ARTICLE |
Correspondence to: Phyllis A. Shaw, Center for Anatomy and Functional Morphology, Box 1007, Mount Sinai School of Medicine, New York, NY 10029. E-mail: phyllis.shaw@mssm.edu
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Summary |
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Homeobox-containing (Hox) genes play important roles in development, particularly in the development of neurons and sensory organs, and in specification of body plan. The Hmx gene family is a new class of homeobox-containing genes defined by a conserved homeobox region and a characteristic pattern of expression in the central nervous system that is more rostral than that of the Hox genes. To date, three closely related members of the Hmx family, Hmx1, Hmx2, and Hmx3, have been described. All three Hmx genes are expressed in the craniofacial region of developing embryos. Here we show, for the first time, the expression of the transcription factor Hmx3 in postnatally developing salivary glands. Hmx3 protein is expressed in a cell type-specific manner in rat salivary glands. Hmx3 is present in both the nuclei and cytoplasm of specific groups of duct cells of the submandibular, parotid, and sublingual glands. Hmx3 expression increases during postnatal development of the submandibular gland. The duct cells show increasing concentrations of Hmx3 protein with progressive development of the submandibular gland. In contrast, the acinar cells of the three salivary glands do not exhibit detectable levels of Hmx3 protein.
(J Histochem Cytochem 51:385396, 2003)
Key Words: homeobox protein, Hmx3, submandibular gland, development, duct cells
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Introduction |
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HOMEOTIC GENES, first described in Drosophila melanogaster (
The major salivary glands (submandibular, parotid, and sublingual) of mammals exist as three pairs of organs that cooperate functionally to produce saliva for the oral cavity. They all share a common developmental pattern in rodents, in that they fully develop postnatally (
The study presented here represents the first examination of the expression and distribution of a transcription factor, Hmx3, in postnatally developing SMG. We find that Hmx3 expression in SMG is increased during postnatal development. We have also examined the expression of the protein in all three salivary glands of adult rats. Hmx3 protein is present in all three salivary glands, (submandibular, parotid, and sublingual). In addition, Hmx3 expression is cell type-specific: The duct cells show increasing concentrations of Hmx3 with progressive SMG development. Hmx3 is present in both the nuclei and cytoplasm of specific sets of duct cells in the submandibular, sublingual, and parotid glands, but not all duct cells show nuclear localization of Hmx3. In contrast, acinar cells do not exhibit detectable levels of Hmx3 protein in any of the salivary glands.
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Materials and Methods |
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Materials
The peroxidase-labeled streptavidinbiotin kit was from DAKO LSAB (Carpinteria, CA). The proteinase inhibitor cocktail was from Roche (Mannheim, Germany). The Bradford protein reageant and horseradish peroxidase-coupled goat anti-rabbit antibody were from Bio-Rad (Hercules, CA). Hybond ECL membranes and enhanced chemiluminescence (ECL) substrate were from Amersham Pharmacia Biotech (Arlington Heights, IL). The protein standards were the BenchMark Prestained Ladder from Gibco (Gaithersburg, MD). All other materials were purchased from commercial sources (Sigma; St. Louis, MO and Fisher, Pittsburgh, PA) and were of the highest purity available.
Animals
Female SpragueDawley rats (Charles River; Wilmington, DE) were kept in a temperature- and humidity-controlled environment (12 hr light/12 hr dark cycle) and had free access to water and standard laboratory chow. All experimental protocols were reviewed by the Mount Sinai Institutional Animal Care and Use committee and conducted in accordance with the NIH guidelines for the care and use of laboratory animals. Female adult rats and pups of 2, 3, 4, 5, 6, 8, 10, 15, 20, 25, 28, and 33 days of age were analyzed. The animals were sacrificed by CO2 and the salivary glands were removed. For morphological studies, the glands were fixed in 4% paraformaldehyde in 1 x PBS (0.01 M phosphate buffer, 2.7 mM potassium chloride, and 0.137 M sodium chloride, pH 7.4) for 2 hr to several days at 4C and embedded in paraffin. The glands for extraction of total proteins were frozen in liquid nitrogen and stored at -80C until used.
