular G land Development (In Situ and Immunohistochemistrv) In situ hybridization from our group revealed that Msx2 mRNA was expressed in the cytoplasm of both epithelial and mesenchymal cells of the submandibular gland (Figure 16). In CMVMsx2 transgenic embryos, which overexpress Msx2 in tissues endogenously expressing the Msx2 gene (Liu et al, 1995; Kundru, unpublished), Msx2 transcripts were detected as well. To examine the expression of the Msx2 transcription factor, immunostaining was performed by our group using an antibody to the Msx2 protein (Antibody was made by injecting the purified Msx2 protein into a chicken and 40 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . then extracting the antibody from the yolk), hnmunohistochemistry revealed that the Msx2 protein was located in the nucleus of both epithelial and mesenchymal cells in the submandibular gland (Figure 17). This is consistent with its role as a transcription factor interacting with DNA sequences through its homeodomain. In order to examine how Msx2 was regulating development o f the submandibular glands, I compared glands from normal mice and their littermates overexpressing Msx2 (CMVMsx2) (Figure 9 A 3 ). The number and size o f the lobules in transgenic and nontransgenic E14.5 embryos (Figure 18) were compared (After E14.5 the tremendous increase in the number of lobules within the glands made them impossible to assess (Figure 19)). Using the Anova test, the areas of the lobules from embryos in each litter (Appendix A) were used to determine if it was possible to consider animals from all five litters to be at the exact same stage of development. The Anova test (as described in Materials and Methods) revealed that wild type (nontransgenic) animals from different litters show a statistically significant difference in their lobule area (P < 0.05)(Figures 22, 23). Therefore, the lobule area from nontransgenic and transgenic animals were only compared within litters. Within each of the 5 litters there is no statistical significance between the nontransgenic and transgenic lobule sizes (P > 0.05)(Figure 20 A 3 ). Therefore, in examining lobule sizes, I was unable to find that Msx2 played any role in developmentally regulating submandibular gland development. To examine the degree error in my estimation of lobule size may have played in my final calculations, I subtracted and added .2 and .1 (Figure 21) to my decimal (%) 41 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . calculations for my nontransgenic animals. I then used the revised areas to recalculate the average nontransgenic lobule areas for each litter. Even if I was off by a factor o f 0.2 in my estimation o f the percent o f lobule in area X, I would still be within the standard deviation of the average transgenic lobule size for each litter. This suggest that my failure to show any significant differences between normal and transgenic animals was an accurate finding. A second factor utilized in comparing the submandibular glands from normal animals and those bearing a copy o f CMVMsx2, was the number of lobules present in the gland. As seen in the figure (Figure 25), the mean and the standard deviation of the number o f lobules in transgenic and nontransgenic embryos from the same litter was very similar. (Northern Analysis ofM sx2 Expression in CMVMsx2 Trcmseenic Animals)) Although it was possible Msx2 is regulating development of other factors besides lobule size and number, I decided to do a northern blot to assess whether there was a significant difference in Msx2 expression between the wild type and transgenic animals. i \ After months of technical problems with Northern blot analysis (Figure 26,13), I was finally able to detect strong signals of Msx2 expression from my positive control (Figure 27). However, when I probed mRNA from 2 ,5 ,1 0 , and 20 submandibular glands, I found no Msx2 expression (Figure 28). This suggests that Msx2 is expressed at low levels in the submandibular glands, and that more sensitive techniques are needed to 4 2 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . examine whether the transgenic animals ate indeed overexpressing Msx2 as previously described (Liu et al, 1995; Kundru, unpublished). t, R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . [ a ^ t a v T M n M T - 1 i m i« b h j H P W B I I ) m y w I I I . . . m u . ■ .< ■ .