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Patterns of cell polarity in the developing mouse limb
DEVELOPMENTAL
BIOLOGY
Patterns
5% 164-173 (1977)
of Cell Polarity LEWIS
in the Developing
Mouse
Limb’
B. HOLMES~ AND ROBERT L. TRELSTAD~
Genetics Unit, Children’s Service, and Department of Pathology, Massachusetts General Hospital, Experimental Pathology Laboratory, Shriners Burns Institute, and Department of Pediatrics and Pathology and Center for Human Genetics, Harvard Medical School, Boston, Massachusetts Received February
11,1977;
accepted in revised form April
29,1977
Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are excreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane. INTRODUCTION
Polarity is an attribute of cells which attracted considerable attention among early cytologists (Wilson, 1925). Although most attention was initially directed at the polarity of individual cells such as ova during meiosis or cells in epithelia, it has also been recognized that mesenchyme cells are anisotropically structured and often behave in a polarized manner (Holtfreter, 1947; Trelstad, 1977). A simple morphological assay for the structural polarity of a cell is to determine the relative intracellular positions of two organelles such as the nucleus and Golgi ’ Supported in part by a grant from the C. H. Hood Foundation and National Institutes of Health Grants HD09689 and HL18714. 2 Reprint requests should be sent to Lewis B. Holmes, Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. :I Recipient of Faculty Research Award PRA-107 from the American Cancer Society.
apparatus. Using silver impregnation as a method for localizing the Golgi apparatus, its intracellular location has been shown to be variable, depending on function. For example, in secretory epithelia such as the developing thyroid (Cowdry, 19221, gall bladder (Wahlin and Schiebler, 19751, tooth (Beams and King, 19331, and cornea1 epithelium (Trelstad, 19701, the Golgi apparatus is located in the pole of the cell from which materials are discharged. Cell polarity has also been demonstrated in amphibian pigment cells which are orienting themselves (Holtfreter, 1947) and in mesenchyme cells of the developing chick vertebral body (Trelstad, 1977). During limb development in the chick embryo an interaction essential for normal development occurs between a portion of the epithelium, the apical ectodermal ridge (AER), and the mesenchyme. We have studied cell polarity in the epithe164
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
HOLMES AND TREI~TAD
lium and mesenchyme of the developing mouse limb to determine whether changes in the polarity or orientation of these two cell types may occur at this interface during their morphogenetic interaction. We found no evidence that the epithelial cells reorient their Golgi apparatuses in a manner that would suggest they are actively involved in secretion. However, the Golgi apparatuses in the mesenchyme cells beneath the epithelium shift toward the epithelium at the time when mesenchyme cells are aggregating into precartilage clusters and when the amount of extracellular matrix at the epithelium-mesenthyme interface is increasing. MATERIALS
AND
METHODS
All studies were done on random-bred Charles River CD-l mice. Fertile females were housed with adult males overnight. The morning the vaginal plug was identified was designated day 0. Pregnant females were killed by cervical dislocation on days lo-15 of gestation. To obtain embryos, ages 9.5 and 12.5 days, the males and females were housed together for a short period of time (2 to 3 hr), so that the pregnancy could be timed more precisely. The impregnation of the Golgi apparatus was accomplished as follows. The limbs were fixed at room temperature for 3 hr 40 min in 6% formaldehyde buffered with 0.28 M glycine at pH 3.2. The tissues were rinsed briefly with an aqueous solution of 1.5% AgNO, in the dark for 4.5 hours. Following silver impregnation, the tissues were rinsed briefly in the reducing solution of 1.5% hydroquinone in 7% aqueous formaldehyde and then were placed in fresh reducing solution in the dark for a minimum of 2 hr, but usually overnight. The limbs were dehydrated in ethanol and embedded in Araldite 6005DDSA (dodecenylsuccinic anhydride, Polysciences, Inc.). Sections 0.5 pm thick were cut on a Porter-Blum MT-2 ultramicrotome (Ivan Sorvall, Norwalk, Connecticut).
Cell Polarity
Patterns
165
Sections of forelimbs and hindlimbs from embryos of ages 10 to 15 days were examined from a number of different litters. The number of limbs in which cell polarity was tabulated ranges from ten on day 10 to two on day 15, reflecting the fact that the older limbs are much larger and have many more cells for quantitative evaluation. In addition, at least ten embryos from each time point were examined qualitatively but were not used for the quantitative analysis. The position of the Golgi apparatus was determined by dividing the cell into four quadrants with respect to the plane of the epithelial basement membrane (Fig. 1). The apical quadrant encompasses the area at the top of the cell 45” on either side of the line drawn perpendicular to the basement membrane (Fig. 1). Similarly, the two lateral and the basal quadrants each cover 90”. Initially the location of the Golgi apparatus in each cell was determined by measurements on photomicrographs. With practice this was subsequently done visually on sections under oil immersion at 1000 x magnification. In a small percentage of cells the Golgi apparatus was on the borderline between quadrants; these were not counted. The location of the Golgi apparatus in the outer layer of epithelial cells, the periderm, was not tabulated. Sections were obtained in the midportion of the limbs in the horizontal plane for the data in Table 1 and in the vertical plane to evaluate polarity in the apical ectodermal ridge. The position of the Golgi apparatus within the cell was quantitated in five areas: the entire epithelium, the basal layer of epithelial cells next to the basement membrane, the layer of the mesenthyme cells adjacent to the epithelium, the layer of mesenchyme cells located at a depth of four cells below the basement membrane, and the apical ectodermal ridge. The entire epithelium of day 10 and 11 embryos was examined. For day 12 to 15 embryos only the epithelium and mesenthyme of the hand plate were evaluated
166
DEVELOPMENTAL BIOLOGY
VOLUME 59, 1977
FIG. 1. Day 10 forelimb showing several cells with silver-impregnated Golgi apparatus in epithelium, but few in mesenchyme cell layer (X 1400). Inset of whole limb with cut on postaxial side for orientation (X 22). The division of an epithelial cell into four quadrants is illustrated. E, Epithelial cell; M, mesenchyme cell. The basement membrane is visible between E and M.