Staining of Submandibular Glands
Five-µm sections were stained with hematoxylin and eosin (H&E) for morphological examination.
Hmx Protein Localization in Salivary Glands
Five-µm paraffin sections were deparaffinized with two changes of xylene and rinsed with two changes of 100% ethanol and double distilled water. For immunostaining, a peroxidase-labeled streptavidinbiotin procedure (DAKO) was used. Sections were incubated with 3% H2O2 for 5 min and washed in water. They were rinsed with 1 x PBS, drained, and then the sections were covered with blocking solution (DAKO LSAB Kit) for 20 min. The primary antibody, rabbit anti-mouse Hmx3 [raised to an MAP-conjugated peptide in the COOH-terminal region of Hmx3 (IVRVPILYHENSAA EGAAAA)] was used at 1:100 dilution (in PBS, 0.1% BSA) for 60 min at room temperature. The specificity of the antibody is shown in Fig 6. As a negative control, BSA was applied in place of the antibody. The slides were washed in PBS, and biotinylated anti-rabbit immunoglobulin (linking solution in the kit) was applied to the sections for 20 min. After washing, freshly made streptavidinperoxide solution was applied to the slides for 20 min. The sections were then washed in PBS. Diaminobenzidine (DAB) was added for approximately 6 min, the slides were washed in water, and then were counterstained with 0.2% Light Green (Fisher). The sections were then dehydrated and coverslipped.
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Extraction of Total Proteins
Total proteins were prepared from the submandibular (SMG), sublingual (SL), and parotid (PA) glands of adult SpragueDawley rats. The tissues were removed, cut into small pieces, and homogenized in 1 ml of buffer (per 160 mg tissue) containing 1 x PBS, 0.25% Triton X-100, and a complete protease inhibitor cocktail (1 tablet per 50 ml buffer; Roche). The homogenates were centrifuged at 12,000 rpm at 4C. Protein concentrations of the supernatants were determined using the Bradford protein assay reagent (Bio-Rad). Extracts containing total proteins were aliquotted and stored at -80C until use.
Specificity of the Hmx3 Antibody
Western blotting analyses were performed using the homeodomain-containing proteins GST-Hmx3, GST-Hmx1, PreScission cleaved (P-cleaved) Hmx1 pure protein, P-cleaved Nkx2.5 pure protein, P-cleaved PITX2C pure protein, P-cleaved PITX2A pure protein, and P-cleaved Msx2 pure protein. These proteins were purified and subjected to PreScission cleavage as previously described (
Western Blotting Analyses of Hmx3 in Salivary Glands
Six micrograms of total protein were loaded into each well of a 12% SDS-polyacrylamide gel, electrophoresed at 100 V for 5060 min at 4C, and electrotransferred (Bio-Rad Mini PROTEAN II Cell) to Hybond ECL membranes (Amersham Pharmacia). As a positive control, 36 and 360 ng of pGST-Hmx 3, which includes amino acids 309458 containing the homeodomain 20-amino-terminal flanking residues and the entire COOH-terminal region, were electrophoresed in the same gel. The membranes were probed with the polyclonal antibody to Hmx3 at a 1:2000 dilution as described above. The blots were then developed using the anti-rabbit HRP antibody at a 1:4000 dilution (Bio-Rad). Immunodetection was performed using the ECL substrate from Amersham according to the manufacturer's instructions. The protein markers were the BenchMark Prestained Ladder and ranged from 9.3 to 172.6 kD (Gibco).
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Results |
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The well-documented postnatal development of rat SMGs is depicted in Fig 1. Particular emphasis on the development of the duct components is described. During days 2, 4, and 6, progressive differentiation of the acinar cell (A) compartment is noted, with increasing elongation of the ductal components of the gland (Fig 1A1C). In addition, there is a large amount of loose connective tissue connecting the newly formed lobes, and an extensive blood supply. Short intercalated ducts (IDs) are observed at day 2 (Fig 1A), but by days 4 and 6, long and branched ducts, whose epithelial cells are more cuboidal, are evident (Fig 1C). A few striated ducts (SDs) are detected at 8 days, and the acinar (A) components are much more numerous (Fig 1D). Many striated ducts are present by day 15, as well as a few granular convoluted tubules (Fig 1F). The granular convoluted tubule components, as well as the excretory ducts, are greatly increased during days 20, 25, and 33 (Fig 1G1I).
