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Morphogenetic determination of the dermoepidermal interface during tail regeneration in Rana catesbeiana
DEVELOPMENTAL
BIOLOGY
Morphogenetic
53, 147-151
Determination of the Dermoepidermal Tail Regeneration in Rana catesbeiana KRISTINE
Cell Science
Laboratories,
(1976)
Department
H.
ATKINSON ofZoology,
Accepted
AND University Canada June
BURR
G.
of Western
Interface
during
ATKINSON Ontario,
London,
Ontario
N6A
5B7,
7,1976
During regeneration of the amputated tadpole tail, reconstruction of the epithelial basal lamina and basement lamella occurs only after the other major morphogenetic processes are well established. At 4 days after tail transection of the bullfrog tadpole, electron microscopy of the internal surface of the basal cell layer of the blastemal epithelium reveals it to be relatively free of extracellular matrix. By 11 days a basal lamina of distinct regularity has formed, and the first rodlets and fibers signaling the replacement of the collagenous basement lamella are identified. At 15 days the basal cells of the epithelium start to exhibit specialization of their internal cell surfaces: Hemidesmosomes and associated tonofilaments appear, and the adepidermal globular layer is formed. Orthogonal packing of collagen plies begins by 19 days after transection, the number of layers exceeding 22 in the latter stages of regeneration.
crofilaments) associated with the dermoepidermal junction in amphibian skin. This sequence of differentiation helps to explain changes in responsiveness of the skin to metamorphic stimuli as development proceeds.
INTRODUCTION
Morphogenesis remains an intriguing field of study because the collective behavior of cells is sometimes unexpected and always complicated by the host of stimuli and interactions influencing a kinetic and temporal phenomenon. To study it, we often stop its progression through time and space. We have employed animals at different stages of development to provide a composite time-lapse record at the ultrastructural level. The results let us pinpoint the temporal onset of purportedly inductive interrelationships and associate biochemical evidence with morphological expression. The basal lamina is a uniform, electron opaque, extracellular layer composed of glycoproteins and a specialized form of collagen (2). Its importance in morphogenesis has been emphasized only recently. Earlier morphologic studies of tissue transitions in frog skin were concerned mainly with the collagenous basement lamella (6, 10, 11, 20,Zl). The purpose of this report is to identify the specific sequence of appearance of microstructures (namely, basal lamina, basement lamella, adepidermal globular layer, hemidesmosomes, and mi-
MATERIALS
All
rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Tails of Rana catesbeiana stage XVII larvae were transected with a sharp blade at a point one-third of the tail length from the distal tip. Tadpoles were placed in individual fingerbowls for periods of 4, 7, 11, 15, 19, 22, or 35 days. Regeneration time varied with season and individual tadpoles, and the stages reported below are based on average rates of growth. Tissue for light microscopy was fixed in Bouin’s fluid for 2 days prior to processing in paraffin. Sections were stained with hematoxylin and eosin or with periodic acidSchiff reagents. The procedure for transmission electron microscopy was to slice off the entire regenerate 1 mm proximal to the wound region, with caution to use only a very sharp blade and minimal pressure. The piece of tissue was bathed in glutaraldehyde fixative for 1 hr before further dissection with sharp clean blades, and three 147
Copyright
AND
148
DEVELOPMENTAL
BIOLOGY
areas were chosen for separate samples of each regenerate (Le., distal, proximal, and wound areas). The tissue was fixed in 5% glutaraldehyde with 1% sucrose in 0.1 M phosphate or cacodylate buffer (pH 7.21, rinsed in buffer with 5% sucrose for 1 hr, postfixed in 1% OsO,, dehydrated, and flat-embedded in Epon. RESULTS
At 4 days after tail transection, the epithelium is intensely involved in the primary stage of the regenerative process, wound healing. The closing-over of the wound is achieved through active migration of epithelial cells from the stump (13), and the sheet of advancing cells does not have the stratified construction of control tissue. Figure la typifies the ultrastructural appearance of the internal surface of the epithelial layer at this time: The plasma membranes are relatively free of extracellular matrix, with only occasional small accumulations of associated material. The cell surfaces are irregular with extensions toward the interior. The cytoplasm of the internal cells is characterized by polyribosomes, and microfilaments that are not organized in the neat skeins characteristic of the figures of Eberth (4, 16). Desmosomes are encountered only occa-