with this area defined as the point where the hand plate first projects laterally on each side of the limb. RESULTS
On day 9.5 the forelimb is barely visible, but not the hindlimb. On day 10 the forelimbs are 0.6 mm long, and the hindlimbs 0.4 mm. By day 12 the forelimb is 1.0 mm long, widening of the hand and foot plates is evident, and there is early demarcation of each digit. By day 14 limbs have five digits and an elbow/knee joint. The width of the epithelium changes from one to two cells on day 10 (Fig. 1) to three to four cells by day 13 (Fig. 3). Stratification of the outer cells is evident by day 14 (Fig. 4). The apical ectodermal ridge (AER) can best be seen in the vertical sections as a widening of the epithelium at the tip of the limb. The AER is identifiable at the tip of
limbs on days 10 to 13 and is narrow, usually containing about 12 to 16 cells at its greatest width. The thickness of the AER decreases from four layers of cells on day 10 to two cells by day 13. The basement membrane is visible in limbs of all ages. There is more extracellular matrix beneath the epithelium in limbs on days 13 to 15 (Figs. 3 and 4) in comparison to days 10 to 12 (Figs. 1 and 2). Cartilage is first visible in the proximal portion of the forelimb on day 12; by day 13 to 13.5 the cartilages of the phalanges, carpals, ulna/radius, and tibia/fibula are visible. The rate of development of the forelimb is about 12 hr ahead of the hindlimb. In the digits of the forelimb we observed the following sequence of events: On day 12 some forelimbs show organization of the mesenchyme with columns of cells showing increased intercellular space
HOLMES AND TREISTAD
Cell Polarity
167
Patterns
TABLE 1 AVERAGE NUMBER OF CELLS WITH GOLGI APPARATUSES IN FOUR QUADRANTS’ Age of embryc (days)
I
Total epithelium
Epithelial cell layer
Mesenchyme cell layer
i t
tf
f
t
4.8
L 0.5
2.5
T
Mesenchyme cells at depth of 4 cells below basement membrane
N
t
tt
5.3
1 6.0
3.8
4.0
-1 3.5
4
tf
F
12.8
6.0
1 0.5
10
F H
28.0 1’7.8
12.9 10.2
2.3 0.7
17.1 10.7
2.9 3.1
0.6 0.0
4.3 2.6
10.1 6.1
6.6 6.2
6.5 4.2
6.1 5.9
3.8 5.3
10 10
11
F H
38.5 60.8
12.3 26.2
2.6 6.0
26.8 41.5
6.0 10.5
0.7 0.8
5.3 3.5
11.3 8.8
9.7 9.2
8.2 5.2
8.2 6.5
6.3 6.8
6 6
12
F H
59.0 62.0
12.0 20.8
0.8 1.3
38.8 39.8
3.8 5.5
0.5 0.3
11.3 5.0
20.3 11.3
13.5 15.5
16.0 8.5
19.0 12.5
11.3 11.3
4 4
12.5
F H
76.0 81.0
25.5 32.0
5.0 4.5
40.5 47.5
11.0 1.5
2.0 3.0
7.0 12.0
10.0 22.5
7.5 13.0
6.5 5.0
12.5 8.5
8.0 7.0
2 2
13
F H
140.5 170.7
69.0 36.7
4.5 2.7
36.0 67.3
16.0 11.3
0.8 0.7
38.0 40.0
50.5 51.0
13.5 21.0
29.0 28.3
49.5 46.0
24.0 33.0
3 3
14
F H
482.5 335.0
110.5 51.0
20.0 8.0
219.5 80.5
38.0 16.5
11.5 4.0
98.5 46.0
79.0 30.0
26.0 11.0
41.5 34.0
55.0 34.0
27.5 23.5
2 2
15
F H
688.0 634.0
181.5 186.5
24.5 19.5
294.5 262.5
37.5 54.0
5.0 9.0
118.5 115.0
119.5 105.5
36.5 35.0
75.0 70.5
101.5 91.0
40.5 37.0
2 2
9.5
12.0
ct
a F, Forelimb; H, hindlimb; t , cell with Golgi apparatus oriented away from basement membrane in the epithelium and toward the basement membrane in the mesenchyme; L cell with Golgi apparatus oriented toward the basement membrane in the epithelium and away from the basement membrane in the mesenchyme; u, cell with Golgi apparatus oriented laterally.
FIG. 2. Day 12 forelimb showing epithelium columns of aggregating mesenchyme cells.
from tip of digit 2 (X 1400). Inset of whole limb (X 27) shows
168
DEVELOPMENTALBIOLOGY
VOLUME 59, 1977
FIG. 3. Day 13 forelimb showing tip of third digit (x 1400). The amount of matrix beneath the basement membrane is greater than in days 10 (Fig. 1) and 12 (Fig. 2). Inset of whole limb with postaxial cut (X 27) showing row of autolyzing cells between each digit.
next to columns of mesenchyme undergoing aggregation (see inset in Fig. 2); by day 12.5 indentation for digits and aggregation of mesenchyme cells in the digits is more apparent. The pattern of mesenthyme cell aggregation suggests the shape of a mushroom, fanning out to fill the entire tip of the region destined to form each digit and narrowing proximally like a stalk (Fig. 2). In the day 13 to 13.5 forelimb, cartilage is visible in the digits, there is less flaring of the aggregation of mesenchyme cells at the tips of the digits, and cell autolysis is visible between the aggregates for each finger (see inset in Fig. 3). By day 14 there is no accentuated concentration of mesenchyme cells at the tips of the digits and the intercellular space between cells on the sides of the
digits is comparable to that at the tip (Fig. 4), i.e., the mushroom-shaped cell aggregate appears to lose the subepithelial cap, but persists as the stalk which subsequently chondrifies. Cell Polarity The percentage of cells in the epithelium and the mesenchyme with a visible Golgi apparatus increases from about 30% on day 10 to about 55% on day 15. The total number of cells counted in the epithelium during the same 5-day period increases twenty-five-fold, reflecting the marked growth in the size of the limb (Table 1). 1. Total epithelium. In day 10 to 15 limbs the Golgi apparatus was in the apiaway from the cal pole, i.e., “pointed” basement membrane in 65 to 80% of the
HOLMES AND TRELSTAD
Cell Polarity
Patterns
FIG. 4. Day 14 forelimb showing tip of third digit with mostly apical Golgi apparatuses and mesenchymal cells (X 1400). Inset of whole limb (x 27).