Immunocytochemical analyses of Hmx3 protein distribution indicate that, as the SMG develops with progressive increase in the duct components of the gland, increased Hmx3 protein is evident in the ducts (Fig 2). It is also interesting to note that, at 4 and 10 days of development of the SMG, Hmx3 protein is present in specific cells of the cell buds (Fig 2A and Fig 2B, arrowheads). These cells could be those that give rise to or are the precursors of duct cells. When the sections are visualized by Nomarski optics, Hmx3 is observed in specific nuclei and the cytoplasm of some duct cells as early as 4 days of development (Fig 3B). By day 10, the duct epithelial cells contain Hmx3 protein in both their nuclei and cytoplasm. However, not all of the nuclei contain Hmx3 (Fig 3D). At 15 days, immunostaining for Hmx3 can be observed in both the nuclei and the cytoplasm of the SDs and the granular convoluted tubule (GCT) cells (Fig 3F). The majority of the duct components contain large amounts of Hmx3 at 20 days of development (Fig 3H). At 25 and 28 days of development of the SMG, there is a large accumulation of Hmx3 in the cytoplasm of the larger ducts, but not all of the nuclei show nuclear staining, as was also observed at the earlier time points (Fig 3J and Fig 3L). Note that there is slight peroxidase staining in the ducts in some BSA-treated SMGs (Fig 3A, Fig 3C, Fig 3E, Fig 3G, Fig 3I, and Fig 3K). Higher magnification using Nomarski imaging of Hmx3 antibody staining of adult female SMGs demonstrates that the nuclei and cytoplasm of only certain duct cells contain Hmx3 protein (Fig 4]). At lower magnification, it is obvious that not all duct cells are stained (Fig 4A). Staining of nuclei and cytoplasm of specific duct cells can be discerned at higher magnification (Fig 4B).
The other two salivary glands from adult rats, the parotid and the sublingual, both show Hmx3 protein in the epithelial cells of the ducts (Fig 5). In the case of the sublingual gland, whose secretory components consist entirely of mucous acini, slight antibody staining is observed in the intercalated ducts. More pronounced staining in the striated duct cells and less staining in a few mature granular duct cells are observed (Fig 5C). Not all ducts are stained, nor are all epithelial cells in the ducts stained. In addition, both the nuclei and the cytoplasm of some cells are stained, but the staining is principally cytoplasmic. Hmx3 protein is also observed in selective cells of the acini (arrow, Fig 5C). The immunocytochemistry of the parotid also shows slight staining in the intercalated duct cells and the striated duct cells (Fig 5I). In both cases, the striated ducts seem to be more heavily stained. The H&E-stained sections of the submandibular, parotid, and sublingual glands demonstrate the typical staining pattern of the acini and ducts (Fig 5A, Fig 5D, and Fig 5G).
