The dependent and independent relationships between cytodifferentiation and morphogenesis in developing salivary gland secretory cells.код для вставкиСкачать
THE ANATOMICAL RECORD 196:341-347 (1980) The Dependent and Independent Relationships Between Cytod iff erent iat i on and Mo r phogenesis in Developing Salivary Gland Secretory Cells LESLIE S. CUTLER Department of Oral Dingnosis, University of Connecticut, School of Dental Medicine, Farmington, Connecticut 06032 ABSTRACT The mesenchymal capsule was removed from the epithelial anlage of 15 day (unbranched) and 16 day (initially branched) fetal rat submandibular gland (SMG) rudiments. The undifferentiated epithelial portion of the SMG rudiments was placed in tissue culture and examined by light and electron microscopy and histochemistry for secretory peroxidase. The 15 day fetal SMG epithelial rudiments failed to attach, and spread over the culture dish and degenerated by 3 days in culture. The 16 day epithelial rudiments attached to the dish and the cells spread radially from the explant. Mitotic activity was minimal. Cells spreading from the 16 day rudiments underwent cytodifferentiation, giving rise to two secretory cell types: 1) peroxidase containing “proacinar cells,” and 2) secretory “terminal tubule” cells. The results suggest that in the developing SMG, morphogenesis and cytodifferentiation are partially coupled but independently regulated processes. The earliest phases of morphogenesis (rudiment down growth and primary branching) seem to be required to initiate cytodifferentiation. Once initiated, cytodifferentiation can proceed in the absence of continued morphogenesis (tissue organization) or significant amounts of connective tissue elements. The fact that one developing tissue can influence the development of another developing tissue was demonstrated by Spemann in 1901, introducing the concept of “inductive tissue interactions.” This idea of two or more tissues of different histogenetic origin, and function becoming closely associated and subsequently effecting their final developmental pathways, has become generally accepted as a major regulatory force in embryonic development (Grobstein, ’56; Lehtonen, ’76). Epithelial-mesenchymal interactions of this nature are required for the development of a wide variety of tissues, including the salivary glands, skin, teeth, mammary glands, ureteric bud, and several others. The mechanism(s)by which these tissue interactions mediate their inductive influences are not fully understood. The organization of chemically unique cells into a functional structural configuration is required for the normal development of a particular tissue. In most cases, cells are first organized into specialized structural units (morphogenesis) and then these cells develop their architectural and biochemical (production of cell-specific molecules)identity (cytodifferentiation) (Rutter et al., ’67).During the de- 000-3276x180/1963-0341$01.400 1980 ALAN R. LISS, INC. velopment of rodent salivary glands and the development of virtually all exocrine organs, the initial phases of morphogenesisprecede the onset of low levels of cell-specific protein production. In most of these systems, morphogenesis is relatively advanced before production of cell-specific protein reaches the level a t which the proteins are packaged into demonstrable secretory granules. It is not known, for salivary glands or for most exocrine glands, to what degree the processes of morphogenesis and subsequent cytodifferentiation are linked with regard to temporal sequencing or with regard to maintenance of cytodifferentiation in the absence of morphogenesis. Numerous studies have shown that a finite (relatively large) quantity of mesenchyme is necessary for the morphogenesis of rodent salivary gland tissue (Borghese, ’50; Grobstein, ’53a, b; Lawson, ’72, ’74). However, no evidence is available with regard to the necessity of the presence of mesenchyme for the cytodifferentiation of rodent salivary gland exocrine cells. The current report describes studies which suggest that morphogenesis and cytodifferentiation are Received May 3, 1979 accepted July 26, 1979. 341 LESLIE S. CUTLER 342 separately controlled processes, which are only partially coupled in the development of the rat submandibular gland (SMG). Further, the results suggest that different types of epithelial-mesenchymal interactions may be required for morphogenesis and cytodifferentiation of the rat SMG. MATERIALS AND METHODS Isolation and culture of the SMG Anlage Sprague-Dawley rats were bred under rigidly controlled conditions as previously reported (Cutler and Chaudhry, '73a). On the 15th and 16th days of gestation, fetuses were aseptically delivered by sterile laparotomy. The SMG rudiments, which show no morphologic signs of cytodifferentiation (Cutler and Chaudhry, '74; Jacoby and Leeson, '591, were excised, washed twice in calcium-magnesium free Tyrode solution, and then incubated for 40 minutes a t room temperature in 0.25% trypsin (Difco 1/250) in calcium-magnesium free Tyrode solution in the well of a glass depression slide. The incubation was terminated by removing the trypsin solution and washing the rudiments with McCoy 5A medium, containing 10%fetal calf serum. The mesenchymal capsule was removed by carefully passing the rudiments in and out of a sterile pasteur pipette and then gently dissecting (under a dissecting microscope with sterile microdissecting instruments) and removing the capsule from the epithelial anlage. The isolated epithelia were cultured in the center well of a plastic organ culture dish (Falcon plastics) directly on the plastic substratum. The cultures were incubated in McCoy 5A medium or Fitton-Jackson modified BGJ medium (GIBCO, Grand Island, NY); both media were supplemented with 10%fetal calf serum, gentamycin, and neomycin. The culture incubations were carried out at 37°C in a humidified 95% air-5% CO, atmosphere. The medium was changed daily. Light and electron microscopy Cultures were monitored daily by phase contrast microscopy. At 3 and 5 days of culture, cells were fixed by removing the medium and flooding the culture dish with cold (4°C) 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 5 minutes. The fixative was removed and the cultures were washed 4 times (10 minutes each) with cold 0.1 M cacodylate buffer (ph 7.3). Some of the dishes were then incubated in 0.05 M tris-HC1 buffer (pH 7.0) containing 10 mM DAB (3,3'-diaminobenzidine tetrahydrochloride, Sigma Chemicals, St. Louis, MO), 5 mM KCN and 1 mM (0.003%)Hz02to demonstrate secretory peroxidase (Graham and Karnovsky, '66). The incubation was carried out for 1hour a t 37°C in the dark. Following the incubation, the cultures were washed twice in 0.1 M cacodylate buffer (pH 7.3) and then postfixed in 1%cacodylate buffered osmium tetroxide. Control experiments included dishes incubated in medium depleted of H,O, or DAB and dishes fixed for 3 hours with 3% cacodylate buffered glutaraldehyde. After postfixation, the cultures were stained for 1hour in 0.25% aqueous uranyl acetate. A t this point, some cultures were lightly stained in 1%methylene blue in 1%borax and examined by light microscopy. Other cells were lifted from the culture dish with a rubber policeman, suspended in 25% acetone, and concentrated by centrifugation (2500 g for 10 minutes). The resulting pellet was dehydrated through a graded series of acetones and embedded in Spurr low viscosity resin (Spurr, '69). Thick (0.5 p ) and thin (6OCk900 A) sections were cut on a n LKB-ultrotome 111. Thick sections were checked by light microscopy for orientation, and thin sections were then examined to confirm the specificity of the histochemical reaction. The thin sections were subsequently counterstained with lead citrate (Venable and Coggeshall, '65) to enhance contrast and examined on a Zeiss EM10 electron microscope. RESULTS Forty-three of 48 explanted 15 day fetal SMG epithelial analgen failed to spread over the culture dish. After 24 hours in culture, these rudiments had rounded into small spheres of cells and by 48 hours in culture, all had detached from the substratum and were floating in the medium. Most of these epithelial spheres had degenerated by 72 hours in culture. The five explants that did attach and spread over the culture dish behaved like the 16 day explants. Thirty-one of 46 explanted 16 day fetal SMG epithelial anlagen attached to the plastic substratum and cells spread radially from the main explant. Cell division was minimal and although there were 7 or 8 rudiments per culture dish, confluence was not reached during the 5 days of culture. By 2-3 days of culture, many cells could be seen with small granules in their cytoplasm and many of these granules contained peroxidase (Fig. 1).The cells did not show any polarity of granule disposition in the cytoplasm and the cells did not show any tendency t~ form acinar or ductal structures (Figs. 1, 2). A t the ultrastructural level, cells which were Fig. 1. Light micrograph of cells from a 16 day fetal SMG epithelial anlage explant after 3 days in culture. The cells were fixed in glutaraldehyde and histochemically stained to demonstrate peroxidase activity. Two epithelial cell types can be identified by the type ofgranule in their cytoplasm (A, B). The granules are generally small in both cases, but the peroxidase containing granules appear much darker because of the oxidized DAB-osmium reaction product (B). Cells without definable granules can also be identified ( X 750). Fig. 2. Light micrograph of cells from a 16 day fetal SMG epithelial anlage explant a f k r 5 days in culture. These cells were fixed and stained to demonstrate peroxidase activity. Two epithelial cell types can be readily identified by the granule type in their cytoplasm (A, B). The majority of cells in this field (B) contain peroxidase positive granules in their cytoplasm. The granules are randomly distributed in the cytoplasm and no polarity of organelle distribution can be seen. The cells do not group into organized acinar or ductal structures ( x 750). Fig. 3. Electron micrograph of a cell from a 16day fetal SMG epithelial anlage explant after 3 days in culture. The cell has been stained to demonstrate peroxidase activity. Weak DAB reaction product (peroxidaseactivity) can be seen in the cisternae of the endoplasmic reticulum (ex.) while more intense reaction product can be seen in the nuclear envelope (arrows). No secretory granules are evident ( X 10,000). 