« » n . p » < ' « DISCUSSION Following decades o f study and speculation, the molecular mechanisms guiding epithelial- mesenchymal interactions in organ development are finally beginning to be understood. Among these organs are the salivary glands, which have been o f particular interest to craniofacial biologist due to their salivary secretions. Saliva is the first digestive fluid secreted into the gastrointestinal path and is important in performing a wide variety o f functions. Although only 10% of saliva is produced by minor salivary glands, including the von Ebner’s gland, the salivary proteins that it secretes are believed to play a critical role in mediating taste sensations. A wide variety of proteins secreted from von Ebner’s gland have recently been characterized and to this list Malcolm Snead & colleagues have added another protein(T61); one that is potentially involved in taste transduction. In the present study, I have further characterized this novel protein which is selectively expressed and released by the von Ebner’s gland. As the only salivary gland providing secretions directly to the taste buds, its protein secretions are favorable candidates to mediate taste reception. The protein we isolated displays substantial homology to a number of other proteins, most notably the olfactory binding proteins, which bind and transport olfactory stimulants (Figure 1). Therefore, it is quite probable that our protein is also able to bind and carry tastants either toward or away from the gustatory receptors. In situ hybridization reveals 44 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . that the mRNA transcripts encoding our VEG protein is expressed in the cytoplasm o f the serous acinar cells o f the gland. T61 mRNA is translated in these serous secretory cells, as immunostaining reveals the expression of the protein in the cytoplasm of these cells as well as in the lumen which enters into the ducts from the secretory region o f the gland. Although we localized the protein to the secretory cells o f the von Ebner’s gland, the function of the protein is still unclear. A major clue leading me to believe this protein is involved in regulating taste sensation is northern blot analysis demonstrating that the messenger RNA is expressed at detectable levels six days after birth, with levels rising into adulthood. Although it is possible that the VEG protein is being expressed in embryonic development, northern blot analysis demonstrates that its levels of expression do not reach detectable levels until 6 days post natal. This is consistent with the development of taste adnexa and the animal’s need to assess the nutritional or deleterious content of the food it eats. These factors, in addition to the sequence homology that this protein shares with the olfactory binding proteins and others which transport lipophilic molecules from one region to another, suggest that this protein may play an integral role in the mediation of taste sensation in the oral cavity. As previously discussed in the introduction, considerably more research has been done on the submandibular gland. Therefore, this major salivary gland posed as an excellent model to study how the transcription factor Msx2 is involved in development. In submandibular glands from E13, E14, and E1S embryos, immunostaining revealed Msx2 expression in the nucleus of both the epithelium and mesenchyme, while in situ 45 p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . hybridization revealed its expression in the cytoplasm of cells from both tissue types. Its presence in both the epithelium and mesenchyme is consistent with its patterns of expression in other tissues. For example, Msx2 expression has been discovered in both the epithelium and mesenchyme during limb development and tooth morphogenesis where it has been implicated in regulating reciprocal interactions between the two tissue types (Davidson, 1995). To understand how Msx2 may regulate development of the submandibular gland, I analyzed glands from transgenic mice bearing the transgene CMVMsx2. I compared morphology of the glands from the wild type and transgenic animals to determine whether there were any differences between the number and size of lobules; there were no significant differences between submandibular glands from the transgenic and non transgenic animals. Concerned that Msx2 was not being overexpressed in the transgenic mice, I used northern blot analysis to examine the expression o f Msx2 in the submandibular gland in normal and transgenic mice. However, I was unable to find Msx2 expression in as many as 20 submandibular glands from normal mice. Northern