VOLUME
53. 1976
sionally and are of small dimensions. Intercellular spaces are large. At 7 days after transection the epithelial cells manifest firm lateral attachments with their neighbors, but there is only one, or at most two, cell layers (except at the apical cap). The basal cell surfaces are still irregular; there is more extracellular material associated with the plasma membranes, but it is not identifiable as a uniform lamina. The basal cytoplasm often contains abundant rough endoplasmic reticulum with large cisternae. By 11 days, significant progress has occurred in the reestablishment of a stratified epithelium. Figure lb is a micrograph depicting the regular lamina that has formed at the basal surface of the epithelium; it is fairly continuous at cell junctions and is separated from the plasma membrane by a discrete space. The basal surface of the epithelium is usually more regular than this micrograph would imply, especially at cellular junctions. The rodlets and fibers which signal collagen deposition also appear at this stage (Fig. lb); they cluster under the epithelium in an unorganized fashion. The basal cytoplasm contains polyribosomes, some rough endoplasmic reticulum, numerous microfilaments, and a few small microfilament
FIG. 1. Development of the basal lamina during tadpole tail regeneration. Surface-associated materials (arrowheads) under the epithelial (e) plasma membranes are amorphous and unevenly distributed at 4 days after tail transection (Fig. la). By 11 days of regeneration (Fig. lb) a symmetrical basal lamina (bl) has formed and is separated from the epithelial plasma membranes by a discrete space (arrows). Collagen rodlets and fibers (c) appear under the epithelium in the internal lumen. (a) x 19,500; (b) x 20,000. FIG. 2. Dermoepidermal interface at the regenerating tip, 15 days. This oblique section illustrates the organization of the collagenous basement lamella (bll) at a time when the basal cytoplasm of the epithelial cells is characterized by free polyribosomes (p) and microfilaments cm). x 23,500. FIG. 3. Differentiation of the dermoepidermal interface, 15 days. These micrographs were taken in close proximity in the same section. Fig. 3a shows an area where the adepidermal space (as) remains relatively clear and the epithelial basal cytoplasm (e) is devoid of organelles. In Fig. 3b, the adepidermal globular layer is identified by lamellated bodies (arrowheads) and fine filaments (arrows). The epithelial basal cytoplasm is characterized by free polyribosomes (p) and microfilaments (ml. In a slightly more proximal area (Fig. 3c), dense plaques (d) at the epithelial plasma membrane are the site of attachment of microfilaments (arrows), and free polyribosomes are abundant in the basal epithelial cytoplasm. x 46,000. FIG. 4. The reconstituted dermoepidermal interface during late regeneration, 35 days. The distinctive strata of epithelial cells (e), hemidesmosomes cd), adepidermal globular layer (ag), basal lamina (bl), and collagenous basement lamella (bll) are indistinguishable from control tissue. Microfilaments (ml are arrayed in the characteristic figures of Eberth, and free polyribosomes are absent from the cytoplasm near the interface. x 17,000.
BRIEF NOTES
149
150
DEVELOPMENTAL BIOLOGY
bundles. Shortly after this stage, notochorda1 outgrowth and myogenesis culminate (13). At 15 days, the products introduced at 11 days are definitively organized. The subepithelial collagen assumes an oriented appearance (Fig. 2). Polyribosomes and microfilaments crowd the basal cytoplasm, and at the basal plasma membrane arise hemidesmosomes which are already the site of the congregation of some microfilaments. The adepidermal globular layer is first identified by the microfilaments and “lamellated bodies” (cf. 5, 9, 15, 17, 18) found in this region in control tissue. These developments are more advanced proximally, as illustrated in Figs. 3a, b, and c; the micrographs were taken at close intervals in the same section, emphasizing the rapidity with which the cytodifferentiation travels along the regenerate. The tail tip is still relatively undifferentiated, as in nonmetamorphic tissue (3); the only notable difference between the ultrastructure of the 4-day and E-day tip epithelium is the presence of a few collagen rodlets subepithelially at 15 days. After 15 days, further morphogenesis of the dermoepidermal zone is limited to the elaboration of the collagen plies. At 19 days at least five uniform plies are evident, the number increasing with time. Advanced specimens (Fig. 4) manifest 22 plies or more. Our data support Kemp’s observation that the number of layers decreases distally (11).
