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Scanning electron microscopic visualization of collagen fibers in embryonic chick skin
DEVEJKIPMENTAL
Scanning
BIOJSGY
48, 80-90
Electron
Department
(1976)
Microscopic Visualization Embryonic Chick Skin of
of Collagen
Fibers in
JANE OVERTON AND JAMES COLLINS Biology, The University of Chicago, Chicago, Illinois 6M37 Accepted July 25,1975
The disposition of collagen fibers in embryonic chick skin can be visualized by use of scanning microscopy @EM). Chick back skin was removed from a-day embryos, the epithelial and mesenchymal components were separated, and the mesenchyme was subjected to 10% trypsin treatment (Stuart, E. S., and Moscona, A. A. (1967) Science 157,947-948), after which it was prepared for SEM by critical point drying and coating. Such preparations were largely free of cellular material. Cavities which presumably had contained the cells were present in a network of fibers. Skin of the scaleless mutant was also studied. In this mutant the collagen network was more irregular and collagen fiber diameter was more variable. These findings are discussed in connection with the formation of feather germ pattern.
skin. There is evidence that the mutant is The spatial arrangement of collagen fi- not deficient in the ability to synthesize bers in embryonic mesenchyme has been collagen (Goetinck and Sekellick, 1972) considered important in morphogenesis, but it seemed possible that a defect in collaparticularly in glandular tissues (Bern- gen arrangement might contribute to interfield and Wessells, 1970; Grobstein and ference with the pattern of cell migration. Cohen, 1965; Wessells and Cohen, 1968), Aspects of tissue organization which probut also in the skin (Stuart and Moscona, mote pattern formation are largely un1967; Stuart et al., 1973; Goetinck and Sek- known so that the idea that collagen organization precedes cellular patterning ellick, 1972). It therefore seemed worth(Stuart, 1967; Stuart and Moscona, 1967) is while to attempt to visualize these fibers of particular interest. by scanning microscopy. With this objective in mind, the method of Stuart and MATERIALS AND METHODS Moscona (1967) was adapted to permit study of fiber arrangement. White Leghorn eggs were incubated at Stuart et al. (1973) outline evidence that 38°C for 7.5-8.5 days, and back skin of the cell migration contributes to feather germ saddle tract was examined when one to formation in the chick skin. Since cells can five or more rows of feather germs were move only over a solid substrate (Harripresent. Mutant eggs of the scaleless stock son, 1914), the fibrous arrangement of col- were kindly provided by Dr. P. F. Goelagen within the skin mesenchyme un- tinck and were incubated under the same doubtedly mediates cell movement. If the conditions. character of this fibrous tissue component For scanning microscopy skin was disis altered by collagenase (Stuart et al., sected free in Hanks’ solution, incubated 6 1973) or by lathyrogens (Goetinck and Sek- min at 4°C in 2% trypsin (Difco, 1:250) in ellick, 1970), feather germs fail to form. calcium-magnesium-free Hanks’ solution Feather germs of the saddle tract also fail after which the epithelium was lifted off to form in the scaleless mutant (Abbott the mesenchyme (Stuart et al., 1973). Mesand Asmundson, 1957; Abbott, 1965). In enchyme was fixed in 70% alcohol for 1 hr the present study the arrangement of the or more, rinsed and incubated in 10% tryp fibrous tissue component of the mutant sin (Difco, 1:250) in CMF Hanks’ at 37°C has been compared with that of normal for 45 min. This treatment was for the INTRODUCTION
80 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
OVERTON
AND
COLLINS
SEM
purpose of removing cellular tissue components (Stuart et al., 1973). The trypsintreated mesenchyme was then fixed in 3% glutaraldehyde and allowed to settle on a thin layer of gelatin in a small foil dish and positioned with either its proximal or distal side upwards (Vial and Porter, 1974). Throughout these procedures the mesenchymal material was transferred gently with a wide-mouthed pipet and was not allowed to come in contact with the air-fluid interface. After 30-60 min of fixation when the tissue adhered firmly to the gelatin, the preparation was rinsed and the sides of the dish were cut off. After dehydration in alcohol and amyl acetate, preparations were dried in a Bomar critical point dryer followed by coating in a vacuum evaporator with gold palladium or with a Hummer gold vapor coater. Preparations were viewed with an ETEC Autoscan or a Hitachi HFS-2 microscope. For polarizing microscopy, intact skin or trypsinized preparations were fixed in glutaraldehyde or 70% alcohol, stained with chromosome red, dehydrated and mounted on slides in Permount. Tissue was transferred from one solution to the next with a wide-mouthed pipet to avoid the liquid-air interface. For transmission microscopy, preparations of whole skin and intact or trypsintreated mesenchyme were fixed in Karnovsky’s fixative, stained en bloc in uranyl acetate and embedded in Araldite following the procedure described by Hay and Revel (1969). Uranyl acetate staining was carried out after sectioning in some cases. Trypsin-treated mesenchyme that had been critical point dried was also prepared for transmission microscopy. Blocks were sectioned with a diamond knife using a Porter MT-2 microtome, stained with lead (Reynolds, 1963) and viewed in a Hitachi HU-11A microscope. RESULTS
Because the accumulation of mesenthyme cells in feather germs cannot be explained entirely as due to cell prolifera-
Visualization
of Collagen
81