The concentration and the approximate size of rat Hmx3 protein were determined by Western blotting analyses of equivalent amounts of total protein from the three salivary glands using the Hmx3-specific antibody. The difference in the concentration of Hmx3 protein in the three salivary glands is most striking. The concentration of Hmx3 protein is significantly higher in the submandibular and sublingual glands than in the parotid gland (Fig 6A). Three major proteins (approximately 77.5, 48.8, and 44 kD) are detected in the SMG protein extract by the Hmx3 antibody (Fig 6A, Lane 1). Two major proteins, approximately 77.5 kD and 72 kD in size, are observed in the sublingual gland (Fig 6A, Lane 2). The parotid shows very faint binding of Hmx3 antibody to two proteins of 77.5 and 72 kD (Fig 6A, Lane 3). The smaller molecular weight proteins are, more than likely, difficult to detect due to low concentration (they show up on much longer exposure) in the sublingual and parotid glands (data not shown). The 48.8- and 44-kD Hmx3 proteins found in the salivary glands are in the range of the deduced size for Hmx3 protein: 51 kD (
The specificity of the Hmx3 antibody is demonstrated by Western blotting analyses of five different homeodomain proteins in addition to Hmx3: Hmx1, Nkx2.5, PITX2C, PITX2A, and Msx2. Fig 6B and Fig 6C show two different exposures of the Western blots performed in the Amendt lab (University of Tulsa) for these homeodomain proteins. The approximate molecular weights of the homeodomain proteins analyzed are GST-Hmx1 64 kD, purified Hmx1 37 kD, purified Nkx2.5 42 kD, purified PITX2C 36 kD, purified PITX2A 32 kD, purified Msx2 30 kD. The Hmx3 antibody is specific for Hmx3 and does not bind GST-Hmx1 (Fig 6B and Fig 6C, Lane 2), purified Hmx1 (Fig 6B and Fig 6C, Lane 3), purified Nkx 2.5 (Fig 6B and Fig 6C, Lane 7), purified PITX2A (Fig 6B and Fig 6C, Lane 9), and purified Msx2 (Fig 6B and Fig 6C, Lane 10). Furthermore, the Hmx3 antibody does not bind GST (Fig 6B and Fig 6C, Lane 4). However, it is of note that PITX2C (Fig 6B and Fig 6C, Lane 8) shows binding of the Hmx3 antibody represented by two bands in molecular weight regions that are not specific for PITX2C or Hmx3. We do not know at this time what these two bands represent.
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Discussion |
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The three major rodent salivary glands develop through a similar pattern of morphogenesis that is regulated by elements in the ECM, hormones, and the autonomic nervous system. Previous studies have indicated that salivary gland development entails temporal and spatial coordination among cell proliferation, cytoarchitectural organization, and the establishment of stable cellsubstratum interactions (
In recent years, a number of homeobox-containing genes have been identified. The homeobox genes in Drosophila show restricted patterns of expression during embryonic development and are believed to encode transcription factors that recognize and bind to specific DNA sequences via their highly conserved homeodomains. Based on their expression patterns, homeobox genes are involved in the development of the sensory organs and the central nervous system (
To date, the only homeobox gene that had been reported to be expressed in mouse submandibular glands is Tlx-1 (
The Hmx family is a new class of homeobox-containing genes. Three new members of the Hmx gene family, Hmx 1, Hmx 2, and Hmx 3, have been identified in humans, mice, and Drosophila (
The study presented here provides the first evidence of the presence of the homeodomain protein Hmx3 in rat salivary glands and, in particular, in the postnatally developing submandibular gland. Our findings are in agreement with previous reports of Hmx1, Hmx2, and Hmx3 expression in other sensory organ-related structures (
The fact that Hmx3 is expressed in the striated, granular, and intercalated ducts suggests that Hmx3 has a common function in these three diverse duct systems. Relatively little is known about the physiology of salivary duct cells. However, what is known is that the duct system of submandibular glands serves as a conduit for the fluid rich in exocrine proteins that is synthesized in the acini. As this primary fluid is carried into the oral cavity via the ducts, it is progressively modified. It is at the level of the epithelial cells of the ducts that a considerable amount of electrolyte flux occurs. Here, most of the sodium and chloride ions are reabsorbed and a small amount of potassium and bicarbonate ions is secreted. In addition, a few members of the aquaporin protein family of water channels that play a fundamental role in transmembrane water transport are expressed in salivary glands (
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Acknowledgments |
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Supported by Grant DE08174 to PAS from the National Institute of Dental and Craniofacial Research, National Institutes of Health, and DE13076 to AFR. Microscopy was performed at the MSSMMicroscopy Shared Resource Facility, supported, in part, by funding from NIH-NCI shared resources grant (1 R24 CA095823-01).
We wish to thank Drs Tibor Barka and Serafín PiñolRoma for critical reading of the manuscript. Thanks also to Konstantin Gaengel for help with the imaging and Jian Luo for technical assistance.
Received for publication June 13, 2002; accepted October 30, 2002.
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