analysis of RNA from transgenic animals is not yet completed. As seen through in situ and immunostaining analysis, it is quite evident that Msx2 is expressed in the submandibular gland. However, it must be expressed at low levels because Msx2 cannot be detected using Northern analysis. Instead Msx2 levels might be analyzed quantitatively using more sensitive techniques discussed below (The Future). If 46 p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Msx2 is expressed at low levels in the submandibular gland, and if it is involved in developmentally regulating submandibular gland morphology, then even the slightest increase in expression would most likely result in significant phenotypic changes. I found no morphologic differences between the submandibular glands of the transgenic mice when compared to non-transgenic littermates. Therefore, my results may suggest that Msx2 does not have a role in submandibular gland development. It is important to realize that this is only a preliminary conclusion. There are other factors in salivary gland development which should be compared morphologically and there are additional experiments which are needed to define Msx2’s role in , morphogenesis. There were some difficulties encountered with the experiment described | i above. For example, it was very difficult to dissect out the glands from E14.5 embryos and only after lots o f practice was I able to successfully microdissect glands without any damage to the soft tissue. Although I was not able to compensate for the lost tissue, I did not use any glands in my calculations that had less than ten intact and clearly visible | lobules inside. Furthermore, lobules superimposed on top of other lobules, as viewed through the microscope and on the slide images, made it difficult to accurately assess the t r size of all the lobules visible. For this reason, I only traced over lobules with well defined £ perimeters, and therefore many lobules were not used in my calculations. ! fr ,1 i. 47 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . THE FUTURE If I was able to continue on forever as a Master’s student (Which is only a hypothetical situation!), I contemplated what direction I might head with my research experiments, hi characterizing the von Ebner’s gland protein (T61) that we have isolated, I would like to examine mRNA and protein levels in other salivary gland tissue for T 61 to verify that the protein is only being released by VEG. If the other salivary glands do not express the protein, and it’s therefore only expressed by the VEG gland which directly bathes the taste buds with their salivary secretions, then I would have additional evidence that this protein is involved in taste transduction. If possible, I would also stimulate the tongues of anesthetized mice, lay sweet, sour, bitter and salty tastants on their papillae, and then collect their salivary secretions. Using an antibody to the VEG protein, I could then examine which stimuli, if any, were responsible for triggering its release. In addition, I could try using antibodies for other von Ebner’s gland proteins to examine their presence. Along with this experiment, I might also do northern blot analysis to examine the times o f expression o f other proteins secreted by VEG. All of these experiments could provide additional information about the function of VEG proteins, as well as additional clues to the factors regulating taste reception. Although very little is known about the molecular mechanisms regulating von Ebner’s gland development, submandibular gland morphogenesis can be used as a 48 p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . developmental model. There are many areas of research left uncovered in examining Msx2’s role in possibly regulating salivary gland development. It is possible, that Msx2 is time dependently expressed in the submandibular glands. However, in order to characterize its patterns o f expression, more sensitive techniques such as poly A RNA, RNAse protection assays, or RT-PCR are needed to quantitatively assess Msx2 gene transcripts. Furthermore, in order to rule out Msx2’s role in submandibular gland development as my conclusions suggest, Msx2 transgenic mice lacking the functional homeodomain should be compared with normal mice for any phenotypic changes in submandibular gland morphogenesis. A third set of experiments that I could do, if I had time and money, would be to take a sampling of the lobules from salivary glands of E13.5 wild type