tion (Stuart et al., 1973) and because cells are oriented in a manner suggesting directed migration in a well-defined pattern throughout the mesenchyme which is associated with feather germ formation (Stuart et al., 19731, evidence for a systematically oriented substrate has been sought (Stuart, 1967; Stuart and Moscona, 1967; Stuart et al., 1973; Goetinck and Sekellick, 1972; Wessells and Evans, 1968). The present work is a continuation of this effort using SEM and TEM to examine normal skin and using the scaleless mutant for purposes of comparison. Scanning microscopy. The trypsin-digested mesenchyme of white Leghorn back skin prepared for SEM as described above revealed a network of fibers (Figs. 1 and 2). With rare exceptions these preparations were free of cells, though occasional debris was present. Fibers are frequently curved and often delineate rounded cavities that presumably contained cells or cell processes in the live specimen. Attempts to control trypsinization to give preparations at will in which cells were retained were unsuccessful. However, under favorable circumstances some cells remain and their intimate association with the fibers is evident (Fig. 3). No fibers could be seen in samples prepared for SEM when the 10% trypsin treatment was omitted (Fig. 4). No clear indication of the location of feather germs could be discerned in these fibrous networks. Trypsin-treated mesenthyme trimmed to include a known number of feather germ rows was examined by making survey micrographs that were enlarged for detailed examination. Moditications in the fiber network corresponding to the expected location of feather germs could not be found. In most cases the distal side of the fiber network was examined. This side could be distinguished in positioning the sample because of the slight curvature of the body wall from which the mesenchyme was taken. Examination of the proximal side showed the same kind of fiber distribution. When the sample was folded, both sides of
82
DEVELOPMENTAL
BIOLOGY
VOLUME
48,
1976
OVERTON
AND COLLINS
SEM
the same network could be compared and no differences could be found. These fibrous networks are very loose, so that in a scanning preparation one can see some distance into the interior (Fig. 1); however it is unlikely that one can see all the way through. There is some variability in the diameter of the fibers in the mesenchymal network. The smallest fibers commonly observed were -60 nm while the predominant fibers were -100 nm. Occasional larger fibers of up to -400 nm occurred. Fibers tended to be curved rather than straight, and pockets in the network appeared to be either single or clustered. Fiber networks prepared as described here are very delicate and easily distorted by rough handling with forceps or even by pressure around large fragments of debris. Distorted networks have predominantly straight rather than curved fibers and fibers may show marked orientation. Often the artifactual nature of the observed orientation is obvious. In any event, distortions of this type were random. microscopy. Intact skin Polarizing showed a birefringent lattice. Trypsinized preparations typically showed no birefringence or occasional birefringence in no regular pattern. Mutant skin showed no birefringence. Transmission microscopy. The intact 8 day chick skin was studied by transmission microscopy (Fig. 5) to compare the collagen fiber size and distribution with that of the fibrous networks viewed by scanning. Collagen fibers appeared to be oriented randomly and to form a very open net. They could frequently be seen to be curved, and cell processes or cell bodies were often closely applied to them as illustrated by others (Wessells and Evans, 1968). Thus the images seen in thin sections were consistent with the view that the fibrous network seen by scanning was
FIG. 1. Collagen FIG. 2. Collagen
network network
Visualization
of Collagen
83
indeed collagen, preserved in a manner to retain its conformation in intact tissue. To substantiate this point, trypsin-treated mesenchymal components of skin were viewed in thin sections. Such preparations showed fibrous material with the banding pattern characteristic of collagen (Piez and Miller, 1974), as seen in Figs. 6 and 7. Although the arrangement of fibers seen in sections was consistent with the picture obtained by scanning, fiber diameter was not. In sectioned material the predominant fiber size was -180 nm, while in preparations viewed by scanning it was -100 nm. However, since the fibers have been trypsin treated and then dried, some shrinkage might be expected. If a collagen fiber which is cut in cross-section such as that in Fig. 5 is measured and the proportion of the cross-sectional area that is composed of collagen fibrils is calculated, it is found to be approximately 60%. Reduction of the nonfibrillar material could thus account for the discrepancy in fiber diameter between TEM and SEM images. To test this supposition, trypsin-treated and critical point-dried preparations were embedded in Araldite, and sections were viewed. Images indicated that in these preparations collagen fibrils were often in contact with one another (Fig. 8). TEM sections reveal occasional small fibers composed of as few as three fibrils. Small fibers of this type were not conspicuous in SEM preparations, possibly because they are rare or because they did not withstand the preparative procedure. Because it is doubtful that one can see through the fibrous network in scanning images, it seemed worthwhile to try to check for systematic fiber orientation in the central region. Therefore, blocks were trimmed to cut in cross-section selected areas of the skin that have been postulated to contain oriented or unoriented fibers, that is, regions such as b and c or region a
from normal from normal
mesenchyme; mesenchyme;
x 10,000. x3200.
84
FIG. 3. Collagen x 2600.
DEVELOPMENTAL
network
from
normal
BIOLOGY
mesenchyme
VOLUME
with
48, 1976
some cells remaining
after
processing.
C, cells.
OVERTON
FIG.
