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Focal tight junctions between mesenchymal cells of fetal dermis.

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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
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ionic coupling and cell contacts. Nature, 235:262-265.
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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
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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
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lial cells of small capillaries (probably venules) of rat jejunum. J.
fibroblasts in the periodontal ligament of the rat incisor and their
Cell Sci., 18545-551.
possible role in tooth eruption. Archs. Oral Biol., I9:1087-1098.
Trelstad, R.L., D.E. Birk, F.H. Silver (1982)Collagen fibrillogenesis in
Breathnach, A.S. (1978) Development and differentiation of dermal
tissues, in solution and from modeling: A synthesis J. Invest. Dercells in man. J. Invest. Dermatol., 71:2-8.
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Elias, P.M., and D.S. Friend (1975) The permeability barrier in mam- Van der Schueren, B, J.J. Cassiman, H. Van Den Berghe (1976) Aggremalian epidermis. J. Cell Biol., 65:180-191.
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