Spatial Distribution of Mitosis in Mouse Epidermis ' IAN C. MACKENZIE Department of Oral Biology, College of Dentistry, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
The cells of the upper strata of mammalian epidermis are flattened and aligned to form regular columnar units. It has been suggested that the position of the smaller underlying basal cells is related to the overlying cell columns. Examination of the position of metaphase figures in sheets of mouse epidermis indicated that mitosis occurs principally in cells lying just within the periphery of the cell columns but that there is no alignment of interphase basal cells within the columnar peripheries which could account for this position of mitosis.
Maintenance of stratified squamous epithelia is associated with continuous cell proliferation. Although spatially organized patterns of cell proliferation have been demonstrated in some epithelial structures such as hair follicles (Epstein and Maibach, '67) and tongue papillae (Cameron, '66), cell division has generally been thought to occur randomly among the basal cells of other keratinizing epithelia (Frei et al., '63; LeBlond et al., '64). Recently, however, it was shown that mammalian epidermis has an ordered structure in which alignment of the suprabasal cells results in the formation of a regular series of columnar units (Mackenzie, '69, '72; Christophers, '71). The presence of this previously unsuspected pattern of organization suggested the possibility of a related pattern of mitotic activity in the underlying basal cells. Some evidence presented to support this concept (Mackenzie, '70) suggested that mitosis occurred principally beneath the periphery of the overlying cell columns. Although no further attempts to accurately localize the position of greatest mitotic frequency have been reported, the idea of a pattern of mitosis related to epidermal structure has been extended to include concepts of an "epidermal proliferative unit" (Potten, '74) or complex patterns of cell migration from the basal layer (Gertler et al., '73). Therefore, to further investigate such patterns of activity, the relationship of the position of mitotic and non-mitotic basal cell nuclei to the overlying cell columns was examined. Intact ANAT. REC., 281: 705-710.
sheets of mouse epidermis were used to overcome some of the problems previously encountered in an analysis of data from sections of material (Mackenzie, '70). The purpose of the investigation was two-fold: (1 ) to localize more accurately the regions demonstrating a high rate of accumulation of metaphase figures and, (2) to examine the position of non-mitotic, or interphase, basal cells for a pattern of alignment beneath the overlying cell columns which might provide evidence of functionally independent units of basal cells. MATERIALS AND METHODS
Intact sheets of ear epidermis, prepared by treatment with buffered EDTA (Scaletta and McCallum, '72) were taken from six adult male Balb/C mice which were injected with Colchicine (0.2 mg per 100 g body weight) four hours before death to arrest dividing cells in metaphase. The epidermal sheets were fixed in Bouin's solution, washed, stained with hematoxylin, mounted flat on microscope slides, and examined using Nomarski interference microscopy so that the columnar units in the stratum corneum were clearly visible. When viewed from the surface each cell column is hexagonal in outline and because of the marked cell flattening which occurs during column formation (fig. l ) , each column overlies an area containing 10-11 basal cells (fig. 2). By focusing Received July 22, '74. Accepted Sept. 3, '74. 1This work was supported by John A. Hartford Foundation, Inc.
705
706
IAN C. MACKENZIE
Fig. 1 Frozen section of ear epidermis stained with methylene blue and expanded in alkaline buffer (Mackenzie, ’70) to demonstrate the columnar units of structure formed by flattening and alignment of the cells of the upper epidermal strata. The smaller underlying basal cells do not appear to be regularly positioned in relation to the cell columns. Scale = 20 p m .