4. Mesenchyme
component
AND
of skin
SEM Visualization
COLLINS
which
has not been subjected
in Fig. 5 of the report by Stuart et al. (1973). In order to avoid prejudice, blocks were cut by one author and survey micrographs were made blind by the other. No differences could be recorded in these regions of the skin. These observations are in accord with those of Wessells and Evans (1968). Thus the evidence taken as a whole from SEM and TEM studies is consistent with the view that there is no systematic orientation of collagen fibers relative to the locations of developing feather germs in the skin. Comparison
with
the scaleless
mutant.
SEM preparations of the mesenchymal component of the scaleless mutant were examined to see if the fibrous structure differed from that of normal skin. Differences were observed both in the range of diameters of common fibers and in the arrangement of fibers (see Figs. 9-11). In the mutant, a fiber class was prominant which measured only -20 nm and the largest fibers seen were -600 nm. A comparison of
85
of Collagen
to 10% trypsin;
x720;
insert,
x2900.
Figs. 3 and 9 illustrates the difference in appearance of the fibrous network in normal and mutant chicks. Fiber arrangement, when viewed over a wide area, has a coarser texture in the mutant. The normal mesenchyme has a predominant class of -lOO-nm fibers and the spacing of fibers is much more even in appearance. A comparison between mutant and normal networks as seen at a higher magnification is shown in Figs. 1 and 10. It is possible that smaller fibers did not withstand the preparative procedures since, in the -20-nm class, broken fibers were occasionally seen. This would of course apply to normal as well as mutant fiber networks. DISCUSSION
Feather germ formation is a complex process involving localized changes in dermis and epidermis in a precise pattern over the feather tract. Although the local high density of cells in the dermal component of the feather germ may arise in part
86
FIG. tibrils; FIG. FIG. FIG.
DEVELOPMENTAL
5. Normal ~25,000. 6. Collagen 7. Collagen 8. Collagen
mesenchyme;
arrow
BIOLOGY
indicates
fiber from trypsin-treated fiber from trypsin-treated fibers cut in cross-section
VOLUME
collagen
fiber
48, 1976
cut
in cross-section
mesenchyme, normal tissue; mesenchyme, mutant tissue; from dried network; x60,000.
through cell proliferation (Wessells, 1965), cell migration must be a contributing factor since, when mitosis is blocked by colchitine, there is no accumulation of mitotic figures (Stuart et al., 1973). Stuart et al. also point out that feather germs are initiated in 3-6 hr which is too short a period to permit the required doubling of the localized cell population by proliferation. If the dermal component of the feather germ arises through cell migration, then in early stages of the process one should see the migrating cells oriented towards their
showing
spacing
of
~60,000. ~60,000.
destination. Elongated fibroblasts with the appropriate orientation are indeed seen (Sengel and Rusaouen, 1968; Wessells and Evans, 1968; Stuart et al., 1973). This results in a “lattice” pattern in the develop ing feather tract (Stuart and Moscona, 1967). One is thus left with the problem of determining what cues the cells use to achieve this pattern of directed migration. It has been suggested that the spatial organization of collagen might be suitable to serve this purpose (Stuart et al ., 1973). The results of the present report make this
OVERTON
AND COLLINS
FIG.
9. Mutant
SEM
Visualization
mesenchyme;
seem unlikely. SEM as used here permits one to view deep into the collagen network of the mesenchyme from either the distal or proximal side and there is no indication that there is any orientation present which could guide the cells in the direction which they take. On the contrary, fibers are seldom straight but form curved paths outlining pockets where the cells nest.
of Collagen
87
x2600.
Although the present results do not favor collagen fiber direction as a controlling factor in migration, this is not to say that collagen is unimportant in the process. Cells move only over a solid substrate (Harrison, 1914) and fibroblasts will not move from collagen to soft gelatin (Abercrombie, l967), nor, with certain exceptions, will cells move in soft gelatin at all (Strassman
88
DEVELOPMENTAL
FIG. FIG.
10. Mutant 11. Mutant
BIOLOGY
VOLUME
mesenchyme; mesenchyme,
et al., 1973). The present work confirms the observation of Wessells and Evans (1968) who showed a close association between cell processes and collagen fibrils. The collagen network visualized here must be important in permitting cell movement, and presumably its distortion or disruption could interfere with directed migration. It is known, for example, that lathyritic agents which disrupt collagen by interfering with cross-linkages (Nimni, 1968) and cause irregularity in collagen fibrils (van den Hooff et al ., 1959) inhibit feather germ formation (Goetinck and Sekellick, 1970). Development of feather germs is also inhibited by collagenase (Stuart et al., 1973). A relatively normal conformation of the collagen network might thus be considered an essential condition for the migratory process. In this connection, it seemed of interest to study collagen conformation in the scaleless mutant (Abbott and Asmundson, 1957; Abbott, 1965) in which the saddle tract is free
48, 1976
~10,000. ~20,000.
of feathers, though collagen synthesis occurs (Goetinck and Sekellick, 1972). The fibrous network of the mesenchyme of scaleless mutant back skin when viewed by SEM showed differences from that of normal skin. The diameter of common fibers was more variable, ranging from a conspicuous small fiber class of -20 nm to fibers of up to -600 nm, while the conspicuous small fiber class in normal skin was -60 nm and the largest fibers seen were -400 nm. These measurements are consistent with fiber diameters as seen by TEM after conventional preparative procedures, if one takes into account that the mesenthyme has been subjected to enzyme treatment and drying. In addition to variations in fiber diameter there is much more irregularity in fiber disposition (compare Figs. 3 and 9). It is at least conceivable that differences of this kind could alter initiation or rate of fibroblast migration during the few hours when it is critical (see Weiss and Garber, 1952).
