Revised on April 1st 2004
Cell Adhesion, Extracellular Matrix
Epithelial Morphogenesis
(This article is produced with modifications from the original paper by Hieda and Nakanishi published in Develop. Growth & Differ.,39, 1-8, 1997.)
(Related papers)
@Embryonic rudiments of most organs including lung, kidney, submandibular gland, hair and mammary gland are composed of two types of tissues, the epithelium and the mesenchyme, and these two tissues are separated by a basal lamina. Growing epithelial tissue changes and acquires a shape unique to each organ during development. The epithelial shape change is well known to require tissue interactions with the surrounding mesenchyme. It is one of the major issues in developmental biology to understand how shape change of the epithelial tissue is achieved and what is the nature of the tissue interactions between epithelium and mesenchyme.
@The mouse embryonic submandibular gland has been used as a model system for analyzing the mechanisms of epithelial shape change and of the epithelial-mesenchymal interactions (Borghese, 1950; Grobstein, 1953). The organ rudiment can be cultured on a membrane filter or agar, and the epithelial shape change is easily followed by light microscopy. In the organ culture system, the effects of various reagents on the morphogenesis can be tested. Furthermore, the epithelium and mesenchyme can be separated from each other by treatment with collagenase, dispase or other proteases, making various recombination experiments possible.
@Formation of the mouse submandibular gland begins on late day 11 of gestation. The initial epithelial primordium arises from the invagination of the oral epithelium into the mandibular mesenchyme and then undergoes extensive branching morphogenesis during its development.
At the 12-day stage a bulbous lobule is formed at the distal region of a stalk (A) and later, several indentations appear on surface of the epithelial lobule (B) and subsequently a couple of them become narrow and deep clefts (C). Widening of the clefts and epithelial expansion lead to branching of the lobule into smaller ones by the late 13-day stage (D). After repetitive branching morphogenesis, the shape of the epithelium becomes like a bunch of grapes.
@Concomitantly with the branching morphogenesis, the epithelium also exhibits change in tissue architecture (Redman, 1987). The epithelial primordium derived from the oral epithelial sheet is formed as a mass of cells and not as a cell sheet. After substantial branching morphogenesis occurs in this cell mass, definite lumens begin to form in the branched epithelium at the late 14-day stage, leading to a fully cavitated epithelial tissue.
@It has been suggested that epithelial activities such as cell proliferation (Bernfield, Banerjee & Cohn, 1972; Bernfield & Banerjee, 1982; Lawson, 1974) and co-ordinated changes in cell shape (Spooner, 1973; Spooner & Wessells, 1972; Ash, Spooner & Wessells, 1973) may drive branching morphogenesis of the epithelium. In this context, extensive studies have been made on regulatory roles of mesenchymal factors and the extracellular matrix such as interstitial collagens and heparan sulfate proteoglycan, including the basal lamina, in the epithelial morphogenesis. @On the other hand, it has been revealed that the submandibular mesenchyme itself also exhibits dynamic morphogenetic movement (click here for an animation, in preparation) and that the mesenchymal cells can generate physical force in cooperation with the extracellular matrix (Nogawa, 1983; Nogawa & Nakanishi, 1987). These observations raise the possibility that the mesenchyme plays mechanical roles in shape change in epithelial tissue (click here for the hypothesis).
@In an effort to give a mechanistic view of early epithelial shape changes in the developing submandibular gland, in this article, we will mainly focus on the morphogenetic activities of the submandibular mesenchyme and its regulation of epithelial architecture, to which little attention has been paid.
Morphogenetic activities of the submandibular mesenchyme
Inhibition by collagenase and stimulation by collagenase inhibitor
Among various extracellular matrix components present in submandibular glands (Hardman & Spooner, 1992a), involvement of collagens in epithelial branching morphogenesis has been well established. Inhibitors of collagen synthesis and secretion, L-azetidine-2-carboxylic acid and a,a-dipyridyl (Spooner & Faubion, 1980), as well as commercially available bacterial collagenase preparations (Grobstein & Cohen, 1965), have been shown to inhibit branching morphogenesis of epithelium. Although L-azetidine-2-carboxylic acid affected the synthesis of other proteins to some degree and the collagenase preparations possibly contained glycosaminoglycan-degrading enzymes and proteases (Bernfield, Banerjee and Cohn, 1972), a highly-purifiedClostridialcollagenase, which degrades all types of collagens but not other proteins, has been shown to inhibit not only the initiation of cleft formation in epitheium of the 12-day gland but also further epithelial branching in the 13-day gland (Nakanishi et al.,1985; Nakanishi et al., 1986a; Nakanishi et al., 1986b).