through the epidermal sheets and using a projection arm, tracings were made of the relationship of over 3,000 blocked metaphase figures to their overlying cell columns. To reduce parallax error, an attempt was made to focus on the hexagonal cell outline lying deepest in the tissue and, therefore, nearest the basal cell nuclei. From the small intracellular granules present at this plane of focus, this level appeared to be the level of transition between the upper stratum granulosum and lower stratum corneum. On the tracings, the centerpoint of each cell column and each metaphase figure was found by superimposition of a transparent template marked with concentric circles. Measurements were then made of ( a ) the distance from the center of the column to the center of each traced metaphase figure and, ( b ) the length of the radius passing through the center of each metaphase figure (fig. 3 ) . To determine whether the very large majority of basal cells which do not appear
in metaphase at any given time of examination were selectively positioned in relation to the overlying cell columns, the position of all the basal cell nuclei lying beneath 287 columns was examined using the same methods. Assuming a null hypothesis of random distribution of nuclei, there would be an equal probability of the occurrence of nuclei within regions of the basal layer which are of an equal area, Therefore, to determine whether the centers of nuclei occurred with equal frequency beneath various regions of the overlying cell columns, the total area beneath each column was considered to be subdivided by a series of similar concentric hexagons into ten regions of equal area. The series of hexagons which defined such regions was found by subdivision of the measured column radius (r,”) into lengths Ti, rz . . . rlo,such that r, = r z / x . . . rlo//10 (fig. 3 ) . These values were, therefore, calculated for each
SPATIAL DISTRIBUTION OF MITOSIS IN MOUSE EPIDERMIS
707
Fig. 2 A. Sheet of mouse ear epidermis (Nomarski interference microscopy) showing the boundaries of hexagonal units of structure in stratum corneum. B. Same field of vision with plane of focus adjusted to the basal layer of the epidermis showing a blocked metaphase figure (arrow). Projected outlines of the periphery of overlying columns are marked. Scale = 30 fim.
individual pair of measurements, for each type of nucleus using a computer program which also accumulated the number of nuclei occurring in each region. The frequency of occurrence of interphase nuclei
and metaphase figures in the ten regions of equal area from the center to the outer border of the overlying cell columns was then examined for deviations from randomness.
708
IAN C. MACKENZIE CENTRE
PERIPHERY
J 1
4 3 \ \
P' I
Region
Fig. 3 Method of relating the position of a metaphase figure to the overlying cell columns. Right: Measurements were made of ( a ) the distance from the centre of the column to the metaphase figure, and (b) the distance from the centre of the cell column to the periphery along a line through the centre of the metaphase figure. Left: Concentric similar hexagons which defme regions of equal area with the major hexagonn dividing any radius into a series of segments r1, rz, etc., the proportion of which depends upon the number of areas into which the hexagon is divided (see text). From the ratio of a:b, each observed metaphase was allocated to one of ten such regions.
RESULTS
With Nomarski interference microscopy, both the outlines of the cell columns in the stratum corneum and the basal cell nuclei were clearly visible. No obvious arrangement of interphase nuclei to form groups underlying each cell column was observed, but there was a distinct visual impression that the majority of metaphase figures lay beneath the periphery of the cell columns. The visual impression of a lack of alignment of interphase basal cells within each cell column was supported by the results of measurements of their distribution which did not detectably differ from a random distribution. Each of the equal areas produced by the ten concentric subdivisions of the columns contained the centers of approximately 10% of the interphase nuclei (table 1). The observed distribution of metaphase figures (fig. 4) differed markedly and significantly from a random distribution (by
I
I
0 Scale in Frn
5
I
10
I
14.0i0.2
Fig. 4 Distribution of the centres of 3,153 metaphase figures within ten regions of equal area from the centre to the edge of overlying cell columns. Each point represents the mean for that region o f six animals (k S.D.). The scale (based on mean column width) shows that peak distribution occurs at a distance of 4-6 pm from the periphery.
xz, p < 0.05 for each animal, for grouped data p < 0.001). The mean measured radius of cell columns was 14.0 i- 0.2 pm and the mean distance between the centers of basal cell nuclei was 8.6 4 0.7 pm (standard errors based on the means of six animals). The region with the greatest frequency of occurrence of metaphase figures lay at a mean distance of 4.4 2 0.5 pm inside the periphery of overlying cell columns, a distance which corresponded quite closely to half the diameter of a basal cell. Metaphase figures were found less frequently beneath the central region of columns and directly beneath the columnar junctions (fig. 4). It, therefore, appeared that the basal cells lying just within the periphery of the overlying cell columns, showed a higher potential for mitotic activity than those elsewhere. DISCUSSION
Analyses of the distribution of cell division by examination of the spacing between mitotic or DNA-labeled cells in sections of epithelia have usually been interpreted as indicating an essentially random distribution of cell division, though an unexplained tendency for a greater than ex-
709
SPATIAL DISTRIBUTION OF MITOSIS IN MOUSE EPIDERMIS TABLE 1
The distribution of the center points of all basal cell nuclei within ten concentric regions of equal area beneath 287 cell columns in the upper epidermal strata 1
2
3
4
5
6
7
8
9
10
Mean 5%
9.82
10.81
8.95
9.52
10.56
9.36
10.37
10.69
9.72
10.21
S.E.