OVERTON
AND COLLINS
SE !M
Genetically normal ectoderm will produce dermal condensations when combined with scaleless mesoderm (Goetinck and Sekellick, 19721, and morphological changes indicative of feather germ formation may occur in the ectoderm slightly before they appear in the mesoderm (Wessells, 1965) so that the difference in collagen disposition seen here is only one small part of a complex process of tissue interactions. Although the results of this SEM study do not provide an explanation of how feather germs become organized, they do supply concrete information about the spatial organization of collagen in the mesenthyme which must be taken into account in any explanatory design. In addition, because of the recent rapid increase in information concerning disposition, synthesis and function of collagen in development (Konigsberg and Hauschka, 1965; Meier and Hay, 1974; Trelstad, 19731, the method of collagen fiber visualization used here could have a wider application. The Hitachi HFS-2 microscope with a field emission tip is located in the University of Chicago Users Laboratory established with a grant from the Sloan Foundation. Grants from the National Science Foundation (NSF No. GB38669) and the University of Chicago Cancer Research Center (No. I PO CA14599) supported this work. Technical assistance was provided by Ms. Virginia Kriho. REFERENCES ABBOTT, U. K. (1965). Selection for feather number in scaleless chickens. Poultry Sci. 44, 1347. ABBOTT, U. K., and ASMUNDSON, V. S. (1957). Scaleless, an inherited ectodermal defect in the domestic fowl. J. Hered. 48, 63-70. ABERCROMBIE, M. (1967). Contact inhibition: The phenomenon and its biological implications. Nut. Cancer Inst. Mongr. 26, 249-273. BERNFIELD, M. R., and WESSELLS, N. K. (1970). Intra- and extracellular control of epithelial morphogenesis. Develop. Biol. 4(Suppl.), 195-249. G~ETINCK, P. F., and SEKELLICK, M. J. (1970). Early morphogenetic events in normal and mutant skin development in the chick embryo and their relationship to alkaline phosphatase activity. Deuelop. Biol. 21, 349-363. G~ETINCK, P. F., and SEKELLICK, J. (1972). Observations on collagen synthesis, lattice formation, and
Visualization
of Collagen
89
morphology of scaleless and normal embryonic skin. Develop. Biol. 28, 636-648. GROBSTEIN, C., and COHEN, J. (1965). Collagenase: Effect on the morphogenesis of embryonic salivary epithelium in vitro. Science 150, 626-628. HARRISON, R. G. (1914). The reaction of embryonic cells to solid structures. J. Exp. Zool. 17, 521-544. HAY, E., and REVEL, J.-P. (1969). “Fine Structure of Developing Avian Cornea.” Karger, Basel/New York. KONIGSBERG, I. R., and HAUSCHKA, S. D. (1965). Cell and tissue interactions in the reproduction of cell type. In “Reproduction: Molecular, Subcellular and Cellular” (M. Locke, ed.), pp. 243-290. Academic Press, New York. MEIER, S., and HAY, E. D. (1974). Control of cornea1 differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production. Develop. Biol. 38, 249-270. NIMNI, M. E. (1968). A defect in the intramolecular and intermolecular cross-linking of collagen caused by penicillamine. J. Biol. Chem. 243, 1457-1466. PIEZ, K. A., and MILLER, A. (1974). The structure of collagen fibrils. J. Suprumol. Strut. 2. 121-137. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain for electron microscopy. J. Cell Biol. 17, 208-213. SENGEL, P., and RUSAOLJ~N, M. (1968). Aspects histologiques de la differentiation precoce des ebauches plumaires chez le Poulet. Compt. Rend. Acad. Sci. Sec. D, 266, 795-801. STRASSMAN, R. J., LETOURNEAU, P. C., and WE.+ SELLS, N. K. (1973). Elongation of axons in an agar matrix that does not support cell locomotion. Cell Res. 81, 482-487. STUART, E. S. (1967). Experimental studies on the development of feather germs and feather field patterns. Ph.D. thesis, University of Chicago. STUART, E. S., GARBER, B., and MOSCONA, A. A. (1973). An analysis of feather germ formation in the embryo and in uitro, in normal development and in skin treated with hydrocortisone. J. Exp. Zool. 179, 97-118. STUART, E. S., and MOSCONA, A. A. (1967). Emhryonic morphogenesis: Role of fibrous lattice in the development of feathers and feather patterns. Science 157, 947-948. TRELSTAD, R. L. (1973). The developmental biology of vertebrate collagens. J. Histochem. Cytochem. 21, 521-528. VAN DEN HOOFF, A., LEVENE, C. I., and GROSS, J. (1959). Morphologic evidence for collagen changes in chick embryos treated with B-aminopropionitrile. J. Exp. Med. 110, 1017-1021. VIAL, J., and PORTER, K. R. (1974). The surface topography of cells isolated from tissues by maceration. Anat. Rec. 78, 502.