cells in the entire epithelium (Table 1). Less than 3% of the Golgi apparatuses were in the basal pole and the remainder were in the lateral quadrants. 2. Epithelial cells on the basement membrane. From 78 to 90% of the cells adjacent to the basement membrane at all stages examined had the Golgi apparatus in the apical quadrant of the cell (Tables 1 and 2). As with the entire epithelium, less than 5% of the cells had a Golgi apparatus in the basal pole. 3. Mesenchyme cell layer below basement membrane. In day 10 to 12 forelimbs and hindlimbs between 16 and 25% of the mesenchyme cells have their Golgi apparatus oriented toward the epithelium; by
169
in both epithelial
contrast, 30 to 50% are oriented away from the epithelium. A shift in this orientation occurs between days 12 and 13, so that from day 13 to 15 up to 53% (36 to 53%) of the mesenchyme cells have the Golgi apparatus in the pole pointed toward the epithelium; only 12 to 19% are pointed away from the basement membrane (Fig. 5). This shift to a larger percentage of Golgi apparatuses facing the epithelium coincides with the increase in the amount of extracellular matrix that appears between the epithelium and the mesenchyme (Figs. 3 and 4). 4. Mesenchyme cells at a depth of four cells beneath basement membrane. The mesenchyme cells more distant from the
170
DEVELOPMENTAL BIOLOGY
VOLUME 59, 1977
TABLE 2 PERCENTAGE OF CELLS WITH GOLGI APPARATUSES IN APICAL AND BASAL POLE@
Age of embryo (days)
Epithelial
cell layer
Mesenchyme cell layer
Mesenchyme cells at depth of 4 cells below basement membrane
t 69.4
J 2.8
t 18.0
4 43.5
t 33.0
1 31.0
F H
83.0 77.8
2.9 0.0
20.5 17.0
31.4 41.6
39.6 27.0
23.0 34.4
F
-H
80.0 78.6
2.0 1.5
20.0 17.4
36.0 42.2
36.0 28.1
26.0 36.0
F H
89.9 87.2
1.2 0.7
25.0 15.7
29.9 48.7
35.3 26.3
29.2 35.0
12.5 F H
75.7 81.9
3.7 5.2
28.6 25.5
30.6 27.7
24.1 24.4
29.6 34.0
13
F H
78.9 84.9
4.1 0.8
41.9 35.7
13.9 18.8
28.2 26.4
23.4 30.1
14
F H
81.6 79.7
4.2 4.0
48.4 52.9
12.8 12.6
33.5 37.2
22.2 25.7
15
F
87.4 84.2
1.5 2.5
43.2 44.1
13.3 15.4
34.6 35.5
18.7 18.6
9.5 F 10
11
12
H
” F, forelimb; H, hindlimb; T, cell with Golgi apparatus oriented away from basement membrane in the epithelium and toward the basement membrane in the mesenchyme; J , cell with Golgi apparatus oriented toward the basement membrane in the epithelium and away from the basement membrane in the mesenthyme. The percentage of cells oriented laterally is not included for either of these three cell layers. This percentage for each cell layer can be determined by subtracting the percentage of cells with apical and basal polarity from 100%.
basement membrane are as likely to have the Golgi apparatus oriented toward the basement membrane as away in embryos from 10 to 13 days (Tables 1 and 2). However, in day 14 and 15 limbs there is a clear predominance of those cells with Golgi apparatuses pointing toward the epithelium. Interestingly, the shift in the polarity of these more deeply lying cells is evident 1 day later than in the mesenchyme cell layer immediately contiguous with the basement membrane. 5. The apical ectodermal ridge. The polarity of the epithelial cells in the AER, as well as the adjacent mesenchyme cell layer, was similar to the polarity observed in the tissue layers in horizontal sections. More Golgi apparatuses were visible in the
AER than in the adjacent epithelium, reflecting the greater thickness of the AER in day 10 limbs. By day 13 the AER is only slightly thicker than the adjacent epithelium. DISCUSSION
By tracing the movement of labeled grafts in the chick embryo, Rosenquist (1971) found that ectoderm and mesoderm of the limb bud arise near the primitive streak and migrate in an orderly fashion to the region from which the limb bud emerges. Once established as a presumptive limb mesenchyme, however, no migration of cells in the developing limb itself has been demonstrated (Searls, 1967). The early outgrowth and proper development of the chick limb is dependent on an
HOLMES FORELIMB,
AND TREISTAD
Cell Polarity
FORELIMB,
EPITHELIUM
171
Patterns MESENCHYME
100' 80 '
20,
x-
CC----X---
s ‘& ---_
.-x--x----x-_--x
xemx---x----x
0
9
10
11
12
13
14
4
01
15
9
10
11
12
x
14
15
14
15
DAYS
DAYS
HINDLIMB,
13
x ___-
HINDLIMB,
EPITHELIUM
MESENCHYME
100' 80 ' 5 60 ' u : E 40.
9 FIG.
epithelial
10
11
12 DAYS
13
14
15
5. Shows
9
x---=
10
_/--
11
3
12
13
DAYS
shifting pattern of cell polarity in mesenchyme cell layer, but no shift in polarity cell layer. The mesenchyme cell layer is the row of cells immediately below the epithelium.