and transgenic embryos, trace over the lobule as before, but this time employ a computer software program which accurately assesses the area within the outline I had drawn. This would potentially eliminate any error calculation in the estimation of the size o f the lobules within the gland and give a more accurate average to compare the sizes of non transgenic to transgenic lobules. Such software packages are available, but not accessible for this study. In addition, I would look at other factors, such as the size o f the stalks from which the lobules (grape like objects) extended. It is possible that the overexpression of Msx2 is effecting the development of these stalks or perhaps other machinery, rather than the size and number of lobules. 4 9 with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . The opportunity to further examine proteins which are involved in taste transduction and factors regulating submandibular gland development, has given me a deeper appreciation for the molecular mechanisms involved in salivary gland biology. Studying the development and secretions o f the submandibular gland, von Ebner’s gland and other salivary glands, is beneficial to understanding the mechanism by which the glands can regulate some of the physiologic functions o f the body. Many congenital defects in salivary gland morphogenesis, as well as pathological conditions will not be fully understood until the mechanisms guiding development are better understood. In characterizing von Ebner’s gland protein (T61) and studying the possible regulation of minor salivary gland development by transcription factors such as Msx2,1 have obtained preliminary data leading to a better understanding of the molecular mechanisms guiding development and the value of salivary secretions which help to maintain our bodies homeostatic state. r i 5 0 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . FIGURES c f \ ! 6 i I t f r f c i ? ? R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Figure 1 A t ’ m . — W 30 30 40 SO <0 T O «0 9Q 300 couomsoq M v m u w u T irrrq rr i q r u w m s q b i u * ^ r ^ m i r c d Q u c n r w o qtmo / w u w y . B t 3 . « U i e t 3X0 X30 U 0 340 ISO U 0 t 370 U 0 t M l (3 i m a n w « n n n r-3 t — c m a r iw rraw rr rrrum m rr m w m . cu*u ev - m u m L .rv.uiuek » i » » * I I i l l 11 U L 1 1 1 « L i u r r hi m m un « « o u» rm rmmvn j w r n a s u r m h v m o k . iw i,rn m » t a b o «l* x.» « ■ * » » ■ « « o i u m » -------------- _„*•»» A t 330 320 330 U 0 ISO ICO ^ 2 v n s w > T 'ivw iiw uesrwJtixv trvB m njA u c v s k k im u u b o C M L S V W M S I B 2 l . < U « U S __________ M 0 M 0___________ 220________ 220 U O _______ 230 J<0 270 C Ml -T im ur ■ mz~mrrzz~ aiierv u wr Ttx-KL-tx* m a — m m i i h i l o flM M iff ar r-nrry ^^m 07 i ti im i » u» i hi ii j it mi ii ii ii ii ii 111 iii 11 i i i i miii A Z on. M a c g w r n a o w r ^ tn m n a t a m u c n o r namgt t x wma niuuacs w z tr jiu c . LB um i r , ™ L ~ U . . r ,« tio iia e t I 210 220 210 2€0 250 2 00 270 200 ISO 300 c m . m s t e n a g s n a m o n t r a n u s o s u n a o m e r lo g p w r o e e / mmm K n o m uui in- u r n a r t x v x u v n t m B 3' l - <i i k c m c m i o im o A t I III III A J cron. ««ac bcfvxaasr A t Translated sequence of coding strand of oar VEG cDNA (T61) compared to the protein sequence form RAT Olfactory Protein ratrm from Rat Olfactory Epithelium (Bock and Axel, 1995). Our VEG protein shows over 32% (100AA/310AA) homology to the rat olfactory protein believed to play a role in binding and transporting olfactory stimulants. All rows marked A are VEG protein sequences; rows marked B are AA sequences from Rat Olfactory Protein. (Sequence comparison was done using the National Institute of Medicine's Blast Search) Title 12A. Abbreviations and Molecular Weights tor Amina Adds. Amina Add Three-tetter Abbreviation One-letter Symbol Malleolar Weight Alanine Ala A 89 Arginine Arg R 174 Asparagine Asn N 132 Aspartic add Asp □ 133 Asparagine or aspartic a d d Asx a — Cysteine Cys c 121 Glutamine Gin 0 146 Glutamic Add GIu E 147 Glutamine or glutamic ad d G bc Z — Glycine Gly G 75 Histidine His H 155 Isoieudne lie 1 131 Leucine Leu L 131 Lysine I.Y * K 146 Methionine Met M 149 Phenylalanine Phe F 165 Praline Pro P 115 Serine Ser S 105 Threonine Thr T 119 Tryptophan Trp W 204 Tyrosine Tyr Y 181 Valine Vai V 1t7 B: Abbreviations for Amino Acids from Figure IA (Taken from Promega catalogue, 1996) 52 R ep r o d u ced with p erm issio n of th e cop yright ow ner. Further