VOLUME 53, 1976
morphogenesis. On the other hand, simple disruption of the microfilaments results in a flat, wafer-like rudiment which resumes morphogenesis where it was interrupted after the inhibitory agent is removed. These theses imply an “if first A, then B” relationship of the primacy of the basal lamina for epithelial integrity. This study on regeneration, and reappraisal of the work of others, clearly defines that the temporal sequence or program of construction toward the morphogenetic steady state is: basal lamina, basement lamella, adepidermal globular layer and microfilaments, and microfilament bundles and hemidesmosomes. Although these transitions were not remarked upon in the original papers, our thorough examination of published micrographs revealed that this is also the sequence for normal amphibian larval ontogeny (10, 15, 181,for larval wound repair (20, 211, and generally for adult urodele and anuran limb regeneration (7, 16). The order is reversed during tail resorption (5, 20), except that the lamina appears to be maintained throughout redifferentiation into adult skin (9, 11). Production of the plicated adult skin (ll), which forms only when the collagenous lamella is removed during metamorphosis, involves playing back the program for construction to the point where morphogenetic alteration is permissible (i.e., a simple basal lamina), then the steadystate structure is sequentially redefined once plication has been accomplished. Bernfield and Wessells further theorize DISCUSSION from autoradiography, histochemistry, In their enquiry into intra- and extraceland logical deduction that regions of aclular control of epithelial morphogenesis, centuated microfilament contractility are Bernfield and Wessells (2) correlate the caused by localized areas of surface-associvarious data which have accumulated in ated mucopolysaccharide-protein, and that this field into an orderly working hypothe- the sites of most active mucopolysacchasis which relates isolated observations into ride accumulation are those that exhibit a basic biological process. Removal of sur- the least amount of collagen. The developface-associated materials-the stuff of the mental sequence we observed substantibasal lamina - produces a round, ball-like ates their theory, since microfilaments are epithelium, which starts again from the scanty before the basal lamina is definibeginning when it is allowed to resume tively constituted, and the collagenous
BRIEF
151
NOTES
basement lamella does not form before organization of the basal lamina. The absence of a basal lamina in the early blastema1 epithelium correlates with the concentrated amounts of collagenase (7) and hyaluronate (19) in early blastemata. The rapid succession of lamina and lamella may serve to protect the basal lamina (2) from the increasing concentration of hyaluronidase (19) which occurs concurrently at the time of precartilage differentiation in amphibians. Our results show that the basal lamina appears at the onset of structural and biochemical differentiation of the mesenthyme. Since mesenchyme can differentiate in vitro in the absence of epithelium (121, we cannot assume that the epithelium is inducing mesenchymal differentiation. Prior to mesenchymal differentiation, the epithelium’s usual response to raw connective tissue-the production of a basal lamina (14) -is suppressed; once stimulated (or released from inhibition), the epithelium quickly produces a basal lamina and controls deposition of a collagenous interface (cf. Results and 1, 2, 8). In future studies we hope to pinpoint the precise nature of the signal which starts the subcellular differentiation of the mesenthyme and/or causes the epithelium to respond to it.
and by an operating grant search Council of Canada.