344 LESLIE S. CUTLER apparently free of granules but showed peroxidase reactivity in the cisternae of the rough endoplasmic reticulum (e.r.1and nuclear envelope, were seen in the 16 day explants after 3 days of culture. Peroxidase activity was more pronounced in the nuclear envelope than in the e.r. (Fig. 3). Several cells were also seen with marked peroxidase reactivity in the cisternae of the e.r. and with several small peroxidasecontaining granules in their cytoplasm (Fig. 4). At 5 days in culture, two cell types were clearly definable on the basis of secretory granule type. One cell type had peroxidase-positivegranules and the other had secretory granules which did not contain peroxidase (Fig. 5). Those 16 day epithelial rudiments which failed to attach and spread on the culture dish behaved similarly to the 15 day fetal rudiment explants. The cytochemical controls were generally nonreactive in all cases. Faint reaction product was seen in the HzO,-depleted control and in the dishes fixed for 3 hours. This sparse reaction product was due, in part, to low levels of endogenous H,O, and to the residual peroxidase activity after prolonged fixation. No reaction product was seen in the DAB-depleted controls. DISCUSSION The results of this study indicate that exocrine cells in the rat SMG can undergo cytodifferentiation in the absence of continued or advanced morphogenesis or substantial amounts of mesenchymal tissue. These observations confirm and extend those of Spooner and coworkers ('77) on the exocrine pancreas and suggest that a large degree of independence exists between cytodifferentiation and morphogenesis. Further, this independencemay be common to all exocrine tissues. As has been pointed out by Spmner et al. ('771, this separation of cytodifferentiation and morphogenesis refers uniquely to the transition of protodifferentiated cells (cellsproducing low levels of cell specific proteins) into more highly differentiated cells. It must be stressed that the initial phases of morphogenesis (up to and including the initiation of rudiment branching) seem to be a prerequisite to cytodifferentiation. Thus, initial morphogenetic events seem to be obligatorily linked to cytodifferentiation. The results of the current study are also in agreement with a recent report by Sakakura and coworkers ('761, which showed that cytodifferentiation of mammary epithelium was unaffected by the morphogenetic organization of the epithelium in heterotypic cultures with sali- vary mesenchyme, and with a report by Lawson ('74) on the regulation of parotid gland cytodifferentiation. These investigators stressed the importance of mesenchymal factors in the regulation of morphogenesis. The specific biosynthetic function of developing epithelium is apparently determined genetically during the developmental history of the epithelium, and once initiated, seems to proceed independently of morphogenesis. It has been shown that a few cells in the end-buds of the initial branches of the 16 day SMG rudiment contain secretory peroxidase within the cisternae of their endoplasmic reticulum. Thus, low levels of specific secretory proteins are present at least 2 days prior to the formation of peroxidase containing secretory granules in the developing proacinar cells (Yamashina and Barka, '72; Cutler and Mooradian, unpublished) indicating that these cells are indeed protodifferentiated. No cells of this type have been seen in 15 day SMG rudiments. The timing of granule appearance in the cultured 16 day SMG epithelial cells is in close agreement with the appearance of secretory granules both in vivo (Yamashina and Barka, '72, '73; Cutler and Chaudhry, '73b, '74) and in organ culture (Rufo and Barka, '76; Cutler, '77) of the developing rat SMG. The observation of two varieties of cells, based on secretory granule type, is also consistent with the developmental sequence seen in vivo and in organ culture. The observation that 15 day fetal SMG epithelium failed to survive in culture in the absence of significant amounts of mesenchyme is consistent with the early classical studies on salivary gland development which showed that at this stage of development the epithelial anlage of the SMG required mesenchyme in order to survive in culture (Borghese,'50; Grobstein, '53a, b). On the other hand, 16 day fetal SMG epithelium will attach, spread, and differentiate into definitive proacinar cells and secretory terminal tubule cells in the absence of significant amounts of mesenchyme.This observation suggests that between days 15 and 16 of