interaction between the ectoderm and mesenchyme, based on a number of different manipulative experiments. For example, removal of the cap of epithelium over the end of the limb called the apical ectodermal ridge (AER) during limb outgrowth results in the loss of distal limb structures (Zwilling, 1949). Adding an extra AER produces limb duplication (Saunders and Gasseling, 1968). Although the same microsurgical experiments have not been carried out on the limbs of mouse embryos, we assume that the AER-mesenthyme interaction is a feature common to limb development in all vertebrates. The stable pattern of epithelial cell polarity we have identified suggests that the epithelium in general, as well as the AER in particular, is never oriented with a subnuclear Golgi apparatus as one would expect for an epithelium actively secreting material such as extracellular matrix ma-
of
terials toward the limb mesenchyme. If secretion occurs from the basal surface of the epithelium, it does not involve relocation of the Golgi apparatus to the basal cell pole as has been observed in other epithelia involved in basal secretion (Beams and King, 1933; Cowdry, 1922; Trelstad, 1970; Wahlin and Schiebler, 1975). In contrast to the epithelium, the pattern of mesenchyma1 cell polarity immediately underneath the epithelium changes significantly during limb outgrowth and early mesenchyme cell aggregation. This period of mesenchyma1 cell reorientation occurs just before cartilage is visible and the shifting cell polarity must be considered in light of the other simultaneous cellular and molecular events that have been identified in studies of limb development over the past 50 years. Fell (1925) showed that the mesenthyme in the limb of the chick embryo condenses to form a compact mass of cells
172
DEVELOPMENTAL
BIOL~CY
which subsequently becomes the site of cartilage formation. She also noted that cartilage cells elongate at right angles to the cartilage. Thorogood and Hinchliffe (1975) showed an increase in mesenchyme cell number per unit area 12 hr prior to matrix formation in chondrogenic regions of the chick limb. They suggested that these condensations are formed by a process of aggregation rather than by a local increase in the rate of mesenchyme cell mitosis. Gould and associates (1974) suggested that matrix secretion in early cartilage caused the pattern of limb mesenthyme cell orientation. With regard to the production of extracellular matrix, several components have been shown to change dramatically just before and during chondrogenesis: There is a pattern of increased uptake of [35S]sulfate presumably into proteoglycans in the region of prospective long bones several hours before cartilage is visible @earls, 1965); the level of hyaluronic acid decreases and the level of chondroitin sulfate increases just before the appearance of cartilage (Toole, 1972); and the type of collagen produced by limb mesenchyme shifts from type I to type II at the time of chondrogenesis (Linsenmayer et al., 1973). These selected features of limb development emphasize the importance of cell aggregation and matrix production in normal morphogenesis, to which we would add cell orientation. The fact that the individual mesenchymal cells are polarized suggests an interrelationship between cellular aggregation, orientation, and ordered matrix deposition. The shift in the polarity of the cells beneath the epithelium occurs just before the appearance of the cartilaginous anlage. The mesenthyme cells further away from the epithelium shift a day later. Possibly this represents an initial and essential orienting of the cells in preparation for the discharge of an oriented matrix in contrast to the theory that matrix secretion causes cell orientation (Gould et al., 1974). Presumably
VOLUME 59. 1977
polarized cells oriented in a common direction will produce an oriented or ordered extracellular matrix. We are currently examining cell polarity throughout the mesenchyme cells during aggregation and early chondrogenesis. The components of the matrix at the AER-mesenchyme interface have not been characterized chemically, but do include ultrastructurally recognizable cross-striated collagen fibrils. This apparent increase in matrix during mouse limb development has also been described in human embryos (Kelley, 1975). The fact that the Golgi apparatus in the AER, and in the epithelial cells in general, remains in the apex throughout limb development suggests that a major secretion of extracellular materials does not occur across the basal surface of the epithelium. However, the AER effect on the underlying mesenchyme might be expressed, in part, by influencing the shift of mesenthyme cell orientation we observed. Our observations are compatible with the model of limb development proposed by Summerbell et al. (1973) who postulated that there is a “progress zone” in the mesenchyme that extends inward from the AER for about 400 pm in the developing chick limb. The course of development of cells depends on the time when they are present in the progress zone. The tissue that emerges from this zone early will be more proximal structures and that which emerges late will form more distal structures. The continued development of new positional values that result in new distal structures was shown to be dependent on the presence of the AER. Regulation of cell orientation in this progress zone could thus be an essential feature in the precise deposition of cell-derived matrix materials and thereby a critical feature of morphogenesis. We thank Kimiko Hayashi for excellent technical assistance. A preliminary report of this work was presented at the Society for Pediatric Research, St. Louis, Missouri, on April 30, 1976.
HOLMES AND TREL~TAD REFERENCES BEAMS, H. W., and KING, R. L. (1933). The Golgi apparatus in the developing tooth, with special reference to polarity. Anat. Rec. 57, 29-39. COWDRY, E. V. (1922). The reticular material as an indicator of physiologic reversal in secretory polarity in the thyroid cells of the guinea pig. Amer. J. Anat. 30, 25-37. FELL, H. B. (1925). The histogenesis of cartilage and bone in the long bones of the embryonic fowl. J. Morphol. Physiol. 40, 417-454. GOULD, R. P., SELWOOD, L., DAY, A., and WOLPERT, L. (1974). The mechanism of cellular orientation during early cartilage formation in the chick limb and regenerating amphibian limb. Exp. Cell. Res. 83, 287-296. HOLTFRETER, J. (1947). Observations of the migration, aggregation and phagocytosis of embryonic cells. J. Morphol. 80, 25-53. KELLEY, R. 0. (1975). Ultrastructural identification of extracellular matrix and cell surface components during limb morphogenesis in man. J. Embryol. Exp. Morphol. 34, 1-18. LINSENMAYER, T., TOOLE, B. P., and TRELSTAD, R. L. (1973). Temporal and spatial transitions in collagen types during embryonic chick limb development. Develop. Biol. 35, 232-239. ROSENQUIST, G. C. (1971). The origin and movement of the limb-bud epithelium and mesenchyme in the chick embryo as determined by radioautographic mapping. J. Embryol. Exp. Morphol. 25, 85-96. SAUNDERS, J. W., and GASSELING, M. T. (1968). Ectodermal-mesodermal interactions in the origin of limb symmetry. In “Epithelial Mesenchymal In-
Cell Polarity
Patterns
173
teractions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 78-97. Williams and Wilkins, Baltimore. SEARLS, R. L. (1965). An autoradiographic study of the uptake of S35-sulfate during the differentiation of limb bud cartilage. Develop. Biol. 11, 155-168. SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick limb bud. J. Exp. 2001. 166, 39-45. SUMMERBELL, D., LEWIS, J. H., and WOLPERT, L. (1973). Positional information in chick limb morphogenesis. Nature (London) 244, 492-49. THOROGOOD, P. V., and HINCHLIFFE, J. R. (1975). An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. J. Embryol. Exp. Morphol. 33, 581-606. TOOLE, B. P. (1972). Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton. Deuelop. Biol. 29, 321-329. TRELSTAD, R. L. (1977). Mesenchyme cell polarity and morphogenesis of chick cartilage. Develop. Biol. 59, 153-163. TRELSTAD, R. L. (1977). Mesenchyme cell polarity and morphogenesis of chick cartilage. Deuel. Biol. 59, 153-163. WAHLIN, T., and SCHIEBLER, T. H. (1975). Zur Entwicklung des Gallenblasenepithels des Meerschweinchens. II. Elektronenmikroskopische und enzymhistochemische Untersuchungen. Histothem. 44, 253-275. WILSON, E. B. (1925). “The Cell in Development and Heredity,” pp. 106-111. Macmillan, London. ZWILLING, E. (1949). The role of epithelial components in the developmental origin of the “wingless” syndrome of chick embryos. J. Erp. 2001. 111, 175-187.