This work was supported from the Muscular Dystrophy
21.
by a research grant Association of Canada
from
the
National
Re-
REFERENCES M. R. (1970). Deu. Biol. 22, 213-231. M. R., and WESSELLS, N. K. (1970). Dev. Bid. Supplement 4 195-249. BRUNS, R. R., and GROSS, J. (1970). Amer. J. Anat. 128, 193-224. CHAPMAN, G. B., and DAWSON, A. B. (1961). J. Biophys. Biochem. Cytol. 10, 425-435. GONA, A. G. (1970). J. Ultrastruc. Res. 30, 103110. GRILLO, H. C., and GROSS, J. (1967). Deu. Biol. 15, 300-317. GRILLO, H. C., LAPIERE, C. M., DRESDEN, M. H., and GROSS, J. (1968). Dev. Biol. 17, 571-583. HAY, E. D., and REVEL, J. P. (1963). Deu. Biol. 7, 303-323. KELLY, D. (1966). J. Cell. Biol. 28, 51-72. KEMP, N. (1959). Dev. Biol. 1, 459-476. KEMP, N. (1961). Deu. Biol. 3, 391-410. KONIGSBERG, U. R., LIPTON, B. H., and KONIGSBERG, I. R. (1975). Deu. Biol. 45, 260-275. LASH, J. (1956). J. Exp. 2001. 131, 239-256. MCLOUGHLIN, C. B. (1968). In “Epithelial-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 244-251. The Williams and Wilkins Company, Baltimore, Md. NAKAO, T. (1974). Amer. J. Anat. 140, 533-550. SALPETER, M. M., and SINGER, M. (1960). Anat. Rec. 136, 27-40. SINGER, M., and SALPETER, M. M. (1961). J. Exp. 2001. 147, 1-19. TARIN, D., and STURDEE, A. P. (1974). J. Embryol. Exp. Morphol. 31, 287-303. TOOLE, B. P., and GROSS, J. (1971). Dev. Biol. 25, 57-77. USUKU, G., and GROSS, J. (1965). Deu. Biol. 11, 352-370. WEISS, P., and FERRIS, W. (1956). J. Biophys. Biochem. Cytol. 2, 275-285.
BERNFIELD, BERNFIELD,
9. 10. 11.
12. 13.
14.
15. 16. 17. 18. 19.
20.
BIOLOGY
Morphogenetic
53, 147-151
Determination of the Dermoepidermal Tail Regeneration in Rana catesbeiana KRISTINE
Cell Science
Laboratories,
(1976)
Department
H.
ATKINSON ofZoology,
Accepted
AND University Canada June
BURR
G.
of Western
Interface
during
ATKINSON Ontario,
London,
Ontario
N6A
5B7,
7,1976
During regeneration of the amputated tadpole tail, reconstruction of the epithelial basal lamina and basement lamella occurs only after the other major morphogenetic processes are well established. At 4 days after tail transection of the bullfrog tadpole, electron microscopy of the internal surface of the basal cell layer of the blastemal epithelium reveals it to be relatively free of extracellular matrix. By 11 days a basal lamina of distinct regularity has formed, and the first rodlets and fibers signaling the replacement of the collagenous basement lamella are identified. At 15 days the basal cells of the epithelium start to exhibit specialization of their internal cell surfaces: Hemidesmosomes and associated tonofilaments appear, and the adepidermal globular layer is formed. Orthogonal packing of collagen plies begins by 19 days after transection, the number of layers exceeding 22 in the latter stages of regeneration.
crofilaments) associated with the dermoepidermal junction in amphibian skin. This sequence of differentiation helps to explain changes in responsiveness of the skin to metamorphic stimuli as development proceeds.
