T H E ANATOMICAL RECORD 214:113-117 (1986) Focal Tight Junctions Between Mesenchymal Cells of Fetal Dermis CLARA V. RIDDLE Department of Biological Structure, School of Medicine, University of Washington, Seattle, W A 98195 ABSTRACT This freeze fracture study shows the presence of focal tight junctions (maculae occludentes) between the mesenchymal cells in the connective tissue matrix of embryonic and fetal dermis. The overall outline of these unique junctions varies from circular to very angular. The junctional elements are most frequently present in a groove on the E fracture face. The corresponding P fracture face has ridges delineating the junction. These intercellular junctions may provide a means of informational or metabolic coupling between cells, may serve a structural role as scaffolding in the deposition and orientation of extracellular materials, or may be involved in the early stages of angiogenesis. Mesenchymal or fibroblastic cells in a connective tissue matrix are usually considered individual cells without contacts to neighboring cells. However, there are reports of intercellular contacts in connective tissue from embryonidfetal (Ross and Greenlee, 1966; Gould et al., 1972; Breathnach, 1978; Holbrook and Smith, 1981),actively growing (Gabbiani et al., 1972; Shore et al., 1981; Squier and Bausch, 19841, and cultured cells (Van der Schueren et al., 1976). These were seen in thin sections examined by transmission electron microscopy and described as either desmosomelike or gap or tight junctions. The junctions described as desmosomelike consisted of an increased density on the cytoplasmic sides of the junctional site and sometimes had a n intercellular density. They have been reported between mesenchymal or fibroblastic cells in fetal dermis (Breathnach, 1978; Holbrook and Smith, 1981), chick limb bud mesenchyme (Gould et al., 19721, fetal tendon (Ross and Greenlee, 1966; Greenlee and Ross, 1967),skin wound healing (Gabbiani et al., 19721, and actively growing tendons (Squier and Bausch, 1984) and ligaments (Shore et al., 1981) and cultured fibroblasts (Van der Schueren et al., 1976). Other very close contacts have been described as either gap junctions (Van der Schueren et al., 1976; Shore et al., 1981; Gilula et al., 1972; Pinto da Silva and Gilula, 1972; Gould et al., 1972; Kelley and Fallon, 1978) or tight junctions (Beertsen, et al., 1974; Van der Schueren et al., 1976). In this paper we utilized freeze fracture techniques and showed unequivocally that focal tight junctions exist between the mesenchymal cells in the connective tissue matrix of human fetal dermis. buffer, pH 7.4, for 15-60 minutes. After several rinses in the buffer, the specimens were immersed in buffered 25% glycerol for 90 minutes and then mounted on gold supports and frozen in Freon 22 slush cooled by liquid nitrogen. Samples were fractured and platinum replicas were prepared in a Balzers BAF 301 freeze fracture unit. Replicas were cleaned with Clorox supplemented with potassium hydroxide, picked up on copper grids and viewed in a Philips 201 transmission electron microscope. All photographs are oriented so that the direction of shadowing is from the bottom of the page. RESULTS During development the fetal dermis changes from a network of mesenchymal cells surrounded by a watery matrix to a more fibrous matrix occupied by fibroblastic cells (Breathnach, 1978; Holbrook and Smith, 1981).The change to the more fibrous condition occurs in the third month of gestation. By the fourth month the reticular and papillary regions of the dermis are delineated. Even though this study utilized dermis from a period (7-10 weeks EGA) when it is still rather homogeneous, the collected data were from tissue restricted to the zone immediately below the epidermis. The freeze fracture images confirmed the previous reports of predominately stellate and elongated cells with few fibers in the extracellular matrix. Replicas show two kinds of close relationships between cells. In some cases rather large areas of plasma membranes of two cells are closely apposed. More frequently two elongated processes are closely apposed and a small portion of their respective plasma membranes is involved in a focal tight junction. Figure 1illustrates a stellate-shaped cell which MATERIALS AND METHODS has numerous elongated processes, some of which have Skin from the proximal portions of limbs from human very close contacts to other processes which presumably fetuses of 7-10 weeks estimated gestation age (EGA) belong to neighboring cells. A focal tight junction is was obtained from the Central Laboratory for Human evident in one area of close contact (Fig. 1inset). Embryology at the University of Washington and processed for freeze fracture electron microscopy. Tissues Received July 1, 1985; accepted September 6, 1985. were fixed in 2% glutaraldehyde in 0.1 M cacodylate 0 1986 ALAN R. LISS, INC Fig. 1. Electron micrograph of a freeze fracture replica of a mesenchymal cell in the dermis. The mesenchymal cell is a stellate-shaped cell with five obvious extensive processes (*I. Three of these processes are in close contact with processes (arrows)presumably from neighbor- ing cells. The inset demonstrates the junctional elements (arrowheads) associated with the processes shown in the lower left corner of Figure 1 (7 weeks EGA: X 19,000; inset X35,OOO). Figure 2. (Legend appears on reverse.) 