Furthermore, a collagenase from bovine dental pulp, which degrades collagens I and III but not collagens IV and V, has been shown to inhibit cleft formation in the epithelium of 12-day submandibular glands (Fukuda et al., 1988). This indicates the involvement of interstitial collagens present in the mesenchyme in the initiation of cleft formation. In contrast, a collagenase inhibitor purified from the culture medium of bovine dental pulp cells, which is a tissue inhibitor of metalloproteases, promoted the formation of more clefts than were formed in control glands (Nakanishi et al., 1986a, b), suggesting the causative role of collagen in cleft formation.

(Scott F. Gilbert, "Developmental Biology"@5th Edition, pp. 684-686, 1997 and Y. Nakanishi et al., Developmental Biology, 113, 201-206, 1986 )
Electron microscopic observations have shown that the basal lamina at the distal part of the lobule is thin and discontinuous while that in the wide clefts and in the stalk region is rather thick and continuous (Coughlin, 1975; Cohn et al., 1977; Spooner and Faubion, 1980; Fukuda et al., 1988). In addition, the rate of turnover of glycosaminoglycans in the basal lamina has been reported to be faster at the tip of the lobule than at the wide clefts (Bernfield & Banerjee, 1982). Thus, stabilization of the basal lamina in the wide clefts and in the stalk region is suggested to be involved in epithelial morphogenesis. In relation to the stability of the basal lamina, it has been suggested that remodeling of the basal lamina is associated with epithelial cell proliferation, a suggestion based on the observation that the proliferation activity of the epithelium was high in the distal region of lobules compared with that in the wide clefts (Bernfield & Banerjee, 1982).
It has been shown that heparan sulfate proteoglycan binds to fibers of interstitial collagens under physiological conditions (Koda et al., 1985); that heparitinase, which inhibits epithelial morphogenesis, brings about the dissociation of collagen fibers from the basal lamina (Nakanishi et al., 1993); and that antibodies against domain E3, a heparin-binding site, of laminin-1 perturb epithelial morphogenesis (Kadoya et al., 1995). It is likely that interactions among collagen networks in the mesenchyme and laminin and heparan sulfate proteoglycans in the basal lamina are required for branching morphogenesis of the epithelium (Nakanishi et al., 1993).
Regulation of epithelial architecture and cell adhesion systems by mesenchyme
Development of epithelial architecture and cell-cell adhesion systems
As described earlier, epithelial morphogenesis in submandibular gland involves a change in the tissue architecture from a mass of cells to a cavitated sheet. It has been shown that this architectural change in the epithelium is accompanied by drastic changes in the organization of cell-cell adhesion systems.
Previous electron microscopic observations have shown that cell-cell junctions including adherens junctions, desmosomes, and tight junctions are rarely seen in the early epithelial tissue making a cell mass but become organized later (Redman, 1987; Kadoya and Yamashina, 1993). These observations have been confirmed and extended by a recent study employing immunofluorescence microscopy with antibodies against components of the cell-cell adhesion systems (Hieda et al., 1996).
In the 12-day and 13-day stages, desmoplakins I/II (major desmosomal proteins) and occludin and ZO-1 (tight junctional proteins), but not the E-cadherin/b-catenin complex, were hardly detected in the epithelial lobules while all these molecules were present in the stalk region and in the oral epithelium. Similar situations were observed in the developing rudiments of hair follicles and mammary gland. It is of note that the E-cadherin/b-catenin complex and the associated microfilaments were uniformly distributed along the cell periphery in the lobules, indicating that adherens junctions had not yet formed. In the 14-day glands, expression of these molecules associated with cell-cell adhesion systems became evident especially in the forming cavitated regions. These data indicate that cell-cell adhesion systems are less developed in the early epithelial lobules packed with cells and that there is a close relationship between changes in tissue architecture of the epithelium and those in the organization of cell-cell adhesion systems during the development.

Regulation by tissue interactions with mesenchyme
Our recent recombination experiments using submanidbular gland and lung rudiments have shown that submandibular mesenchyme plays important roles in the organization of the tissue architecture and cell-cell adhesion systems in the epithelium (Iwai et al., Develop, Growth & Differ, 1998). Lung mesenchyme was able to maintain both the organization of the lung epithelium, in which an epithelial sheet of columnar cells lines a relatively large lumen, and the apico-lateral distribtution of E-cadherin in the cells. However, when lung epithelium was cultured with the submandibular mesenchyme, the regular epithelial organization was disturbed, and E-cadherin lost its apical distribution and became re-distributed to the baso-lateral region of the cells.