0.78
0.86
0.94
0.51
1.1
1.26
0.69
0.86
0.80
1.1
Region adjacent to edge of column = 1, region beneath center of column based on results of measurements of specimens from six mice.
pected frequency of division in adjacent cells has been shown (LeBlond et al., ’64; Cameron et al., ’65; Gibbs and Casarett, ’72). Frei et a1 ( ’ 6 3 ) examined the distribution of mitotic figures in sheets of mouse epidermis and described a random distribution of mitotic activity. They were, however, unaware of patterns of organization in this tissue and used a sampling area which would have contained 70 or more units of epidermal structure. In the previous report of measurement of the distribution of metaphase figures in relation to cell columns (Mackenzie, ’73), which indicated that metaphase figures occurred principally beneath the periphery of cell columns, the relatively small number of metaphase figures examined and the problem of determining the position of section in relation to the cell columns did not allow the position of mitosis to be accurately located. The present results indicate that metaphase figures occur most frequently just within the periphery rather than directly beneath the junctions of overlying cell columns, Allen and Potten (’74) have studied the ultrastructural appearance of epidermal cell columns, which they have termed “epidermal proliferative units,” but they were unable to define a precise alignment of basal cells within the periphery of each column. They confamed the earlier report (Mackenzie, ’72) that the cell situated centrally beneath each cell column in mouse epidermis differs from the surrounding keratinocytes and has features typical of an epidermal Langerhans cell. This finding could explain the low number of metaphase figures found beneath the central region of cell columns (Mackenzie, ’70) and the finding that central cells are labelled less frequently with tritiated thymidine than those elsewhere (Potten, ’74). It would not, however, lead directly to a position of mitosis within,
=
10. Means and standard errors
rather than directly beneath, the column periphery. The presence of functionally independent units or “rosettes” of basal cells within and beneath each cell column (Potten, ’74; Gertler et al., ’73) might be expected to lead to a position of mitosis similar to that observed. However, using the same methods as those used to demonstrate a non-random position of metaphase figures, no pre-existing regular position of interphase basal cells could be detected which might account for the position of metaphase. There is no direct evidence to support the concept of such functional units and this concept may not be a valid one. Larger spatially-organized epithelial structures such as hair follicles and tongue papillae show patterns of mitotic activity which are related to the cellular architecture of the overlying strata and the organization of such structures has been considered the result of connective tissue control of the micro-environment adjacent to the epithelium through differentiating or other influences (Cameron, ’66; Epstein and Maibach, ’67). An explanation of control of the rate of cell proliferation in the epidermis, however, has been given in terms of an intraepithelial negative feedback mechanism operating through a diffusable mitosis-inhibiting substance produced by post-mitotic differentiating cells (Bullough, ’72). There is, therefore, no clear indication from previous work as to the type of mechanism which may be controlling the patterns of cell division and spatial organization found in the small columnar units of structure in the epidermis. The existence of spatially organized units of cells within the epidermis points to patterns of behavior of epithelial cells which may be of interest to descriptions of pathological epithelial changes. For ex-
710
IAN C. MACKENZIE
ample, the spread of epithelial tumors has been considered without reference to spatial organization of basal cell activity (Williams and Bjerknes, '72) and, concerning the growth behavior of tumors, it has been assumed that cells within a n intact epithelium are without locomotive capabilities and possess no sense of direction other than that imposed by physical pressure (Van Scott, '64). Contrary to these working hypotheses, mouse epidermis shows a non-random position of mitosis, and movement of cells into alignment following mitosis beneath the periphery of the cell columns indicates that there is not complete contact inhibition of movement within the intact epithelium and that the movement which occurs is directional in nature. The small, simple and continuously regenerated units of structure which are present in the epidermis may also form a useful model for examining the mechanisms controlling the organization of larger, more complex epithelial structures. LITERATURE CITED Allen, T. D., and C. S. Potten 1974 Fine structural identification and organization of the epidermal proliferative unit. J. Cell Sci., 15: 291. Bullough, W. S. 1972 The control of epidermal thickness. Br. J. Derm., 87: 187. Cameron, I. L. 1966 Cell proliferation, migration, and specialization in the epithelium of the mouse tongue. J. Exp. Zool., 163: 271. Cameron, I. L., D. G. Gosslee and C. Pilgrim 1965 The spatial distribution of dividing and DNA-synthesizing cells in mouse epithelium. J. Cell and Comp. Physiol., 66: 431.