116 C.V. RIDDLE mis they have been postulated to function as a scaffolding for intercellular lipid lamellae (Elias and Friend, 1975). The intramembranous particle partitioning characteristic of the focal tight junction in the fetal dermis is unusual. Generally, in freeze-fractured tissue which has been pretreated with glutaraldehyde, intramembranous particles tend to partition with the P fracture face presumably because of some interaction of the junctional particles with cytoskeletal elements of the cell. This is especially true of tight junctions. In the case of these focal tight junctions between mesenchymal cells, the DISCUSSION particles tend to stay with the E fracture face. This This paper reports the first unequivocal demonstra- partitioning pattern has been reported for zonular tight tion of focal tight junctions (maculae occludentes) be- junctions between endothelial cells of postcapillary ventween mesenchymal cells in a connective tissue matrix. ules (Simionescu et al., 1976, 1978; Staehelin, 1975). The discontinuous nature of the rows of junctional These junctions have specific characteristics revealed by freeze fracture techniques. The junctions are 1)“focal” particles in dermal focal tight junctions is also unusual. or localized to a small area; and 2) the junctional ele- Without true complementary replicas of these dermal ments partition mostly with the E fracture face (P face junctions, it cannot be determined whether the sum of partitioning is the usual case for junctional elements in the particles on both the P and the E fracture faces freeze fracture); 3) the junctional elements on the E would form a continuous strand. In the endothelial cells fracture face sit in a groove; 4)the elements are noncon- of postcapillary venules where a similar “loose” organitiguous; and 5) the P fracture face has complementary zation of intramembranous particles has been described, Simionescu et al. (1978) concluded, on the basis of thin ridges with a few junctional elements present. Such a junction is difficult to label since junctions with sections and tracers, that 70% of the junctions could be these exact characteristics have not been reported. How- classified as tight and 30%as open. The overall shape of the focal tight junctions between ever, as increasingly more tissues are subjected to freeze fracture studies, it becomes evident that the “standard” fetal dermal mesenchymal cells is quite variable. This types of intercellular junctions are heterogeneous. Until variability may be a characteristic for this category of more evidence is available on the functions of specific focal tight junctions or it may reflect the fact that these junctions, we are forced to categorize junctions on the junctions do not all belong to one class of junctions. They basis of their structural characteristics. For the purposes may represent differential interactions between homoof this report we are categorizing the junctions between typic or heterotypic cells. Several cell types have been reported for the human mesenchymal cells as focal tight junctions because they are focal and are similar in structure to those reported fetal dermis 7-10 weeks EGA. Breathnach (1978) has for some occluding junctions (Staehelin, 1975; Simi- described three main cell types: “undifferentiated general mesenchymal cells,” which are the most numerous onescu, et al., 1976, 1978). The primary characteristic of these focal tight junc- cell type and can be stellate or elongated; a phagocytic tions in the developing dermis is their localized, nonzon- cell type; and a cell type with numerous small vesicles. ular character. Focal tight junctions with characteristics Elements of the nervous system such as neurons and common to zonular tight junctions have been reported Schwann cells are also present. Small blood vessels are in a variety of epithelia where they have usually been also evident a t this period (Smith and Holbrook, 1982). Some of the junctions illustrated in this paper may be associated with the breakdown or formation of continuous zonular tight junctions. In adult mammalian epider- between mesenchymal cells only or between mesenchyma1 cells and those of the other classes since cell type could not always be identified. Considering the close similarity of these focal tight junctions to the “loose” tight junctions present between endothelial cells of postFig. 2. Freeze fracture electron micrographs of focal tight junctions capillary venules (Staehlin, 1975; Simionescu et al., (maculae occludentes) between mesenchymal cells. Junctional ele1976, 19781, it is possible that these junctions might be ments are more frequently present on the E fracture face (EF) than the P fracture face (PF). The junctional elements on the E fracture face the initial interactions between mesenchymal cells are usually noncontiguous and are lined up in a groove. The corre- which eventually are seen as capillary sprouts. In angisponding P fracture face has ridges delineating the junction. a) Focal ogenesis numerous capillary sprouts are formed, but not tight junction with circular outline. The P fracture face has slightly all end up as capillaries since many are broken down elevated continuous ridges on which are a few noncontiguous particles. A large portion of the E fracture face from the adjacent cell is also (Folkman and Haudenschild, 1980). Thus some of these present. A larger proportion of the particles is associated with shallow focal tight junctions may possibly represent different grooves on the E fracture face (8 weeks EGA; ~50,000.b) Focal tight stages in this process. junction consists of irregularly arranged linear segments in groove on The mesenchymal cells of the dermis are part of a the E fracture face. A contacting