BIOLOGY
Patterns
5% 164-173 (1977)
of Cell Polarity LEWIS
in the Developing
Mouse
Limb’
B. HOLMES~ AND ROBERT L. TRELSTAD~
Genetics Unit, Children’s Service, and Department of Pathology, Massachusetts General Hospital, Experimental Pathology Laboratory, Shriners Burns Institute, and Department of Pediatrics and Pathology and Center for Human Genetics, Harvard Medical School, Boston, Massachusetts Received February
11,1977;
accepted in revised form April
29,1977
Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are excreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane. INTRODUCTION
Polarity is an attribute of cells which attracted considerable attention among early cytologists (Wilson, 1925). Although most attention was initially directed at the polarity of individual cells such as ova during meiosis or cells in epithelia, it has also been recognized that mesenchyme cells are anisotropically structured and often behave in a polarized manner (Holtfreter, 1947; Trelstad, 1977). A simple morphological assay for the structural polarity of a cell is to determine the relative intracellular positions of two organelles such as the nucleus and Golgi ’ Supported in part by a grant from the C. H. Hood Foundation and National Institutes of Health Grants HD09689 and HL18714. 2 Reprint requests should be sent to Lewis B. Holmes, Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. :I Recipient of Faculty Research Award PRA-107 from the American Cancer Society.
apparatus. Using silver impregnation as a method for localizing the Golgi apparatus, its intracellular location has been shown to be variable, depending on function. For example, in secretory epithelia such as the developing thyroid (Cowdry, 19221, gall bladder (Wahlin and Schiebler, 19751, tooth (Beams and King, 19331, and cornea1 epithelium (Trelstad, 19701, the Golgi apparatus is located in the pole of the cell from which materials are discharged. Cell polarity has also been demonstrated in amphibian pigment cells which are orienting themselves (Holtfreter, 1947) and in mesenchyme cells of the developing chick vertebral body (Trelstad, 1977). During limb development in the chick embryo an interaction essential for normal development occurs between a portion of the epithelium, the apical ectodermal ridge (AER), and the mesenchyme. We have studied cell polarity in the epithe164
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
HOLMES AND TREI~TAD
lium and mesenchyme of the developing mouse limb to determine whether changes in the polarity or orientation of these two cell types may occur at this interface during their morphogenetic interaction. We found no evidence that the epithelial cells reorient their Golgi apparatuses in a manner that would suggest they are actively involved in secretion. However, the Golgi apparatuses in the mesenchyme cells beneath the epithelium shift toward the epithelium at the time when mesenchyme cells are aggregating into precartilage clusters and when the amount of extracellular matrix at the epithelium-mesenthyme interface is increasing. MATERIALS
AND
METHODS
All studies were done on random-bred Charles River CD-l mice. Fertile females were housed with adult males overnight. The morning the vaginal plug was identified was designated day 0. Pregnant females were killed by cervical dislocation on days lo-15 of gestation. To obtain embryos, ages 9.5 and 12.5 days, the males and females were housed together for a short period of time (2 to 3 hr), so that the pregnancy could be timed more precisely. The impregnation of the Golgi apparatus was accomplished as follows. The limbs were fixed at room temperature for 3 hr 40 min in 6% formaldehyde buffered with 0.28 M glycine at pH 3.2. The tissues were rinsed briefly with an aqueous solution of 1.5% AgNO, in the dark for 4.5 hours. Following silver impregnation, the tissues were rinsed briefly in the reducing solution of 1.5% hydroquinone in 7% aqueous formaldehyde and then were placed in fresh reducing solution in the dark for a minimum of 2 hr, but usually overnight. The limbs were dehydrated in ethanol and embedded in Araldite 6005DDSA (dodecenylsuccinic anhydride, Polysciences, Inc.). Sections 0.5 pm thick were cut on a Porter-Blum MT-2 ultramicrotome (Ivan Sorvall, Norwalk, Connecticut).
Cell Polarity
Patterns
165
Sections of forelimbs and hindlimbs from embryos of ages 10 to 15 days were examined from a number of different litters. The number of limbs in which cell polarity was tabulated ranges from ten on day 10 to two on day 15, reflecting the fact that the older limbs are much larger and have many more cells for quantitative evaluation. In addition, at least ten embryos from each time point were examined qualitatively but were not used for the quantitative analysis. The position of the Golgi apparatus was determined by dividing the cell into four quadrants with respect to the plane of the epithelial basement membrane (Fig. 1). The apical quadrant encompasses the area at the top of the cell 45” on either side of the line drawn perpendicular to the basement membrane (Fig. 1). Similarly, the two lateral and the basal quadrants each cover 90”. Initially the location of the Golgi apparatus in each cell was determined by measurements on photomicrographs. With practice this was subsequently done visually on sections under oil immersion at 1000 x magnification. In a small percentage of cells the Golgi apparatus was on the borderline between quadrants; these were not counted. The location of the Golgi apparatus in the outer layer of epithelial cells, the periderm, was not tabulated. Sections were obtained in the midportion of the limbs in the horizontal plane for the data in Table 1 and in the vertical plane to evaluate polarity in the apical ectodermal ridge. The position of the Golgi apparatus within the cell was quantitated in five areas: the entire epithelium, the basal layer of epithelial cells next to the basement membrane, the layer of the mesenthyme cells adjacent to the epithelium, the layer of mesenchyme cells located at a depth of four cells below the basement membrane, and the apical ectodermal ridge. The entire epithelium of day 10 and 11 embryos was examined. For day 12 to 15 embryos only the epithelium and mesenthyme of the hand plate were evaluated
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DEVELOPMENTAL BIOLOGY
VOLUME 59, 1977
FIG. 1. Day 10 forelimb showing several cells with silver-impregnated Golgi apparatus in epithelium, but few in mesenchyme cell layer (X 1400). Inset of whole limb with cut on postaxial side for orientation (X 22). The division of an epithelial cell into four quadrants is illustrated. E, Epithelial cell; M, mesenchyme cell. The basement membrane is visible between E and M.