reproduction prohibited w ithout p erm issio n . Figure 2 1 © @ t S W O T II II u Diagrammatic representation o f taste transduction mechanisms. Sour transduction involves a d d block o f voltage-dependent < * channels, which are restricted to the apical membrane. Salt (Na~) transduction involves the passage o f N a* into taste cells through passive. amSoride- blockable Na * channels on the apical membrane o f taste cells: N a* is then pum ped o ut b y a (Na *. K~)-ATPase on the basolateral membrane. Sw eet transduction involves receptor-mediated stimulation o f adenylate cydase: voltage-independent K* channels on the basolateral membrane are then dosed in response to cAM P-dependent phosphorylation. However, the link between increased adenylate cydase activity in response to sw eet stim uli and dosure o f K* channels by cAM P-dependent phosphorylation has not y e t been show n in the same taste receptor celts. Transduction by all these pathways involves one final common pathw ay: depolarization and influx o f Ca1* through voltage-dependent Ca2* channels. Transduction mechanisms for other taste modalities have n o t been illustrated because evidence fo r them is still preliminary, it is n o t y e t dear i f all these mechanisms are present on a tingle taste cell, a s illustrated here, or if different taste cells are specialized to detect particular taste modalities. (taken from Kinnamon, S, 1988) 5 3 R ep ro d u ced with p erm issio n o f th e copyrigh t ow ner. Further reproduction prohibited w ithout p erm issio n . Figure 3 A: HE stain of the von Ebner’s Gland from an adult mouse. a-secretory region of the gland (stained red due to the increased number of nuclei in the glandular cells as compared to the surrounding muscle cells) b-ductal regions of gland leading to the circumvallate papillae c-circumvallate papillae B: Immunostaining using VEG Protein Antibody. Serous acinar cells react with the antibody and turn red. 5 4 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . Figure 4:(taken from Scieebay, 1987) Diagram of steps in morphogenesis of submandibular glands, a-initiatioa b-primary cord and terminal bulb formation. c-clefting of terminal lobuIe;beginning of branching morhphogenesis. d-lumenizadon in cords. e-lumenizadon o f terminal bulbs; beginning of cytodifferentiation. f-ducts and terminal lobules consist of a bilayer of cells around a central lumen. Soron a t OamflvMof wroui ofli (odhar nlli) M i r a m i M m b r a i M biptaa SM M and ucrwory dacM Amylaio-cont omin g PRIMARY SECRETION (naan* (Manic; ■ rah at Na’. It*. Q ~ and [probably] H CO fiM tario piasma) M odSkation of fonfcaMtfwir Figure 5: (Berne and Levy, 1996) Structure of human submandibular gland. Figure 6: (Beme and Levy, 1996) 2 Stage Model of Salivary secredon:Primary secretion is by acinar cells, while the striated and excretory ducts modify the saliva composition. 55 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . OgMBamHt Exon 1 B afflM - Ccan EeoHtBunW B w l l u—H -O iX -----i r - s u - ■«“ Exon 2 h r W * - 1C HtfldOt Figure 7 : (takea fiom Bell et al, 1993) Sr;h«»marie map of Msx2 gene based on restriction enzyme digest sites. Boxes indicate exons: open box=transcribed only; darkened box=protein coding sequence; x=homeobox in 2nd exon. bpiS bpto j Figure 8 : (taken from Pabo and Sauer, 1992) i Sketch of homeodomain contacting a DNA comlex, summarizing the relationship of the alpha- | helices & N-terminal arm with respect to the DNA. Helices 2 & 3 form the HTH unit. Notice it contacts the major and minor grooves in order to “communicate” with the DNA R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . C M V MnZ U T * C M V M ssZ W T Pronuciear injactiort i col (•nilizid agg QNA in soiuticn B Figure 9 A 3 (above): CMVMsx2 construct (A) which was inserted into the pronuclei of a fertilized egg (B) to establish a transgenic mouse line. I Tfli lit AdllJt l* Figure 10 A3-‘ Southern Blot of genomic DNA from the tails of mouse embryos. Hybridization was done with a probe specific for CMV I order to asses whether the embryos bear a copy of the transgene CMVMsx2. 5 7 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . i Ms*2 insert A m p " * - Ndel E c o R I Figure 11: pcKyMsr? Plasmid cloned by Yi Liu. The 1 idlobase Msx2 cDNA insert was cloned into this vector (pBluescript H SK-). Msx2 cDNA was cut out with BaxnHl & EcoRI, and used as a template to synthesize a probe specific for Msx2 mRNA. Figure 12: A-Size seperation of Msx2 insert and plasmid after cutting with enzymes BamHl & EcoRI B-Size analysis of 1 kb insert (Msx2 cDNA insen) 58 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . 3 MM Paper 2 3 • • V A B C • • • • • ■B,' T Figure 13-1: A- 1 drop from newly synthesized probe before DNA precipitation (incorporated + unincorporated) B- 1 drop from probe after precipitation & resuspension (probe*incorporated) C- 1 drop from supernatant after precipitation of synthesized probe (unincorporated) Figure 13-2:3M M Paper was placed into KH2P04 Buffer (0.75 M ) Rgure 13-3: i j - A,B,C-Lower dot corresponds to the radiolabeled dCTP incorporated into the DNA ^ immediately after synthesis. Upper dot corresponds to unincorporated isotope. [ Rgure 13-4: Capillary reaction experiment i ' unincorporated incorpocatea 5 9 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . Rgure 14 A: I first traced the perimeter of a cross section of the lobules as seen through the obliquely lit microscope (10X Magnification) Large circle Spr _______lobuli all circle drawn inside lobule Jobule B: Using a template ruler with increasingly larger circles, I drew a Small circle inside the lobule and a Large circle outside. Blackened region = lobule. Difference in area between Large circle area and Small circle area. X=AREA(large circ!e)-AREA(small circle) C : I then calculated the area of region X (the difference in area between the Large circle and the Small circle')_____________ 25% % of lobule in X-20+20+12.5+10=60% 25%s'^ p ^ 2 5 % D : I then estimated the % or lobule in area X . E : I then calculated the total area of the lobule. TOTAL AREA O F LOBULE- Small Area +%(Large Area-Small Area) 6 0 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm ission . Figure 1 6 : In situ hybridization demonstrating expression o f Msx2 mRNA in the cytoplasm o f mesenchymal and epithelial tissue o f the submandibular gland. E 14 % E14.5 I, 6 2 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Figure 17 : Immunostaining demonstrating expression o f Msx2 protein in nuclei of mesenchymal and epithelial tissue o f the submandibular gland. E14.5 t E lSJ R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Figure 18 : rnmparicnn o f Submandibular Glands from E14.5 Wild Type and Transgenic Mice E14.5 Wad Type E14.5 Transgenic fCMVMsx2) R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Figure 19 : C o m p a ris o n o f Submandibular Glands from E15.5 Wild Type and Transgenic Mice EI5. 5 Wild Type E1S.S Transgenic fCMVMsx2) 6 5 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . i * i Figure - 20 A 400 300 N g g 200 4 4 N 3 3 1 0 0 M ice f Bar graph showing the average lobule | area in nontransaenic and transgenic mice Uttar# ava size (mm2) standard deviation P-value Utter# avg size (mm2) standard deviation NTg 1 183.5 89.7 0.15 Tfl1 227.3 93.4 NTa 2 184.9 90.7 0.93 Tfl2 188.8 101 NTs 3 164.8 71.9 0.8 Tfl3 . 1623 63.5 NTa 4 132 46.8 0.21 Tfl4 1426 63.2 NTa 5 154.2* 58.2 0.45 Tfl5 _ . 168 75.8 Raura 20 B : Chart of tha avaraoa lobule area In each of the 5 litters examined. Also Included is the Standard Deviation and P value from the Anova test comcarina lobule sizes from transaenic and nontransaenie animals IP > 0.05 * not statistically significant). I I I I I . . I " Anova test results for litter to litter comnarison are in Aooendlx B. 1 1 6 6 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . A.) Utter# M*4V2)*(LA-SA) SA+<%'.1)*(L4rSA) STUtWOA-SA) 5A*<Vf.1)fm-SA) S A * T M r (L A -S A ) I Si I 143.6 1 63.2 1 8 3 2 2 0 2 4 2 2 2 NTg 2 avg (mm2) 148.4 172.4 1 9 4 .5 2 2 0 .6 2 4 4 .7 NTg 3 avg (m m 21 119.6 1 4 2 2 1 6 4 .8 1 8 7 2 2 1 0 .7 NTg 4 avg (mm2) 92.4 1 1 2 2 1 3 2 1 5 1 .8 171.6 NTg5avg(mm2) 116.1 1 3 5 2 1 5 4 2 1 7 3 .3 1 9 2 2 1 SrU W O JM A ) B.) Tg 1 avg (nun2) 2 2 7 2 Tg2avg(mm2) 1 88.8 Tg 3 avg (mm2) 1 62.3 Tg4avg(mm2) 1 42.6 Tg 5 avg (mm2) 168 Rgure 21: Error calculation: The total area of tlta lobules in each of non transgenic IR ta ra was recalculated to assess any error which may have been involved in estimating the % (2,.4,.6,.8) of lobule in between the small and. large circles. Subtracting and adding as much as 2 from the decimal percent value, and then recalculating my average areas while accounting for this error margin, gave me revised (error assessed) average values ( A ) for each litter. These values were then comoared to the average lobule area from the transgenic animals (B). f f f I { ; 6 7 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . Anova: Single Factor Variance and P value for NTg Litter 