INTRODUCTION
Morphogenesis remains an intriguing field of study because the collective behavior of cells is sometimes unexpected and always complicated by the host of stimuli and interactions influencing a kinetic and temporal phenomenon. To study it, we often stop its progression through time and space. We have employed animals at different stages of development to provide a composite time-lapse record at the ultrastructural level. The results let us pinpoint the temporal onset of purportedly inductive interrelationships and associate biochemical evidence with morphological expression. The basal lamina is a uniform, electron opaque, extracellular layer composed of glycoproteins and a specialized form of collagen (2). Its importance in morphogenesis has been emphasized only recently. Earlier morphologic studies of tissue transitions in frog skin were concerned mainly with the collagenous basement lamella (6, 10, 11, 20,Zl). The purpose of this report is to identify the specific sequence of appearance of microstructures (namely, basal lamina, basement lamella, adepidermal globular layer, hemidesmosomes, and mi-
MATERIALS
All
rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Tails of Rana catesbeiana stage XVII larvae were transected with a sharp blade at a point one-third of the tail length from the distal tip. Tadpoles were placed in individual fingerbowls for periods of 4, 7, 11, 15, 19, 22, or 35 days. Regeneration time varied with season and individual tadpoles, and the stages reported below are based on average rates of growth. Tissue for light microscopy was fixed in Bouin’s fluid for 2 days prior to processing in paraffin. Sections were stained with hematoxylin and eosin or with periodic acidSchiff reagents. The procedure for transmission electron microscopy was to slice off the entire regenerate 1 mm proximal to the wound region, with caution to use only a very sharp blade and minimal pressure. The piece of tissue was bathed in glutaraldehyde fixative for 1 hr before further dissection with sharp clean blades, and three 147
Copyright
AND
148
DEVELOPMENTAL
BIOLOGY
areas were chosen for separate samples of each regenerate (Le., distal, proximal, and wound areas). The tissue was fixed in 5% glutaraldehyde with 1% sucrose in 0.1 M phosphate or cacodylate buffer (pH 7.21, rinsed in buffer with 5% sucrose for 1 hr, postfixed in 1% OsO,, dehydrated, and flat-embedded in Epon. RESULTS
At 4 days after tail transection, the epithelium is intensely involved in the primary stage of the regenerative process, wound healing. The closing-over of the wound is achieved through active migration of epithelial cells from the stump (13), and the sheet of advancing cells does not have the stratified construction of control tissue. Figure la typifies the ultrastructural appearance of the internal surface of the epithelial layer at this time: The plasma membranes are relatively free of extracellular matrix, with only occasional small accumulations of associated material. The cell surfaces are irregular with extensions toward the interior. The cytoplasm of the internal cells is characterized by polyribosomes, and microfilaments that are not organized in the neat skeins characteristic of the figures of Eberth (4, 16). Desmosomes are encountered only occa-
VOLUME
53. 1976
sionally and are of small dimensions. Intercellular spaces are large. At 7 days after transection the epithelial cells manifest firm lateral attachments with their neighbors, but there is only one, or at most two, cell layers (except at the apical cap). The basal cell surfaces are still irregular; there is more extracellular material associated with the plasma membranes, but it is not identifiable as a uniform lamina. The basal cytoplasm often contains abundant rough endoplasmic reticulum with large cisternae. By 11 days, significant progress has occurred in the reestablishment of a stratified epithelium. Figure lb is a micrograph depicting the regular lamina that has formed at the basal surface of the epithelium; it is fairly continuous at cell junctions and is separated from the plasma membrane by a discrete space. The basal surface of the epithelium is usually more regular than this micrograph would imply, especially at cellular junctions. The rodlets and fibers which signal collagen deposition also appear at this stage (Fig. lb); they cluster under the epithelium in an unorganized fashion. The basal cytoplasm contains polyribosomes, some rough endoplasmic reticulum, numerous microfilaments, and a few small microfilament