with this area defined as the point where the hand plate first projects laterally on each side of the limb. RESULTS
On day 9.5 the forelimb is barely visible, but not the hindlimb. On day 10 the forelimbs are 0.6 mm long, and the hindlimbs 0.4 mm. By day 12 the forelimb is 1.0 mm long, widening of the hand and foot plates is evident, and there is early demarcation of each digit. By day 14 limbs have five digits and an elbow/knee joint. The width of the epithelium changes from one to two cells on day 10 (Fig. 1) to three to four cells by day 13 (Fig. 3). Stratification of the outer cells is evident by day 14 (Fig. 4). The apical ectodermal ridge (AER) can best be seen in the vertical sections as a widening of the epithelium at the tip of the limb. The AER is identifiable at the tip of
limbs on days 10 to 13 and is narrow, usually containing about 12 to 16 cells at its greatest width. The thickness of the AER decreases from four layers of cells on day 10 to two cells by day 13. The basement membrane is visible in limbs of all ages. There is more extracellular matrix beneath the epithelium in limbs on days 13 to 15 (Figs. 3 and 4) in comparison to days 10 to 12 (Figs. 1 and 2). Cartilage is first visible in the proximal portion of the forelimb on day 12; by day 13 to 13.5 the cartilages of the phalanges, carpals, ulna/radius, and tibia/fibula are visible. The rate of development of the forelimb is about 12 hr ahead of the hindlimb. In the digits of the forelimb we observed the following sequence of events: On day 12 some forelimbs show organization of the mesenchyme with columns of cells showing increased intercellular space
HOLMES AND TREISTAD
Cell Polarity
167
Patterns
TABLE 1 AVERAGE NUMBER OF CELLS WITH GOLGI APPARATUSES IN FOUR QUADRANTS’ Age of embryc (days)
I
Total epithelium
Epithelial cell layer
Mesenchyme cell layer
i t
tf
f
t
4.8
L 0.5
2.5
T
Mesenchyme cells at depth of 4 cells below basement membrane
N
t
tt
5.3
1 6.0
3.8
4.0
-1 3.5
4
tf
F
12.8
6.0
1 0.5
10
F H
28.0 1’7.8
12.9 10.2
2.3 0.7
17.1 10.7
2.9 3.1
0.6 0.0
4.3 2.6
10.1 6.1
6.6 6.2
6.5 4.2
6.1 5.9
3.8 5.3
10 10
11
F H
38.5 60.8
12.3 26.2
2.6 6.0
26.8 41.5
6.0 10.5
0.7 0.8
5.3 3.5
11.3 8.8
9.7 9.2
8.2 5.2
8.2 6.5
6.3 6.8
6 6
12
F H
59.0 62.0
12.0 20.8
0.8 1.3
38.8 39.8
3.8 5.5
0.5 0.3
11.3 5.0
20.3 11.3
13.5 15.5
16.0 8.5
19.0 12.5
11.3 11.3
4 4
12.5
F H
76.0 81.0
25.5 32.0
5.0 4.5
40.5 47.5
11.0 1.5
2.0 3.0
7.0 12.0
10.0 22.5
7.5 13.0
6.5 5.0
12.5 8.5
8.0 7.0
2 2
13
F H
140.5 170.7
69.0 36.7
4.5 2.7
36.0 67.3
16.0 11.3
0.8 0.7
38.0 40.0
50.5 51.0
13.5 21.0
29.0 28.3
49.5 46.0
24.0 33.0
3 3
14
F H
482.5 335.0
110.5 51.0
20.0 8.0
219.5 80.5
38.0 16.5
11.5 4.0
98.5 46.0
79.0 30.0
26.0 11.0
41.5 34.0
55.0 34.0
27.5 23.5
2 2
15
F H
688.0 634.0
181.5 186.5
24.5 19.5
294.5 262.5
37.5 54.0
5.0 9.0
118.5 115.0
119.5 105.5
36.5 35.0
75.0 70.5
101.5 91.0
40.5 37.0
2 2
9.5
12.0
ct
a F, Forelimb; H, hindlimb; t , cell with Golgi apparatus oriented away from basement membrane in the epithelium and toward the basement membrane in the mesenchyme; L cell with Golgi apparatus oriented toward the basement membrane in the epithelium and away from the basement membrane in the mesenchyme; u, cell with Golgi apparatus oriented laterally.
FIG. 2. Day 12 forelimb showing epithelium columns of aggregating mesenchyme cells.
from tip of digit 2 (X 1400). Inset of whole limb (X 27) shows
168
DEVELOPMENTALBIOLOGY
VOLUME 59, 1977
FIG. 3. Day 13 forelimb showing tip of third digit (x 1400). The amount of matrix beneath the basement membrane is greater than in days 10 (Fig. 1) and 12 (Fig. 2). Inset of whole limb with postaxial cut (X 27) showing row of autolyzing cells between each digit.
next to columns of mesenchyme undergoing aggregation (see inset in Fig. 2); by day 12.5 indentation for digits and aggregation of mesenchyme cells in the digits is more apparent. The pattern of mesenthyme cell aggregation suggests the shape of a mushroom, fanning out to fill the entire tip of the region destined to form each digit and narrowing proximally like a stalk (Fig. 2). In the day 13 to 13.5 forelimb, cartilage is visible in the digits, there is less flaring of the aggregation of mesenchyme cells at the tips of the digits, and cell autolysis is visible between the aggregates for each finger (see inset in Fig. 3). By day 14 there is no accentuated concentration of mesenchyme cells at the tips of the digits and the intercellular space between cells on the sides of the
digits is comparable to that at the tip (Fig. 4), i.e., the mushroom-shaped cell aggregate appears to lose the subepithelial cap, but persists as the stalk which subsequently chondrifies. Cell Polarity The percentage of cells in the epithelium and the mesenchyme with a visible Golgi apparatus increases from about 30% on day 10 to about 55% on day 15. The total number of cells counted in the epithelium during the same 5-day period increases twenty-five-fold, reflecting the marked growth in the size of the limb (Table 1). 1. Total epithelium. In day 10 to 15 limbs the Golgi apparatus was in the apiaway from the cal pole, i.e., “pointed” basement membrane in 65 to 80% of the
HOLMES AND TRELSTAD
Cell Polarity
Patterns
FIG. 4. Day 14 forelimb showing tip of third digit with mostly apical Golgi apparatuses and mesenchymal cells (X 1400). Inset of whole limb (x 27).