gestation, changes occur in the developing SMG epithelia which permit cytodifferentiation to proceed in the absence of mesenchyme. Some of the changes in the SMG cells, which occur during this period may be at the cell surface and may be reflected in the increased ability of cells from the 16 day rudiments to attach to the culture dish. Attachment to substrata is known to be required for differentiation of some cell types. However, a wide variety of cytoplasmic and/or nuclear changes may also be involved in the MORPHOGENESIS AND CYTODIFFERENTIATION 345 Fig. 4. Electron micrograph of portions of three cells from the outgrowth of a 16 day fetal SMG epithelial anlage which was in culture for 3 days. The cells have been stained to demonstrate peroxidase activity. Two ofthe cells (A, B) show DAB reaction product indicative of peroxidase activity in their endoplasmic reticulum (e.r.1and nuclear envelope (arrowheads). These cells also contain several small DAB positive secretory granules (small arrows). The third cell (C) contains several large granules (g)which do not show peroxidase activity. Strands of endoplasmic reticulum that do not contain peroxidase activity can also be seen (large arrows) ( x 4,000). Fig. 5. Electron micrograph of portions of two cells (A, B) from the outgrowth of a 16 day fetal SMG epithelial anlage which was in culture for 5 days. Cell A contains numerous, large pale granules (g). Cell B contains many electron dense granules (arrows) ( x 4,000). Inset: Higher magnification electron micrograph showing the different types of granules ( x 10,000). 346 LESLTE S. CUTLER developmental changes which take place i n the SMG cells between the 15th and 16th days of gestation. Independent of the nature of these changes, it appears that the epithelial cells must be in close association with the mesenchyme during this brief but important time period for the changes required for cytodifferentiation to occur. These observations suggest that epithelialmesenchymal interactions of some type are required for cytodifferentiationto proceed. There appear to be at least two types of epithelialmesenchymal interactions involved i n SMG development. There are epithelial-mesenchyma1 interactions involved in initiating and maintaining morphogenesis of the SMG. These interactions require that relatively large quantities of mesenchyme remain in close association with the developing epithelium for relatively prolonged periods in order to maintain morphogenesis (Borghese, '50; Grobstein, '53a, b). The epithelial-mesenchymal interactions which appear to be involved in the cytodifferention of the SMG are apparently of a more transient nature and do not require continued association of epithelium and mesenchyme once they have occurred. The transient, direct heterotypic, cell-cell (epithelial-mesenchymal) interactions known to occur in the developing SMG rudiment prior to cytodifferentiation in vivo and in vitro could represent this second type of interaction (Cutler and Chaudhry, '73; Cutler, '77). Transient epithelial-mesenchymal contacts of this type have been described in a variety of developing tissues other than salivary glands including teeth, duodenum, skin, and lung (see Lehtonen, '76; and Cutler, '77 for review). The exact mechanism(s) of transmission of developmental signals by contact-mediated tissue interactions is not known, but current theories have recently been reviewed by Lehtonen ('76). The current report represents only the second experimental study dealing with the relationship between morphogenesis and cytodifferentiation in developing exocrine systems. The results suggest that in the development of the rat SMG, morphogenesis and cytodifferentiation are partially coupled, independently regulated processes and support the data of Spooner et al. ('77) in the developing exocrine pancreas. This confirmation suggests that the observation may represent a general biologic phenomenon in the development of secretory organs. ACKNOWLEDGMENTS A preliminary report of this study has been presented Journal of Dental Research58A:288, 1979. The author wishes to thank Ms. M. Reid and Mrs. C. Christian for their technical assistance and Mrs. V. Every for her secretarial help. This study was supported in part by N.I.H. grant DE-03933. LITERATURE CITED Borghese, E. (1950)The development in vitro of the submandibular and sublingual glands of Mus musculm. J. Anat., 84:287-302. Cutler, L.S. (1977) Intercellular contacts a t the epithelialmesenchymal interface of the developing rat submandibular gland in vitro. J. Embryol. Exp. Morphol., 39:71-77. Cutler, L.S., and A.P. Chaudhry (1973a) Intercellular contact a t the epithelial-mesenchymal interface during the prenatal development of the rat submandibular gland. Dev. Biol., 33r229-240. Cutler, L.S., and A.P. Chaudhry (1973b) Differentiation of the mywpithelial cells of the rat submandibular gland in vivo and in vitro: An ultrastructural study. J. Morphol. 140:34%354. 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