FIG. 1. Development of the basal lamina during tadpole tail regeneration. Surface-associated materials (arrowheads) under the epithelial (e) plasma membranes are amorphous and unevenly distributed at 4 days after tail transection (Fig. la). By 11 days of regeneration (Fig. lb) a symmetrical basal lamina (bl) has formed and is separated from the epithelial plasma membranes by a discrete space (arrows). Collagen rodlets and fibers (c) appear under the epithelium in the internal lumen. (a) x 19,500; (b) x 20,000. FIG. 2. Dermoepidermal interface at the regenerating tip, 15 days. This oblique section illustrates the organization of the collagenous basement lamella (bll) at a time when the basal cytoplasm of the epithelial cells is characterized by free polyribosomes (p) and microfilaments cm). x 23,500. FIG. 3. Differentiation of the dermoepidermal interface, 15 days. These micrographs were taken in close proximity in the same section. Fig. 3a shows an area where the adepidermal space (as) remains relatively clear and the epithelial basal cytoplasm (e) is devoid of organelles. In Fig. 3b, the adepidermal globular layer is identified by lamellated bodies (arrowheads) and fine filaments (arrows). The epithelial basal cytoplasm is characterized by free polyribosomes (p) and microfilaments (ml. In a slightly more proximal area (Fig. 3c), dense plaques (d) at the epithelial plasma membrane are the site of attachment of microfilaments (arrows), and free polyribosomes are abundant in the basal epithelial cytoplasm. x 46,000. FIG. 4. The reconstituted dermoepidermal interface during late regeneration, 35 days. The distinctive strata of epithelial cells (e), hemidesmosomes cd), adepidermal globular layer (ag), basal lamina (bl), and collagenous basement lamella (bll) are indistinguishable from control tissue. Microfilaments (ml are arrayed in the characteristic figures of Eberth, and free polyribosomes are absent from the cytoplasm near the interface. x 17,000.
BRIEF NOTES
149
150
DEVELOPMENTAL BIOLOGY
bundles. Shortly after this stage, notochorda1 outgrowth and myogenesis culminate (13). At 15 days, the products introduced at 11 days are definitively organized. The subepithelial collagen assumes an oriented appearance (Fig. 2). Polyribosomes and microfilaments crowd the basal cytoplasm, and at the basal plasma membrane arise hemidesmosomes which are already the site of the congregation of some microfilaments. The adepidermal globular layer is first identified by the microfilaments and “lamellated bodies” (cf. 5, 9, 15, 17, 18) found in this region in control tissue. These developments are more advanced proximally, as illustrated in Figs. 3a, b, and c; the micrographs were taken at close intervals in the same section, emphasizing the rapidity with which the cytodifferentiation travels along the regenerate. The tail tip is still relatively undifferentiated, as in nonmetamorphic tissue (3); the only notable difference between the ultrastructure of the 4-day and E-day tip epithelium is the presence of a few collagen rodlets subepithelially at 15 days. After 15 days, further morphogenesis of the dermoepidermal zone is limited to the elaboration of the collagen plies. At 19 days at least five uniform plies are evident, the number increasing with time. Advanced specimens (Fig. 4) manifest 22 plies or more. Our data support Kemp’s observation that the number of layers decreases distally (11).
VOLUME 53, 1976
morphogenesis. On the other hand, simple disruption of the microfilaments results in a flat, wafer-like rudiment which resumes morphogenesis where it was interrupted after the inhibitory agent is removed. These theses imply an “if first A, then B” relationship of the primacy of the basal lamina for epithelial integrity. This study on regeneration, and reappraisal of the work of others, clearly defines that the temporal sequence or program of construction toward the morphogenetic steady state is: basal lamina, basement lamella, adepidermal globular layer and microfilaments, and microfilament bundles and hemidesmosomes. Although these transitions were not remarked upon in the original papers, our thorough examination of published micrographs revealed that this is also the sequence for normal amphibian larval ontogeny (10, 15, 181,for larval wound repair (20, 211, and generally for