90
DEVELOPMENTAL BIOLOGY
WEISS, P., and GARBER, B. (1952). Shape and movement of mesenchyme cells as functions of the physical structure of the medium. Proc. Nut. Acad. Sci. 38, 264-280. WESSELLS, N. K. (1965). Morphology and proliferation during early feather development. Develop. Biol. 12, 131-153. WESSELLS, N. K., and COHEN, J. H. (1968). Effects of
VOLUME 48, 1976
collagenase on developing epithelia in vitro lung, ureteric bud, and pancrease. Develop. Biol. 18, 294-309. WESSELLS, N. K., and EVANS, J. (1968). The ultrastructure of oriented cells and extracellular materials between developing feathers. Develop. Biol. l&42-61.
Scanning
BIOJSGY
48, 80-90
Electron
Department
(1976)
Microscopic Visualization Embryonic Chick Skin of
of Collagen
Fibers in
JANE OVERTON AND JAMES COLLINS Biology, The University of Chicago, Chicago, Illinois 6M37 Accepted July 25,1975
The disposition of collagen fibers in embryonic chick skin can be visualized by use of scanning microscopy @EM). Chick back skin was removed from a-day embryos, the epithelial and mesenchymal components were separated, and the mesenchyme was subjected to 10% trypsin treatment (Stuart, E. S., and Moscona, A. A. (1967) Science 157,947-948), after which it was prepared for SEM by critical point drying and coating. Such preparations were largely free of cellular material. Cavities which presumably had contained the cells were present in a network of fibers. Skin of the scaleless mutant was also studied. In this mutant the collagen network was more irregular and collagen fiber diameter was more variable. These findings are discussed in connection with the formation of feather germ pattern.
skin. There is evidence that the mutant is The spatial arrangement of collagen fi- not deficient in the ability to synthesize bers in embryonic mesenchyme has been collagen (Goetinck and Sekellick, 1972) considered important in morphogenesis, but it seemed possible that a defect in collaparticularly in glandular tissues (Bern- gen arrangement might contribute to interfield and Wessells, 1970; Grobstein and ference with the pattern of cell migration. Cohen, 1965; Wessells and Cohen, 1968), Aspects of tissue organization which probut also in the skin (Stuart and Moscona, mote pattern formation are largely un1967; Stuart et al., 1973; Goetinck and Sek- known so that the idea that collagen organization precedes cellular patterning ellick, 1972). It therefore seemed worth(Stuart, 1967; Stuart and Moscona, 1967) is while to attempt to visualize these fibers of particular interest. by scanning microscopy. With this objective in mind, the method of Stuart and MATERIALS AND METHODS Moscona (1967) was adapted to permit study of fiber arrangement. White Leghorn eggs were incubated at Stuart et al. (1973) outline evidence that 38°C for 7.5-8.5 days, and back skin of the cell migration contributes to feather germ saddle tract was examined when one to formation in the chick skin. Since cells can five or more rows of feather germs were move only over a solid substrate (Harripresent. Mutant eggs of the scaleless stock son, 1914), the fibrous arrangement of col- were kindly provided by Dr. P. F. Goelagen within the skin mesenchyme un- tinck and were incubated under the same doubtedly mediates cell movement. If the conditions. character of this fibrous tissue component For scanning microscopy skin was disis altered by collagenase (Stuart et al., sected free in Hanks’ solution, incubated 6 1973) or by lathyrogens (Goetinck and Sek- min at 4°C in 2% trypsin (Difco, 1:250) in ellick, 1970), feather germs fail to form. calcium-magnesium-free Hanks’ solution Feather germs of the saddle tract also fail after which the epithelium was lifted off to form in the scaleless mutant (Abbott the mesenchyme (Stuart et al., 1973). Mesand Asmundson, 1957; Abbott, 1965). In enchyme was fixed in 70% alcohol for 1 hr the present study the arrangement of the or more, rinsed and incubated in 10% tryp fibrous tissue component of the mutant sin (Difco, 1:250) in CMF Hanks’ at 37°C has been compared with that of normal for 45 min. This treatment was for the INTRODUCTION
80 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
OVERTON
AND
COLLINS
SEM
purpose of removing cellular tissue components (Stuart et al., 1973). The trypsintreated mesenchyme was then fixed in 3% glutaraldehyde and allowed to settle on a thin layer of gelatin in a small foil dish and positioned with either its proximal or distal side upwards (Vial and Porter, 1974). Throughout these procedures the mesenchymal material was transferred gently with a wide-mouthed pipet and was not allowed to come in contact with the air-fluid interface. After 30-60 min of fixation when the tissue adhered firmly to the gelatin, the preparation was rinsed and the sides of the dish were cut off. After dehydration in alcohol and amyl acetate, preparations were dried in a Bomar critical point dryer followed by coating in a vacuum evaporator with gold palladium or with a Hummer gold vapor coater. Preparations were viewed with an ETEC Autoscan or a Hitachi HFS-2 microscope. For polarizing microscopy, intact skin or trypsinized preparations were fixed in glutaraldehyde or 70% alcohol, stained with chromosome red, dehydrated and mounted on slides in Permount. Tissue was transferred from one solution to the next with a wide-mouthed pipet to avoid the liquid-air interface. For transmission microscopy, preparations of whole skin and intact or trypsintreated mesenchyme were fixed in Karnovsky’s fixative, stained en bloc in uranyl acetate and embedded in Araldite following the procedure described by Hay and Revel (1969). Uranyl acetate staining was carried out after sectioning in some cases. Trypsin-treated mesenchyme that had been critical point dried was also prepared for transmission microscopy. Blocks were sectioned with a diamond knife using a Porter MT-2 microtome, stained with lead (Reynolds, 1963) and viewed in a Hitachi HU-11A microscope. RESULTS