Christophers, E. 1971 Cellular architecture of the stratum corneum. J. Invest. Derm., 56: 3-165. Epstein, W. L., and H. I. Maibach 1967 Cell proliferation and movement of human hair bulbs. Adv. Biol. Skin, 9: 83. Frei, J. V., W. 0. Waugh and A. C. Ritchie 1963 Mitoses: Distribution in mouse ear epidermis. Science, 140: 487. Gertler, K., M. Reuter and H. E. Strahmer 1973 Morphologishe Untersuchsingen zur Proliferationskinetik der Mausehaut. Z. Zellforsch., 142: 131. Gibbs, S. J., and G. W. Casarett 1972 Spatial distribution of cells in mitotic and DNA-synthetic phases of the cell cycle in hamster cheek pouch epithelium. J. Dent. Res., 51: 30. LeBlond, C. P., R. C. Greulich and J. P. M. Pereira 1964 Relationship of cell formation and cell migration in the renewal of stratified squamous epithelia. Adv. Biol. Skin, 5: 39. Mackenzie, I. C. 1969 Ordered structure of the stratum corneum of mammalian skin. Nature, 222: 881. 1970 Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis. Nature, 226: 653. 1972 The ordered structure of mammalian epidermis. In: Epidermal Wound Healing. Year Book Medical Publishers, Chicago, p. 5. Potten, C. S. 1974 The epidermal proliferative unit: The possible role of the central basal cell. Cell Tiss. Kinet., 7: 77. Scaletta, L. J., and D. K. McCallum 1972 A fine structural study of divalent cation-mediated epithelial union with connective tissue in human oral mucosa. Am. J. Anat., 133: 431. Van Scott, E. J. 1964 Definition of epidermal cancer. In: The Epidermis. Academic Press, London and New York, p. 573. Williams, T., and R. Bjerknes 1972 Stochastic model for abnormal clone spread through epithelial basal layers. Nature, 236: 19.
ABSTRACT
The cells of the upper strata of mammalian epidermis are flattened and aligned to form regular columnar units. It has been suggested that the position of the smaller underlying basal cells is related to the overlying cell columns. Examination of the position of metaphase figures in sheets of mouse epidermis indicated that mitosis occurs principally in cells lying just within the periphery of the cell columns but that there is no alignment of interphase basal cells within the columnar peripheries which could account for this position of mitosis.
Maintenance of stratified squamous epithelia is associated with continuous cell proliferation. Although spatially organized patterns of cell proliferation have been demonstrated in some epithelial structures such as hair follicles (Epstein and Maibach, '67) and tongue papillae (Cameron, '66), cell division has generally been thought to occur randomly among the basal cells of other keratinizing epithelia (Frei et al., '63; LeBlond et al., '64). Recently, however, it was shown that mammalian epidermis has an ordered structure in which alignment of the suprabasal cells results in the formation of a regular series of columnar units (Mackenzie, '69, '72; Christophers, '71). The presence of this previously unsuspected pattern of organization suggested the possibility of a related pattern of mitotic activity in the underlying basal cells. Some evidence presented to support this concept (Mackenzie, '70) suggested that mitosis occurred principally beneath the periphery of the overlying cell columns. Although no further attempts to accurately localize the position of greatest mitotic frequency have been reported, the idea of a pattern of mitosis related to epidermal structure has been extended to include concepts of an "epidermal proliferative unit" (Potten, '74) or complex patterns of cell migration from the basal layer (Gertler et al., '73). Therefore, to further investigate such patterns of activity, the relationship of the position of mitotic and non-mitotic basal cell nuclei to the overlying cell columns was examined. Intact ANAT. REC., 281: 705-710.