cells in the entire epithelium (Table 1). Less than 3% of the Golgi apparatuses were in the basal pole and the remainder were in the lateral quadrants. 2. Epithelial cells on the basement membrane. From 78 to 90% of the cells adjacent to the basement membrane at all stages examined had the Golgi apparatus in the apical quadrant of the cell (Tables 1 and 2). As with the entire epithelium, less than 5% of the cells had a Golgi apparatus in the basal pole. 3. Mesenchyme cell layer below basement membrane. In day 10 to 12 forelimbs and hindlimbs between 16 and 25% of the mesenchyme cells have their Golgi apparatus oriented toward the epithelium; by
169
in both epithelial
contrast, 30 to 50% are oriented away from the epithelium. A shift in this orientation occurs between days 12 and 13, so that from day 13 to 15 up to 53% (36 to 53%) of the mesenchyme cells have the Golgi apparatus in the pole pointed toward the epithelium; only 12 to 19% are pointed away from the basement membrane (Fig. 5). This shift to a larger percentage of Golgi apparatuses facing the epithelium coincides with the increase in the amount of extracellular matrix that appears between the epithelium and the mesenchyme (Figs. 3 and 4). 4. Mesenchyme cells at a depth of four cells beneath basement membrane. The mesenchyme cells more distant from the
170
DEVELOPMENTAL BIOLOGY
VOLUME 59, 1977
TABLE 2 PERCENTAGE OF CELLS WITH GOLGI APPARATUSES IN APICAL AND BASAL POLE@
Age of embryo (days)
Epithelial
cell layer
Mesenchyme cell layer
Mesenchyme cells at depth of 4 cells below basement membrane
t 69.4
J 2.8
t 18.0
4 43.5
t 33.0
1 31.0
F H
83.0 77.8
2.9 0.0
20.5 17.0
31.4 41.6
39.6 27.0
23.0 34.4
F
-H
80.0 78.6
2.0 1.5
20.0 17.4
36.0 42.2
36.0 28.1
26.0 36.0
F H
89.9 87.2
1.2 0.7
25.0 15.7
29.9 48.7
35.3 26.3
29.2 35.0
12.5 F H
75.7 81.9
3.7 5.2
28.6 25.5
30.6 27.7
24.1 24.4
29.6 34.0
13
F H
78.9 84.9
4.1 0.8
41.9 35.7
13.9 18.8
28.2 26.4
23.4 30.1
14
F H
81.6 79.7
4.2 4.0
48.4 52.9
12.8 12.6
33.5 37.2
22.2 25.7
15
F
87.4 84.2
1.5 2.5
43.2 44.1
13.3 15.4
34.6 35.5
18.7 18.6
9.5 F 10
11
12
H
” F, forelimb; H, hindlimb; T, cell with Golgi apparatus oriented away from basement membrane in the epithelium and toward the basement membrane in the mesenchyme; J , cell with Golgi apparatus oriented toward the basement membrane in the epithelium and away from the basement membrane in the mesenthyme. The percentage of cells oriented laterally is not included for either of these three cell layers. This percentage for each cell layer can be determined by subtracting the percentage of cells with apical and basal polarity from 100%.
basement membrane are as likely to have the Golgi apparatus oriented toward the basement membrane as away in embryos from 10 to 13 days (Tables 1 and 2). However, in day 14 and 15 limbs there is a clear predominance of those cells with Golgi apparatuses pointing toward the epithelium. Interestingly, the shift in the polarity of these more deeply lying cells is evident 1 day later than in the mesenchyme cell layer immediately contiguous with the basement membrane. 5. The apical ectodermal ridge. The polarity of the epithelial cells in the AER, as well as the adjacent mesenchyme cell layer, was similar to the polarity observed in the tissue layers in horizontal sections. More Golgi apparatuses were visible in the
AER than in the adjacent epithelium, reflecting the greater thickness of the AER in day 10 limbs. By day 13 the AER is only slightly thicker than the adjacent epithelium. DISCUSSION
By tracing the movement of labeled grafts in the chick embryo, Rosenquist (1971) found that ectoderm and mesoderm of the limb bud arise near the primitive streak and migrate in an orderly fashion to the region from which the limb bud emerges. Once established as a presumptive limb mesenchyme, however, no migration of cells in the developing limb itself has been demonstrated (Searls, 1967). The early outgrowth and proper development of the chick limb is dependent on an
HOLMES FORELIMB,
AND TREISTAD
Cell Polarity
FORELIMB,
EPITHELIUM
171
Patterns MESENCHYME
100' 80 '
20,
x-
CC----X---
s ‘& ---_
.-x--x----x-_--x
xemx---x----x
0
9
10
11
12
13
14
4
01
15
9
10
11
12
x
14
15
14
15
DAYS
DAYS
HINDLIMB,
13
x ___-
HINDLIMB,
EPITHELIUM
MESENCHYME
100' 80 ' 5 60 ' u : E 40.
9 FIG.
epithelial
10
11
12 DAYS
13
14
15
5. Shows
9
x---=
10
_/--
11
3
12
13
DAYS
shifting pattern of cell polarity in mesenchyme cell layer, but no shift in polarity cell layer. The mesenchyme cell layer is the row of cells immediately below the epithelium.