Because the accumulation of mesenthyme cells in feather germs cannot be explained entirely as due to cell prolifera-
Visualization
of Collagen
81
tion (Stuart et al., 1973) and because cells are oriented in a manner suggesting directed migration in a well-defined pattern throughout the mesenchyme which is associated with feather germ formation (Stuart et al., 19731, evidence for a systematically oriented substrate has been sought (Stuart, 1967; Stuart and Moscona, 1967; Stuart et al., 1973; Goetinck and Sekellick, 1972; Wessells and Evans, 1968). The present work is a continuation of this effort using SEM and TEM to examine normal skin and using the scaleless mutant for purposes of comparison. Scanning microscopy. The trypsin-digested mesenchyme of white Leghorn back skin prepared for SEM as described above revealed a network of fibers (Figs. 1 and 2). With rare exceptions these preparations were free of cells, though occasional debris was present. Fibers are frequently curved and often delineate rounded cavities that presumably contained cells or cell processes in the live specimen. Attempts to control trypsinization to give preparations at will in which cells were retained were unsuccessful. However, under favorable circumstances some cells remain and their intimate association with the fibers is evident (Fig. 3). No fibers could be seen in samples prepared for SEM when the 10% trypsin treatment was omitted (Fig. 4). No clear indication of the location of feather germs could be discerned in these fibrous networks. Trypsin-treated mesenthyme trimmed to include a known number of feather germ rows was examined by making survey micrographs that were enlarged for detailed examination. Moditications in the fiber network corresponding to the expected location of feather germs could not be found. In most cases the distal side of the fiber network was examined. This side could be distinguished in positioning the sample because of the slight curvature of the body wall from which the mesenchyme was taken. Examination of the proximal side showed the same kind of fiber distribution. When the sample was folded, both sides of
82
DEVELOPMENTAL
BIOLOGY
VOLUME
48,
1976
OVERTON
AND COLLINS
SEM
the same network could be compared and no differences could be found. These fibrous networks are very loose, so that in a scanning preparation one can see some distance into the interior (Fig. 1); however it is unlikely that one can see all the way through. There is some variability in the diameter of the fibers in the mesenchymal network. The smallest fibers commonly observed were -60 nm while the predominant fibers were -100 nm. Occasional larger fibers of up to -400 nm occurred. Fibers tended to be curved rather than straight, and pockets in the network appeared to be either single or clustered. Fiber networks prepared as described here are very delicate and easily distorted by rough handling with forceps or even by pressure around large fragments of debris. Distorted networks have predominantly straight rather than curved fibers and fibers may show marked orientation. Often the artifactual nature of the observed orientation is obvious. In any event, distortions of this type were random. microscopy. Intact skin Polarizing showed a birefringent lattice. Trypsinized preparations typically showed no birefringence or occasional birefringence in no regular pattern. Mutant skin showed no birefringence. Transmission microscopy. The intact 8 day chick skin was studied by transmission microscopy (Fig. 5) to compare the collagen fiber size and distribution with that of the fibrous networks viewed by scanning. Collagen fibers appeared to be oriented randomly and to form a very open net. They could frequently be seen to be curved, and cell processes or cell bodies were often closely applied to them as illustrated by others (Wessells and Evans, 1968). Thus the images seen in thin sections were consistent with the view that the fibrous network seen by scanning was
FIG. 1. Collagen FIG. 2. Collagen
network network
Visualization
of Collagen
83
indeed collagen, preserved in a manner to retain its conformation in intact tissue. To substantiate this point, trypsin-treated mesenchymal components of skin were viewed in thin sections. Such preparations showed fibrous material with the banding pattern characteristic of collagen (Piez and Miller, 1974), as seen in Figs. 6 and 7. Although the arrangement of fibers seen in sections was consistent with the picture obtained by scanning, fiber diameter was not. In sectioned material the predominant fiber size was -180 nm, while in preparations viewed by scanning it was -100 nm. However, since the fibers have been trypsin treated and then dried, some shrinkage might be expected. If a collagen fiber which is cut in cross-section such as that in Fig. 5 is measured and the proportion of the cross-sectional area that is composed of collagen fibrils is calculated, it is found to be approximately 60%. Reduction of the nonfibrillar material could thus account for the discrepancy in fiber diameter between TEM and SEM images. To test this supposition, trypsin-treated and critical point-dried preparations were embedded in Araldite, and sections were viewed. Images indicated that in these preparations collagen fibrils were often in contact with one another (Fig. 8). TEM sections reveal occasional small fibers composed of as few as three fibrils. Small fibers of this type were not conspicuous in SEM preparations, possibly because they are rare or because they did not withstand the preparative procedure. Because it is doubtful that one can see through the fibrous network in scanning images, it seemed worthwhile to try to check for systematic fiber orientation in the central region. Therefore, blocks were trimmed to cut in cross-section selected areas of the skin that have been postulated to contain oriented or unoriented fibers, that is, regions such as b and c or region a
from normal from normal
mesenchyme; mesenchyme;
x 10,000. x3200.