sheets of mouse epidermis were used to overcome some of the problems previously encountered in an analysis of data from sections of material (Mackenzie, '70). The purpose of the investigation was two-fold: (1 ) to localize more accurately the regions demonstrating a high rate of accumulation of metaphase figures and, (2) to examine the position of non-mitotic, or interphase, basal cells for a pattern of alignment beneath the overlying cell columns which might provide evidence of functionally independent units of basal cells. MATERIALS AND METHODS
Intact sheets of ear epidermis, prepared by treatment with buffered EDTA (Scaletta and McCallum, '72) were taken from six adult male Balb/C mice which were injected with Colchicine (0.2 mg per 100 g body weight) four hours before death to arrest dividing cells in metaphase. The epidermal sheets were fixed in Bouin's solution, washed, stained with hematoxylin, mounted flat on microscope slides, and examined using Nomarski interference microscopy so that the columnar units in the stratum corneum were clearly visible. When viewed from the surface each cell column is hexagonal in outline and because of the marked cell flattening which occurs during column formation (fig. l ) , each column overlies an area containing 10-11 basal cells (fig. 2). By focusing Received July 22, '74. Accepted Sept. 3, '74. 1This work was supported by John A. Hartford Foundation, Inc.
705
706
IAN C. MACKENZIE
Fig. 1 Frozen section of ear epidermis stained with methylene blue and expanded in alkaline buffer (Mackenzie, ’70) to demonstrate the columnar units of structure formed by flattening and alignment of the cells of the upper epidermal strata. The smaller underlying basal cells do not appear to be regularly positioned in relation to the cell columns. Scale = 20 p m .
through the epidermal sheets and using a projection arm, tracings were made of the relationship of over 3,000 blocked metaphase figures to their overlying cell columns. To reduce parallax error, an attempt was made to focus on the hexagonal cell outline lying deepest in the tissue and, therefore, nearest the basal cell nuclei. From the small intracellular granules present at this plane of focus, this level appeared to be the level of transition between the upper stratum granulosum and lower stratum corneum. On the tracings, the centerpoint of each cell column and each metaphase figure was found by superimposition of a transparent template marked with concentric circles. Measurements were then made of ( a ) the distance from the center of the column to the center of each traced metaphase figure and, ( b ) the length of the radius passing through the center of each metaphase figure (fig. 3 ) . To determine whether the very large majority of basal cells which do not appear
in metaphase at any given time of examination were selectively positioned in relation to the overlying cell columns, the position of all the basal cell nuclei lying beneath 287 columns was examined using the same methods. Assuming a null hypothesis of random distribution of nuclei, there would be an equal probability of the occurrence of nuclei within regions of the basal layer which are of an equal area, Therefore, to determine whether the centers of nuclei occurred with equal frequency beneath various regions of the overlying cell columns, the total area beneath each column was considered to be subdivided by a series of similar concentric hexagons into ten regions of equal area. The series of hexagons which defined such regions was found by subdivision of the measured column radius (r,”) into lengths Ti, rz . . . rlo,such that r, = r z / x . . . rlo//10 (fig. 3 ) . These values were, therefore, calculated for each
SPATIAL DISTRIBUTION OF MITOSIS IN MOUSE EPIDERMIS
707
Fig. 2 A. Sheet of mouse ear epidermis (Nomarski interference microscopy) showing the boundaries of hexagonal units of structure in stratum corneum. B. Same field of vision with plane of focus adjusted to the basal layer of the epidermis showing a blocked metaphase figure (arrow). Projected outlines of the periphery of overlying columns are marked. Scale = 30 fim.
individual pair of measurements, for each type of nucleus using a computer program which also accumulated the number of nuclei occurring in each region. The frequency of occurrence of interphase nuclei
and metaphase figures in the ten regions of equal area from the center to the outer border of the overlying cell columns was then examined for deviations from randomness.
708
IAN C. MACKENZIE CENTRE
PERIPHERY
J 1
4 3 \ \
P' I
Region
Fig. 3 Method of relating the position of a metaphase figure to the overlying cell columns. Right: Measurements were made of ( a ) the distance from the centre of the column to the metaphase figure, and (b) the distance from the centre of the cell column to the periphery along a line through the centre of the metaphase figure. Left: Concentric similar hexagons which defme regions of equal area with the major hexagonn dividing any radius into a series of segments r1, rz, etc., the proportion of which depends upon the number of areas into which the hexagon is divided (see text). From the ratio of a:b, each observed metaphase was allocated to one of ten such regions.