process (p) presumably from a neighsystem undergoing differentiation and morphogenesis. c) Focal tight junction boring cell is evident (10 weeks EGA: ~50,000). is linear without any branching or angularity. Fragmentary particles The focal tight junctions could be responsible for comare present in a groove on the E fracture face (10 weeks EGA, ~35,000). munication or metabolic coupling between adjacent cells d) Focal tight junction consists of linear portions with several interconnecting bars. Process (p) from adjacent cell is present (9 weeks EGA; or a network of cells. Although such coupling is gener~35,000).e) Focal tight junction is linear with sharp angles present. ally considered the function of gap junctions (Kelley and Adjacent process (p) is present (9 weeks EGA: ~35,000). Fallon, 19831, Sheridan (1980) showed that fluorescent These intercellular junctions are recognized by several characteristics. Their overall form varies from circular (Fig. 2a), to rectangular (Figs. 2b,d), to mere linear in outline (Figs. 2c,e). Some of the linear junctions have distinctive sharp angles (Fig. 2e). In all cases the junctions are delineated by continuous grooves on the E fracture face and by continuous ridges on the P fracture face. Some irregular and noncontiguous intramembranous particles are present on both these grooves and ridges, although the majority of them are associated with the grooves of the E face. FOCAL TIGHT JUNCTIONS dye is transferred between endothelial cells of postcapillary venules and capillaries. Gap junctions are absent between endothelial cells in these portions of the vasculature. The only intercellular junctions are tight junctions (Larson and Sheridan, 1982) with the structural characteristics of the focal tight junctions reported here. Sheridan’s suggestion that tight junctions might be involved in intercellular exchange between endothelial cells might also be valid for the mesenchymal cells of the dermis. The dermis in fetal skin is fast growing and continuously remodeled. The junctions could be linking cells together as a temporary scaffold for modeling and remodeling of the dermis. In this regard, it has been proposed by Trelsted (1982) that collagen-secreting cells in tendon line up with specific orientations to direct the deposition and alignment of the appropriate collagen lattice. A cellular and fibrous scaffold in the fetal dermis could provide a template for the orientation and movement of cells. It may also provide a structure onto which other cell types or extracellular matrix components may attach. 117 Gabbiani, G., B.J. Hirschel, G.B. Ryan, P.R. Statkov, and G. Majno (1972) Granulation tissue as a contractile organ. J. Exp. Med., 135:719-734. Gilula, N.B., O.R. Reeves, A. Steinbach (1972) Metabolic coupling, ionic coupling and cell contacts. Nature, 235:262-265. Gould, R.P., A. Day, L. Wolpert (1972)Mesenchymal condensation and contact in early morphogenesis of the chick limb. Exp. Cell Res., 72:325-336. Greenlee, T.K., R. Ross (1967) The development of the rat flexor digital tendon, a fine structure study. J. Ultrastruct. Res., 18t354-376. Holbrook, K.A., and L.T. Smith (1981) Ultrastructural aspects of human skin during the embryonic, fetal, premature, neonatal, and adult periods of life. Birth Defects: March of Dimes Series 17t9-38. Kelley, R.O., and J.F. Fallon (19781 Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud. J. Embryol. Exp. Morphol. 46:99-110. Kelley, R.O., and J.F. Fallon (1983)A freeze-fractureand morphometric analysis of gap junctions of limb bud cells: Initial studies on a possible mechanism for morphogenetic signalling durring development. Prog. Clin. Biol. Res., 101At119-130. Larson, D.M., and J.D. Sheridan (1982) Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures. J. Cell Biol., 92183-191. Pinto da Silva, P., and N.B. Gilula (1972) Gap junctions in normal and transformed fibroblasts in culture. Exp. Cell Res., 71t393-401. Ross, R., and T.K. Greenlee (1966) Electron microscopy: attachment sites between connective tissue cells. Science, 253:997-999. Sheridan, J.D. (1980)Dye transfer in small vessels from the rat omenturn: Homologous and heterologous junctions. J. Cell Biol., 87:61a. ACKNOWLEDGMENTS Shore, R.C., B.K.B. Berkovitz, B.J. Moxham (1981) Intercellular conThis study was supported by NIH grants HD17664 tacts between fibroblasts in the periodontal connective tissues of and AM21557 and the Dermatology Foundation. I thank , the rat. J. Anat., 133t67-76. M., N. Simionescu, G.E. Palade (1976) Segmental differDrs. K.A. Holbrook and L.T. Smith for critical reading Simionescu, entiations of cell junctions in the vascular endothelium. Arteries of the manuscript, Drs. J. Luft, S. Schwartz, and S. and veins. J. Cell Biol., 68:705-723. Bordin for interesting discussions, Cynthia Stahl for Simionescu, N., M. Simionescu, G.E. Palade (1978) Open junctions in the endothelium of the postcapillary venules of the diaphragm. J. excellent technical assistance, Robert Underwood for Cell Biol., 79:27-44. excellent photography, and Dr. J. Koehler for use of the Smith, L.T., and K.A. Holbrook (19821Development of dermal connecBalzer’s. I also thank Dr. G.F. Odland and Ms. M. Hoff tive tissue in human embryonic and fetal skin. SEM, 411745-1751. for various aspects of this paper. Squier, C.A., and W.H. Bausch (1984)Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon. Cell Tissue LITERATURE CITED Res., 238:319-327. Staehelin, L.A. (1975) A new occludens-like junction linking endotheBeertsen, W., V. 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