1,2,3,4,5 SU M M A RY Groups Count Sum Average Variance Litter 1 76 13893.3 182.8068 8037.605 Litter2 98 19102.1 194.9197 8233.889 Litter 3 83 13681.3 164.8353 5175.012 Litter4 87 11487.1 132.0352 2186.483 Litter 5 29 4472.37 154.2197 3385.583 A N O V A Source o f Variation SS df MS F P-value F ait Between Groups Within Groups 2065482 2108692 4 368 51637.05 5730.142 9.011478 5.94E-07 2396199 Total 2315241 372 Figure 22 : Anova test to compare the average and variance in lobule area between the 5 nontransgenic litters. Notice the P-value (P < 0.05) signifying that the lobule areas from wild type mice significantly vary between litters. Anova: Single Factor Variance and P value for Tg Litter! ,2,3,4,5 S U M M A R Y Groups Count Sum Average Variance Litter 1 10 2272574 2272574 8714.342 Litter 2 150 29067.89 193.7858 6489.963 Litter 3 59 9578272 1623436 4028.09 Litter 4 152 21677.05 1426122 3999.898 Litter5 26 4367208 167.9695 5742113 A N O V A ouneofVariatfo s i or MS F P-value F ait Between Groups W ithin Groups 234529.8 2473600 4 392 5863246 6310205 9291689 3.5E-Q7 2394707 Total 2708130 396 Figure 23 : Anova test to compare average and variance in lobule area between the 5 transgenic litters. Notice the P-value (P < 0.05) signifying that the lobule areas from transgenic animaU significantly vary between litters. 6 8 with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . Figure - 25 A 100- 7 5 - 50- I 25- * O * o f lobules Mice Bar graph showing the average f-QfJfltaites in nontransoenic and transgenic mice. Figure 25B Average * of lobule (SO) N T S _ To Utter 1 24(0.7) 24(3.4) Utter 2 35(13.9) 41 (6.4) Utter 3 26.6 (4) 30(6.7) Utter 4 61 (9.1) 69 (13.7) UtlerS 28(1.4) 24(0.7) 6 9 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . Figure 26 B > f \ A 3 : Northern blots demonstating technical difficulties that were causing me to constantly £ reavaluate all of my techniques. t I f 7 0 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . A; Formaldehyde-agarose gel of RNA from sutures and underlying neural tissue (Positive control for Msx2 expression) (Lanes: 1A=140 ug RNA; 2A=70 ug RNA; 3 A=35 ug RNA) B: Hybridization of Msx2 cDNA radiolabeled probe to + control RNA in A. 71 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm ission . I Figure 28 A: Fonnaldehyde-Agarose gel of wild type E16.5 submandibular gland RNA (Lanes: 1= 2 submandibular glands (SG); 2= 5 SG; 3 * 10 SG; 4*20SG;5*+ control (RNA from sutures of E16 moose) B: Nylon filter to which RNA was transferee! OHybridization of Msx2 probe to RNA immobilized on nylon filter 7 2 R ep ro d u ced with p erm issio n o f th e copyright ow n er. Further reproduction prohibited w ithout p erm issio n . BIBLIOGRAPHY Beidler, L. M. and Smallman, R. L. (1965) Renewal o f cells within taste buds. X Cell. Bio. 27:263-272. Bell, J.R., Noveen, A., Liu, Y, Ma, L.., Dobias, S. Kundu, R., Luo, W ., Xia, Y., Lusis, A.J., Snead, M X., and Maxson, R. (1993) Genomics, 16, pp 123-131. Beme, R. And Levy, M. (1996) Principles o f Physiology. Mosby, New York. Brand, R.W. and Isselhard, D. (1985) Anatomy of Orofacial Structures. Mosby, » I New York. [ Buck, L and Axel, R. (1991) A Novel Multigene Gamily May Encode Odorant Receptors: A Molecular Basis for Odor Recognition. Cell. 75:175-187. Cowart, B. J. (1989) Relationships Between Taste and Smell Across the Adult Life Span. Nutrition and the Chemical Senses in Aging. Ann N.Y. Acad. Sci. 561: 31-55. Delay, R.J., Kinamon, J.C. and Roper, S.D. (1986) Ultrastructure of Mouse Vallate Taste Buds. Cell Types and Cell Lineage. J. Comp. Neurol. 253:242-252. i Davidson, D. (1995) The function and evolution of Msx genes: pointers and paradoxes. TIG. 11:405-411. 73 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . 1 i Fleming, T. (1992) Epithelial O rganisation and Development Chapman and Hall, New York. Frank, M E. and Hettinger, T P . (1992) The Sense o f TasterNeurobiology, Aging, and Medication Effects. Critical Reviews in Oral Biology and Medicine 3:371- 393. Gurkan,S and Bradley, R. (1988) Secretions of von Ebner’s gland influence responses from taste buds in rat circumvallate papilla. Chem. Senses 13:655- 661. > Hamosh, M and Bums, W.A. (1977) Lypolytic activity of human lingual glands i t I (Ebner) Lab. Investigations 37:603-608. Hand, A.R. (1970) The Fine Structure of von Ebner’s Gland of the Rat. J.C ell. Bio. 44:340-353. Hieda. Y., Iwai, K., Toshiteru, M., and Nakanishi, Y. (1996) Mouse Embryonic Submandibular Gland Epithelium Loses Its Tissue Integrity During Early Branching Morphogenesis. Developmental Dynamics. 