interaction between the ectoderm and mesenchyme, based on a number of different manipulative experiments. For example, removal of the cap of epithelium over the end of the limb called the apical ectodermal ridge (AER) during limb outgrowth results in the loss of distal limb structures (Zwilling, 1949). Adding an extra AER produces limb duplication (Saunders and Gasseling, 1968). Although the same microsurgical experiments have not been carried out on the limbs of mouse embryos, we assume that the AER-mesenthyme interaction is a feature common to limb development in all vertebrates. The stable pattern of epithelial cell polarity we have identified suggests that the epithelium in general, as well as the AER in particular, is never oriented with a subnuclear Golgi apparatus as one would expect for an epithelium actively secreting material such as extracellular matrix ma-
of
terials toward the limb mesenchyme. If secretion occurs from the basal surface of the epithelium, it does not involve relocation of the Golgi apparatus to the basal cell pole as has been observed in other epithelia involved in basal secretion (Beams and King, 1933; Cowdry, 1922; Trelstad, 1970; Wahlin and Schiebler, 1975). In contrast to the epithelium, the pattern of mesenchyma1 cell polarity immediately underneath the epithelium changes significantly during limb outgrowth and early mesenchyme cell aggregation. This period of mesenchyma1 cell reorientation occurs just before cartilage is visible and the shifting cell polarity must be considered in light of the other simultaneous cellular and molecular events that have been identified in studies of limb development over the past 50 years. Fell (1925) showed that the mesenthyme in the limb of the chick embryo condenses to form a compact mass of cells
172
DEVELOPMENTAL
BIOL~CY
which subsequently becomes the site of cartilage formation. She also noted that cartilage cells elongate at right angles to the cartilage. Thorogood and Hinchliffe (1975) showed an increase in mesenchyme cell number per unit area 12 hr prior to matrix formation in chondrogenic regions of the chick limb. They suggested that these condensations are formed by a process of aggregation rather than by a local increase in the rate of mesenchyme cell mitosis. Gould and associates (1974) suggested that matrix secretion in early cartilage caused the pattern of limb mesenthyme cell orientation. With regard to the production of extracellular matrix, several components have been shown to change dramatically just before and during chondrogenesis: There is a pattern of increased uptake of [35S]sulfate presumably into proteoglycans in the region of prospective long bones several hours before cartilage is visible @earls, 1965); the level of hyaluronic acid decreases and the level of chondroitin sulfate increases just before the appearance of cartilage (Toole, 1972); and the type of collagen produced by limb mesenchyme shifts from type I to type II at the time of chondrogenesis (Linsenmayer et al., 1973). These selected features of limb development emphasize the importance of cell aggregation and matrix production in normal morphogenesis, to which we would add cell orientation. The fact that the individual mesenchymal cells are polarized suggests an interrelationship between cellular aggregation, orientation, and ordered matrix deposition. The shift in the polarity of the cells beneath the epithelium occurs just before the appearance of the cartilaginous anlage. The mesenthyme cells further away from the epithelium shift a day later. Possibly this represents an initial and essential orienting of the cells in preparation for the discharge of an oriented matrix in contrast to the theory that matrix secretion causes cell orientation (Gould et al., 1974). Presumably
VOLUME 59. 1977
polarized cells oriented in a common direction will produce an oriented or ordered extracellular matrix. We are currently examining cell polarity throughout the mesenchyme cells during aggregation and early chondrogenesis. The components of the matrix at the AER-mesenchyme interface have not been characterized chemically, but do include ultrastructurally recognizable cross-striated collagen fibrils. This apparent increase in matrix during mouse limb development has also been described in human embryos (Kelley, 1975). The fact that the Golgi apparatus in the AER, and in the epithelial cells in general, remains in the apex throughout limb development suggests that a major secretion of extracellular materials does not occur across the basal surface of the epithelium. However, the AER effect on the underlying mesenchyme might be expressed, in part, by influencing the shift of mesenthyme cell orientation we observed. Our observations are compatible with the model of limb development proposed by Summerbell et al. (1973) who postulated that there is a “progress zone” in the mesenchyme that extends inward from the AER for about 400 pm in the developing chick limb. The course of development of cells depends on the time when they are present in the progress zone. The tissue that emerges from this zone early will be more proximal structures and that which emerges late will form more distal structures. The continued development of new positional values that result in new distal structures was shown to be dependent on the presence of the AER. Regulation of cell orientation in this progress zone could thus be an essential feature in the precise deposition of cell-derived matrix materials and thereby a critical feature of morphogenesis. We thank Kimiko Hayashi for excellent technical assistance. A preliminary report of this work was presented at the Society for Pediatric Research, St. Louis, Missouri, on April 30, 1976.
HOLMES AND TREL~TAD REFERENCES BEAMS, H. W., and KING, R. L. (1933). The Golgi apparatus in the developing tooth, with special reference to polarity. Anat. Rec. 57, 29-39. COWDRY, E. V. (1922). The reticular material as an indicator of physiologic reversal in secretory polarity in the thyroid cells of the guinea pig. Amer. J. Anat. 30, 25-37. FELL, H. B. (1925). The histogenesis of cartilage and bone in the long bones of the embryonic fowl. J. Morphol. Physiol. 40, 417-454. GOULD, R. P., SELWOOD, L., DAY, A., and WOLPERT, L. (1974). The mechanism of cellular orientation during early cartilage formation in the chick limb and regenerating amphibian limb. Exp. Cell. Res. 83, 287-296. HOLTFRETER, J. (1947). Observations of the migration, aggregation and phagocytosis of embryonic cells. J. Morphol. 80, 25-53. KELLEY, R. 0. (1975). Ultrastructural identification of extracellular matrix and cell surface components during limb morphogenesis in man. J. Embryol. Exp. Morphol. 34, 1-18. LINSENMAYER, T., TOOLE, B. P., and TRELSTAD, R. L. (1973). Temporal and spatial transitions in collagen types during embryonic chick limb development. Develop. Biol. 35, 232-239. ROSENQUIST, G. C. (1971). The origin and movement of the limb-bud epithelium and mesenchyme in the chick embryo as determined by radioautographic mapping. J. Embryol. Exp. Morphol. 25, 85-96. SAUNDERS, J. W., and GASSELING, M. T. (1968). Ectodermal-mesodermal interactions in the origin of limb symmetry. In “Epithelial Mesenchymal In-
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Patterns
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teractions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 78-97. Williams and Wilkins, Baltimore. SEARLS, R. L. (1965). An autoradiographic study of the uptake of S35-sulfate during the differentiation of limb bud cartilage. Develop. Biol. 11, 155-168. SEARLS, R. L. (1967). The role of cell migration in the development of the embryonic chick limb bud. J. Exp. 2001. 166, 39-45. SUMMERBELL, D., LEWIS, J. H., and WOLPERT, L. (1973). Positional information in chick limb morphogenesis. Nature (London) 244, 492-49. THOROGOOD, P. V., and HINCHLIFFE, J. R. (1975). An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. J. Embryol. Exp. Morphol. 33, 581-606. TOOLE, B. P. (1972). Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton. Deuelop. Biol. 29, 321-329. TRELSTAD, R. L. (1977). Mesenchyme cell polarity and morphogenesis of chick cartilage. Develop. Biol. 59, 153-163. TRELSTAD, R. L. (1977). Mesenchyme cell polarity and morphogenesis of chick cartilage. Deuel. Biol. 59, 153-163. WAHLIN, T., and SCHIEBLER, T. H. (1975). Zur Entwicklung des Gallenblasenepithels des Meerschweinchens. II. Elektronenmikroskopische und enzymhistochemische Untersuchungen. Histothem. 44, 253-275. WILSON, E. B. (1925). “The Cell in Development and Heredity,” pp. 106-111. Macmillan, London. ZWILLING, E. (1949). The role of epithelial components in the developmental origin of the “wingless” syndrome of chick embryos. J. Erp. 2001. 111, 175-187.