84
FIG. 3. Collagen x 2600.
DEVELOPMENTAL
network
from
normal
BIOLOGY
mesenchyme
VOLUME
with
48, 1976
some cells remaining
after
processing.
C, cells.
OVERTON
FIG.
4. Mesenchyme
component
AND
of skin
SEM Visualization
COLLINS
which
has not been subjected
in Fig. 5 of the report by Stuart et al. (1973). In order to avoid prejudice, blocks were cut by one author and survey micrographs were made blind by the other. No differences could be recorded in these regions of the skin. These observations are in accord with those of Wessells and Evans (1968). Thus the evidence taken as a whole from SEM and TEM studies is consistent with the view that there is no systematic orientation of collagen fibers relative to the locations of developing feather germs in the skin. Comparison
with
the scaleless
mutant.
SEM preparations of the mesenchymal component of the scaleless mutant were examined to see if the fibrous structure differed from that of normal skin. Differences were observed both in the range of diameters of common fibers and in the arrangement of fibers (see Figs. 9-11). In the mutant, a fiber class was prominant which measured only -20 nm and the largest fibers seen were -600 nm. A comparison of
85
of Collagen
to 10% trypsin;
x720;
insert,
x2900.
Figs. 3 and 9 illustrates the difference in appearance of the fibrous network in normal and mutant chicks. Fiber arrangement, when viewed over a wide area, has a coarser texture in the mutant. The normal mesenchyme has a predominant class of -lOO-nm fibers and the spacing of fibers is much more even in appearance. A comparison between mutant and normal networks as seen at a higher magnification is shown in Figs. 1 and 10. It is possible that smaller fibers did not withstand the preparative procedures since, in the -20-nm class, broken fibers were occasionally seen. This would of course apply to normal as well as mutant fiber networks. DISCUSSION
Feather germ formation is a complex process involving localized changes in dermis and epidermis in a precise pattern over the feather tract. Although the local high density of cells in the dermal component of the feather germ may arise in part
86
FIG. tibrils; FIG. FIG. FIG.
DEVELOPMENTAL
5. Normal ~25,000. 6. Collagen 7. Collagen 8. Collagen
mesenchyme;
arrow
BIOLOGY
indicates
fiber from trypsin-treated fiber from trypsin-treated fibers cut in cross-section
VOLUME
collagen
fiber
48, 1976
cut
in cross-section
mesenchyme, normal tissue; mesenchyme, mutant tissue; from dried network; x60,000.
through cell proliferation (Wessells, 1965), cell migration must be a contributing factor since, when mitosis is blocked by colchitine, there is no accumulation of mitotic figures (Stuart et al., 1973). Stuart et al. also point out that feather germs are initiated in 3-6 hr which is too short a period to permit the required doubling of the localized cell population by proliferation. If the dermal component of the feather germ arises through cell migration, then in early stages of the process one should see the migrating cells oriented towards their
showing
spacing
of
~60,000. ~60,000.
destination. Elongated fibroblasts with the appropriate orientation are indeed seen (Sengel and Rusaouen, 1968; Wessells and Evans, 1968; Stuart et al., 1973). This results in a “lattice” pattern in the develop ing feather tract (Stuart and Moscona, 1967). One is thus left with the problem of determining what cues the cells use to achieve this pattern of directed migration. It has been suggested that the spatial organization of collagen might be suitable to serve this purpose (Stuart et al ., 1973). The results of the present report make this
OVERTON
AND COLLINS
FIG.
9. Mutant
SEM
Visualization
mesenchyme;
seem unlikely. SEM as used here permits one to view deep into the collagen network of the mesenchyme from either the distal or proximal side and there is no indication that there is any orientation present which could guide the cells in the direction which they take. On the contrary, fibers are seldom straight but form curved paths outlining pockets where the cells nest.
of Collagen
87
x2600.
Although the present results do not favor collagen fiber direction as a controlling factor in migration, this is not to say that collagen is unimportant in the process. Cells move only over a solid substrate (Harrison, 1914) and fibroblasts will not move from collagen to soft gelatin (Abercrombie, l967), nor, with certain exceptions, will cells move in soft gelatin at all (Strassman
88
DEVELOPMENTAL
FIG. FIG.
10. Mutant 11. Mutant
BIOLOGY
VOLUME
mesenchyme; mesenchyme,
et al., 1973). The present work confirms the observation of Wessells and Evans (1968) who showed a close association between cell processes and collagen fibrils. The collagen network visualized here must be important in permitting cell movement, and presumably its distortion or disruption could interfere with directed migration. It is known, for example, that lathyritic agents which disrupt collagen by interfering with cross-linkages (Nimni, 1968) and cause irregularity in collagen fibrils (van den Hooff et al ., 1959) inhibit feather germ formation (Goetinck and Sekellick, 1970). Development of feather germs is also inhibited by collagenase (Stuart et al., 1973). A relatively normal conformation of the collagen network might thus be considered an essential condition for the migratory process. In this connection, it seemed of interest to study collagen conformation in the scaleless mutant (Abbott and Asmundson, 1957; Abbott, 1965) in which the saddle tract is free
48, 1976
~10,000. ~20,000.