RESULTS
With Nomarski interference microscopy, both the outlines of the cell columns in the stratum corneum and the basal cell nuclei were clearly visible. No obvious arrangement of interphase nuclei to form groups underlying each cell column was observed, but there was a distinct visual impression that the majority of metaphase figures lay beneath the periphery of the cell columns. The visual impression of a lack of alignment of interphase basal cells within each cell column was supported by the results of measurements of their distribution which did not detectably differ from a random distribution. Each of the equal areas produced by the ten concentric subdivisions of the columns contained the centers of approximately 10% of the interphase nuclei (table 1). The observed distribution of metaphase figures (fig. 4) differed markedly and significantly from a random distribution (by
I
I
0 Scale in Frn
5
I
10
I
14.0i0.2
Fig. 4 Distribution of the centres of 3,153 metaphase figures within ten regions of equal area from the centre to the edge of overlying cell columns. Each point represents the mean for that region o f six animals (k S.D.). The scale (based on mean column width) shows that peak distribution occurs at a distance of 4-6 pm from the periphery.
xz, p < 0.05 for each animal, for grouped data p < 0.001). The mean measured radius of cell columns was 14.0 i- 0.2 pm and the mean distance between the centers of basal cell nuclei was 8.6 4 0.7 pm (standard errors based on the means of six animals). The region with the greatest frequency of occurrence of metaphase figures lay at a mean distance of 4.4 2 0.5 pm inside the periphery of overlying cell columns, a distance which corresponded quite closely to half the diameter of a basal cell. Metaphase figures were found less frequently beneath the central region of columns and directly beneath the columnar junctions (fig. 4). It, therefore, appeared that the basal cells lying just within the periphery of the overlying cell columns, showed a higher potential for mitotic activity than those elsewhere. DISCUSSION
Analyses of the distribution of cell division by examination of the spacing between mitotic or DNA-labeled cells in sections of epithelia have usually been interpreted as indicating an essentially random distribution of cell division, though an unexplained tendency for a greater than ex-
709
SPATIAL DISTRIBUTION OF MITOSIS IN MOUSE EPIDERMIS TABLE 1
The distribution of the center points of all basal cell nuclei within ten concentric regions of equal area beneath 287 cell columns in the upper epidermal strata 1
2
3
4
5
6
7
8
9
10
Mean 5%
9.82
10.81
8.95
9.52
10.56
9.36
10.37
10.69
9.72
10.21
S.E.
0.78
0.86
0.94
0.51
1.1
1.26
0.69
0.86
0.80
1.1
Region adjacent to edge of column = 1, region beneath center of column based on results of measurements of specimens from six mice.
pected frequency of division in adjacent cells has been shown (LeBlond et al., ’64; Cameron et al., ’65; Gibbs and Casarett, ’72). Frei et a1 ( ’ 6 3 ) examined the distribution of mitotic figures in sheets of mouse epidermis and described a random distribution of mitotic activity. They were, however, unaware of patterns of organization in this tissue and used a sampling area which would have contained 70 or more units of epidermal structure. In the previous report of measurement of the distribution of metaphase figures in relation to cell columns (Mackenzie, ’73), which indicated that metaphase figures occurred principally beneath the periphery of cell columns, the relatively small number of metaphase figures examined and the problem of determining the position of section in relation to the cell columns did not allow the position of mitosis to be accurately located. The present results indicate that metaphase figures occur most frequently just within the periphery rather than directly beneath the junctions of overlying cell columns, Allen and Potten (’74) have studied the ultrastructural appearance of epidermal cell columns, which they have termed “epidermal proliferative units,” but they were unable to define a precise alignment of basal cells within the periphery of each column. They confamed the earlier report (Mackenzie, ’72) that the cell situated centrally beneath each cell column in mouse epidermis differs from the surrounding keratinocytes and has features typical of an epidermal Langerhans cell. This finding could explain the low number of metaphase figures found beneath the central region of cell columns (Mackenzie, ’70) and the finding that central cells are labelled less frequently with tritiated thymidine than those elsewhere (Potten, ’74). It would not, however, lead directly to a position of mitosis within,
=
10. Means and standard errors