Little information is available on the molecular mechanisms of the mesenchymal regulation. Novel distribution patterns revealed by immunofluorescence microscopy, however, suggest the possible involvement of epimorphin, which is a mesenchymal factor required for epithelial morphogenesis and for maintenance of cell adhesion systems and polarity of epithelial cells (Hirai et al., 1992). In submandibular glands, epimorphin distribution was hardly detected at the 12-day stage but became evident in the mesenchyme and the basal lamina region at the 13-day stage (Kadoya et al., 1995; Nanba et al., unpublished data, 1998). Noticeably, accumulation of epimorphin around the basal lamina was restricted to the stalk and not observed in the lobular region (Nanba et al., unpublished data, 1996). The appearance of the less-organized submandibular epithelium appears to involve regulation of the epimorphin deposition at the basal lamina. The lack of apparent inhibition of the submandibular gland morphogenesis by a monoclonal antibody against epimorphin may be due to the absence of epimorphin from the lobular region exhibiting active morphogenesis (Kadoya et al., 1995; Nakanishi, unpublished data, 1993).
Recently the E-cadherin distribution in submandibular epithelium has been shown to be affected by a monoclonal antibody against the integrin a6 subunit, which is distributed at the basal surface of the epithelium (Kadoya et al., 1995). The antibody inhibited the branching morphogenesis to some extent and brought about loss of the E-cadherin distribution in the inner but not peripheral area of epithelial lobules packed with cells. Since integrin a6b1 recognizes domain E8 of laminin-1 (Hall et al, 1990), constitutive expression of E-cadherin in the epithelial lobules may require signals due to interactions of epithelial cells with laminin-1, which is specifically localized in the basal lamina (Kadoya et al., 1995).
It has been well known that epithelial branching morphogenesis during the early development of the mouse embryonic submandibular gland requires tissue interactions with the surrounding mesenchyme. The mesenchymal cell population shows active flowing movement and can generate physical forces in cooperation with collagen fibers, while the epithelial organization is likely to become loose because of down-regulation of intercellular adhesion systems, as described above. Changes in the epithelial contour would therefore be dependent on such tissue properties of the epithelium and the mesenchyme, which are kept in contact in intact organ.
Some previous studies have attributed the formation of clefts to the epithelial cell proliferation and to co-ordinated contraction of actin-containing microfilaments leading to epithelial shape changes (Hardman & Spooner, 1992b). However, cleft formation seems to be much more complex, involving dynamics of tissue organization of both the mesenchyme and the epithelium. We have proposed a model in which clefts on epithelial surfaces are formed by mesenchymal forces that result from the ability of the moving mesenchymal cells to contract collagen fibers and to promote the alignment of these fibers (Nakanishi et al., 1986c; Nakanishi & Ishii, 1989).The model could be extended by taking into consideration the following notion concerning responses of cell aggregates to external forces: When external forces are applied, cell aggregates initially respond as elastic bodies with internal tensions, and if the forces persist, the internal tensions dissipate due to the ability of the cells to slip past one another (Steinberg, 1978; Foty et al., 1996).
As has been reported, before definite clefts are formed on early epithelial lobules of the submandibular gland, many tiny indentations appear and disappear on the epithelial surfaces (Nogawa, 1983; Hieda and Nakanishi, unpublished data). When the mesenchymal forces exerted on the epithelial surfaces are transient, probably due to the instability of nascent collagen bundles and of their interactions with the mesenchymal cells, such unstable indentations will occur if the epithelium has elastic properties. The elastic responses of the early epithelial tissue during initial cleft formation is also suggested by the observations that shallow but not deep clefts disappear after collagenase treament of the whole glands or the removal of the mesenchyme by protease treatment. However, as the submandibular gland development proceeds, the mesenchymal forces become persistent at certain sites of the epithelial surfaces, probably because of stable supracellular complexes containing collagen bundles and mesenchymal cells. This may be accompanied by dissipation of internal tensions because of the ability of cells to slip past one another which ability could be facilitated by the down-regulation of intercellular adhesion systems (Hieda et al., 1996). This, in turn, would help shallow clefts deepen more into the epithelium, and increasing deposition of various types of collagens including the basal lamina network would make the clefts more stable.
Even if the contraction of actin-containing microfilaments of basal epithelial cells is directly related to the cleft formation as had been proposed, the properties of the early epithelial lobules with loose cell-cell adhesion systems would be an essential prerequisite for stable cleft formation.
Finally, it is noteworthy that cell adhesion systems are also less developed in early epithelial buds of hair follicle and mammary gland (Nanba et al., submitted) and in the inner cell mass in early mouse embryos (Fleming et al., 1994). Furthermore, accumulation of epimorphin at the basal lamina region is not observed aroung the early epithelial buds of hair follicle and mammary gland (Nanba et al., submitted). These observations suggest that common mechanisms work during formation of early epithelial tissues in various organ rudiments.

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