adult urodele and anuran limb regeneration (7, 16). The order is reversed during tail resorption (5, 20), except that the lamina appears to be maintained throughout redifferentiation into adult skin (9, 11). Production of the plicated adult skin (ll), which forms only when the collagenous lamella is removed during metamorphosis, involves playing back the program for construction to the point where morphogenetic alteration is permissible (i.e., a simple basal lamina), then the steadystate structure is sequentially redefined once plication has been accomplished. Bernfield and Wessells further theorize DISCUSSION from autoradiography, histochemistry, In their enquiry into intra- and extraceland logical deduction that regions of aclular control of epithelial morphogenesis, centuated microfilament contractility are Bernfield and Wessells (2) correlate the caused by localized areas of surface-associvarious data which have accumulated in ated mucopolysaccharide-protein, and that this field into an orderly working hypothe- the sites of most active mucopolysacchasis which relates isolated observations into ride accumulation are those that exhibit a basic biological process. Removal of sur- the least amount of collagen. The developface-associated materials-the stuff of the mental sequence we observed substantibasal lamina - produces a round, ball-like ates their theory, since microfilaments are epithelium, which starts again from the scanty before the basal lamina is definibeginning when it is allowed to resume tively constituted, and the collagenous
BRIEF
151
NOTES
basement lamella does not form before organization of the basal lamina. The absence of a basal lamina in the early blastema1 epithelium correlates with the concentrated amounts of collagenase (7) and hyaluronate (19) in early blastemata. The rapid succession of lamina and lamella may serve to protect the basal lamina (2) from the increasing concentration of hyaluronidase (19) which occurs concurrently at the time of precartilage differentiation in amphibians. Our results show that the basal lamina appears at the onset of structural and biochemical differentiation of the mesenthyme. Since mesenchyme can differentiate in vitro in the absence of epithelium (121, we cannot assume that the epithelium is inducing mesenchymal differentiation. Prior to mesenchymal differentiation, the epithelium’s usual response to raw connective tissue-the production of a basal lamina (14) -is suppressed; once stimulated (or released from inhibition), the epithelium quickly produces a basal lamina and controls deposition of a collagenous interface (cf. Results and 1, 2, 8). In future studies we hope to pinpoint the precise nature of the signal which starts the subcellular differentiation of the mesenthyme and/or causes the epithelium to respond to it.
and by an operating grant search Council of Canada.
This work was supported from the Muscular Dystrophy
21.
by a research grant Association of Canada
from
the
National
Re-
REFERENCES M. R. (1970). Deu. Biol. 22, 213-231. M. R., and WESSELLS, N. K. (1970). Dev. Bid. Supplement 4 195-249. BRUNS, R. R., and GROSS, J. (1970). Amer. J. Anat. 128, 193-224. CHAPMAN, G. B., and DAWSON, A. B. (1961). J. Biophys. Biochem. Cytol. 10, 425-435. GONA, A. G. (1970). J. Ultrastruc. Res. 30, 103110. GRILLO, H. C., and GROSS, J. (1967). Deu. Biol. 15, 300-317. GRILLO, H. C., LAPIERE, C. M., DRESDEN, M. H., and GROSS, J. (1968). Dev. Biol. 17, 571-583. HAY, E. D., and REVEL, J. P. (1963). Deu. Biol. 7, 303-323. KELLY, D. (1966). J. Cell. Biol. 28, 51-72. KEMP, N. (1959). Dev. Biol. 1, 459-476. KEMP, N. (1961). Deu. Biol. 3, 391-410. KONIGSBERG, U. R., LIPTON, B. H., and KONIGSBERG, I. R. (1975). Deu. Biol. 45, 260-275. LASH, J. (1956). J. Exp. 2001. 131, 239-256. MCLOUGHLIN, C. B. (1968). In “Epithelial-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 244-251. The Williams and Wilkins Company, Baltimore, Md. NAKAO, T. (1974). Amer. J. Anat. 140, 533-550. SALPETER, M. M., and SINGER, M. (1960). Anat. Rec. 136, 27-40. SINGER, M., and SALPETER, M. M. (1961). J. Exp. 2001. 147, 1-19. TARIN, D., and STURDEE, A. P. (1974). J. Embryol. Exp. Morphol. 31, 287-303. TOOLE, B. P., and GROSS, J. (1971). Dev. Biol. 25, 57-77. USUKU, G., and GROSS, J. (1965). Deu. Biol. 11, 352-370. WEISS, P., and FERRIS, W. (1956). J. Biophys. Biochem. Cytol. 2, 275-285.
BERNFIELD, BERNFIELD,
9. 10. 11.
12. 13.
14.
15. 16. 17. 18. 19.
20.