rather than directly beneath, the column periphery. The presence of functionally independent units or “rosettes” of basal cells within and beneath each cell column (Potten, ’74; Gertler et al., ’73) might be expected to lead to a position of mitosis similar to that observed. However, using the same methods as those used to demonstrate a non-random position of metaphase figures, no pre-existing regular position of interphase basal cells could be detected which might account for the position of metaphase. There is no direct evidence to support the concept of such functional units and this concept may not be a valid one. Larger spatially-organized epithelial structures such as hair follicles and tongue papillae show patterns of mitotic activity which are related to the cellular architecture of the overlying strata and the organization of such structures has been considered the result of connective tissue control of the micro-environment adjacent to the epithelium through differentiating or other influences (Cameron, ’66; Epstein and Maibach, ’67). An explanation of control of the rate of cell proliferation in the epidermis, however, has been given in terms of an intraepithelial negative feedback mechanism operating through a diffusable mitosis-inhibiting substance produced by post-mitotic differentiating cells (Bullough, ’72). There is, therefore, no clear indication from previous work as to the type of mechanism which may be controlling the patterns of cell division and spatial organization found in the small columnar units of structure in the epidermis. The existence of spatially organized units of cells within the epidermis points to patterns of behavior of epithelial cells which may be of interest to descriptions of pathological epithelial changes. For ex-
710
IAN C. MACKENZIE
ample, the spread of epithelial tumors has been considered without reference to spatial organization of basal cell activity (Williams and Bjerknes, '72) and, concerning the growth behavior of tumors, it has been assumed that cells within a n intact epithelium are without locomotive capabilities and possess no sense of direction other than that imposed by physical pressure (Van Scott, '64). Contrary to these working hypotheses, mouse epidermis shows a non-random position of mitosis, and movement of cells into alignment following mitosis beneath the periphery of the cell columns indicates that there is not complete contact inhibition of movement within the intact epithelium and that the movement which occurs is directional in nature. The small, simple and continuously regenerated units of structure which are present in the epidermis may also form a useful model for examining the mechanisms controlling the organization of larger, more complex epithelial structures. LITERATURE CITED Allen, T. D., and C. S. Potten 1974 Fine structural identification and organization of the epidermal proliferative unit. J. Cell Sci., 15: 291. Bullough, W. S. 1972 The control of epidermal thickness. Br. J. Derm., 87: 187. Cameron, I. L. 1966 Cell proliferation, migration, and specialization in the epithelium of the mouse tongue. J. Exp. Zool., 163: 271. Cameron, I. L., D. G. Gosslee and C. Pilgrim 1965 The spatial distribution of dividing and DNA-synthesizing cells in mouse epithelium. J. Cell and Comp. Physiol., 66: 431.
Christophers, E. 1971 Cellular architecture of the stratum corneum. J. Invest. Derm., 56: 3-165. Epstein, W. L., and H. I. Maibach 1967 Cell proliferation and movement of human hair bulbs. Adv. Biol. Skin, 9: 83. Frei, J. V., W. 0. Waugh and A. C. Ritchie 1963 Mitoses: Distribution in mouse ear epidermis. Science, 140: 487. Gertler, K., M. Reuter and H. E. Strahmer 1973 Morphologishe Untersuchsingen zur Proliferationskinetik der Mausehaut. Z. Zellforsch., 142: 131. Gibbs, S. J., and G. W. Casarett 1972 Spatial distribution of cells in mitotic and DNA-synthetic phases of the cell cycle in hamster cheek pouch epithelium. J. Dent. Res., 51: 30. LeBlond, C. P., R. C. Greulich and J. P. M. Pereira 1964 Relationship of cell formation and cell migration in the renewal of stratified squamous epithelia. Adv. Biol. Skin, 5: 39. Mackenzie, I. C. 1969 Ordered structure of the stratum corneum of mammalian skin. Nature, 222: 881. 1970 Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis. Nature, 226: 653. 1972 The ordered structure of mammalian epidermis. In: Epidermal Wound Healing. Year Book Medical Publishers, Chicago, p. 5. Potten, C. S. 1974 The epidermal proliferative unit: The possible role of the central basal cell. Cell Tiss. Kinet., 7: 77. Scaletta, L. J., and D. K. McCallum 1972 A fine structural study of divalent cation-mediated epithelial union with connective tissue in human oral mucosa. Am. J. Anat., 133: 431. Van Scott, E. J. 1964 Definition of epidermal cancer. In: The Epidermis. Academic Press, London and New York, p. 573. Williams, T., and R. Bjerknes 1972 Stochastic model for abnormal clone spread through epithelial basal layers. Nature, 236: 19.