207: 395-403. Ignelzi, M., Liu, Yi., Maxson, R., and Snead, M. (1995) Genetically Engineered Mice:Tools to Understand Craniofacial Development Crit Rev Oral Biol Med. 613:181-201. 7 4 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Jabs, E.W., Muller, M., Li, X., Ma, L., Luo, W., Haworth, L, Klisak, I., Sparkes, R., Warman, M., Mulliken, J., Snead, M., and Maxson, R. (1993) A Mutation in the Homeodomain of the Human Msx2 Gene in a Family Affected with Autosomal Dominant Craniosynostosis. Cell. 75:443-450. Jowett, A., Vaino, S., Ferguson, m., Sharpe, P and Thesleff, I. (1993) Epithelial- mesenchymal interaction are required for msxl and msx2 expression in the developming murine molar tooth. Development. 117:461-470. Kinnamon, J.C. (1987) Organization and Innervation of Taste Buds. Neurobiology of Taste and Smell. John Wiley and Sons, Ney York. Kinnamon, S. (1988) Taste transduction: a diversity of mechanisms. TINS. 11(11): 491-496. LePage, T., Ghigliones,C., and Gache, C. (1992) Developement 114:147-163. Leydig, F. (1851) Z. Wiss. Zool. 3:1. Li, X.J. and Snyder, D.H. (1995) Molecular Cloning of Ebnerin, a von Ebner’s Gland Protein associated with the Taste Buds. J. O f Biological Chemistry 270:17674-17679. < Liu, Y., Ma, L., Wu, L.Y., Luo, W., Kundu, R., Sangiorgi, F., Snead, M., and Maxson, R. (1994) Mechanisms of Development 48:187-197. 75 R ep ro d u ced with p erm issio n o f th e copyright ow ner. Further reproduction prohibited w ithout p erm issio n . Liu, Y., Kundu, R., Wu, L., Luo, W., Ignelzi, M., Snead, M ., Maxson, R. (1995) Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull. Proc. Natl. Acad. Sci. 92:6137-6141. Liu, Y., Ma, L., Kundu, EL, Ignelzi, M., Sangiori, F., Wu, L., Luo, W., Snead, M., and Maxson, E L (1996) Function o f the Msx2 Gene in Morphogenesis of the Skull. Ann. New York Academy o f Sciencesl3:48-58. Mason, D and Chisholm, D. (1975) Salivary Glands in Health and Disease. WB Saunders Company Ltd, Philadelphia. ' ■ Miller, LJ. (1977) Gustatory Receptors o f the Palate. Food Intake and the Chemical | Senses. Univ. of Tokyo, Tokyo pp173-178. t 5 Pabo, C and Sauer, ELT, (1992) Transcription Factors. Ann. Rev. Biochem. i 61:1053-1095. r ! Pevsner, J., Reed, R.R., Feinstein, P.G. and Snyder, S.H. (1988) Science. * 241:336-339. f Phippard, D., Hall, S. Sharpe, P., Naylor, S., Jayatalake, H., Maas, R., Woo, I., Clark, D. West, PH, Liu, Yi, Maxson, Hill, and Dale, T. Regulation of Msx-1, | Msx-2, Mbp-2, and Bmp-4 during foetal and postnatal mammary gland, j Development 122:2729-2737. 7 6 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n . Rankow, R and Polayes, I. (197ff> Diseases o f the Salivary Glands, W.B. Saunders Company, Philadelphia. Riva, F.T., Cassu, M., Lantini, M.S., Riva, A. (1985) Fine structure o f human deep posterior lingual glands. J. Anat 142:103*115. Sambrook, J., Fritsch, E J 7 ., and Maniatis (1989) Molecular Clonic: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York. Schmale, H., Grez, H.H., Christiansen, Heid, E. (1990) Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins. Nature 343:366-369. Screebny, L M . (1987) The Salivary System. CRC Press, Inc, Florida. Schwalbe, G. (1867) Arch. Mikr. A nat. 3:504. Teeter, J.H. and Brand, J.G. (1987) Peripheral Mechanisms of Gustation: Physiology and Biochemistry. Neurobiology of Taste and Smell. John Wiley and Sons, New York, 299-329. Von Ebner, V. (1873) Die acinosen Drusen der Zunge und ihre Beziehungen zu den Geschmacksorganen. Graz. 7 7 R ep ro d u ced with p erm issio n o f th e copyrigh t ow n er. Further reproduction prohibited w ithout p erm issio n .

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Asset Metadata

Creator Burstein, Jeffrey Michael (author)

Core Title Development and secretions of salivary glands using mouse models

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, animal physiology,biology, molecular,health sciences, human development,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Tokes, Zoltan A. (committee chair), [illegible] (committee member), Stellwagen, Robert H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-14827

Unique identifier UC11337577

Identifier 1387814.pdf (filename),usctheses-c16-14827 (legacy record id)

Legacy Identifier 1387814.pdf

Dmrecord 14827

Document Type Thesis

Rights Burstein, Jeffrey Michael

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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