of feathers, though collagen synthesis occurs (Goetinck and Sekellick, 1972). The fibrous network of the mesenchyme of scaleless mutant back skin when viewed by SEM showed differences from that of normal skin. The diameter of common fibers was more variable, ranging from a conspicuous small fiber class of -20 nm to fibers of up to -600 nm, while the conspicuous small fiber class in normal skin was -60 nm and the largest fibers seen were -400 nm. These measurements are consistent with fiber diameters as seen by TEM after conventional preparative procedures, if one takes into account that the mesenthyme has been subjected to enzyme treatment and drying. In addition to variations in fiber diameter there is much more irregularity in fiber disposition (compare Figs. 3 and 9). It is at least conceivable that differences of this kind could alter initiation or rate of fibroblast migration during the few hours when it is critical (see Weiss and Garber, 1952).
OVERTON
AND COLLINS
SE !M
Genetically normal ectoderm will produce dermal condensations when combined with scaleless mesoderm (Goetinck and Sekellick, 19721, and morphological changes indicative of feather germ formation may occur in the ectoderm slightly before they appear in the mesoderm (Wessells, 1965) so that the difference in collagen disposition seen here is only one small part of a complex process of tissue interactions. Although the results of this SEM study do not provide an explanation of how feather germs become organized, they do supply concrete information about the spatial organization of collagen in the mesenthyme which must be taken into account in any explanatory design. In addition, because of the recent rapid increase in information concerning disposition, synthesis and function of collagen in development (Konigsberg and Hauschka, 1965; Meier and Hay, 1974; Trelstad, 19731, the method of collagen fiber visualization used here could have a wider application. The Hitachi HFS-2 microscope with a field emission tip is located in the University of Chicago Users Laboratory established with a grant from the Sloan Foundation. Grants from the National Science Foundation (NSF No. GB38669) and the University of Chicago Cancer Research Center (No. I PO CA14599) supported this work. Technical assistance was provided by Ms. Virginia Kriho. REFERENCES ABBOTT, U. K. (1965). Selection for feather number in scaleless chickens. Poultry Sci. 44, 1347. ABBOTT, U. K., and ASMUNDSON, V. S. (1957). Scaleless, an inherited ectodermal defect in the domestic fowl. J. Hered. 48, 63-70. ABERCROMBIE, M. (1967). Contact inhibition: The phenomenon and its biological implications. Nut. Cancer Inst. Mongr. 26, 249-273. BERNFIELD, M. R., and WESSELLS, N. K. (1970). Intra- and extracellular control of epithelial morphogenesis. Develop. Biol. 4(Suppl.), 195-249. G~ETINCK, P. F., and SEKELLICK, M. J. (1970). Early morphogenetic events in normal and mutant skin development in the chick embryo and their relationship to alkaline phosphatase activity. Deuelop. Biol. 21, 349-363. G~ETINCK, P. F., and SEKELLICK, J. (1972). Observations on collagen synthesis, lattice formation, and
Visualization
of Collagen
89
morphology of scaleless and normal embryonic skin. Develop. Biol. 28, 636-648. GROBSTEIN, C., and COHEN, J. (1965). Collagenase: Effect on the morphogenesis of embryonic salivary epithelium in vitro. Science 150, 626-628. HARRISON, R. G. (1914). The reaction of embryonic cells to solid structures. J. Exp. Zool. 17, 521-544. HAY, E., and REVEL, J.-P. (1969). “Fine Structure of Developing Avian Cornea.” Karger, Basel/New York. KONIGSBERG, I. R., and HAUSCHKA, S. D. (1965). Cell and tissue interactions in the reproduction of cell type. In “Reproduction: Molecular, Subcellular and Cellular” (M. Locke, ed.), pp. 243-290. Academic Press, New York. MEIER, S., and HAY, E. D. (1974). Control of cornea1 differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production. Develop. Biol. 38, 249-270. NIMNI, M. E. (1968). A defect in the intramolecular and intermolecular cross-linking of collagen caused by penicillamine. J. Biol. Chem. 243, 1457-1466. PIEZ, K. A., and MILLER, A. (1974). The structure of collagen fibrils. J. Suprumol. Strut. 2. 121-137. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain for electron microscopy. J. Cell Biol. 17, 208-213. SENGEL, P., and RUSAOLJ~N, M. (1968). Aspects histologiques de la differentiation precoce des ebauches plumaires chez le Poulet. Compt. Rend. Acad. Sci. Sec. D, 266, 795-801. STRASSMAN, R. J., LETOURNEAU, P. C., and WE.+ SELLS, N. K. (1973). Elongation of axons in an agar matrix that does not support cell locomotion. Cell Res. 81, 482-487. STUART, E. S. (1967). Experimental studies on the development of feather germs and feather field patterns. Ph.D. thesis, University of Chicago. STUART, E. S., GARBER, B., and MOSCONA, A. A. (1973). An analysis of feather germ formation in the embryo and in uitro, in normal development and in skin treated with hydrocortisone. J. Exp. Zool. 179, 97-118. STUART, E. S., and MOSCONA, A. A. (1967). Emhryonic morphogenesis: Role of fibrous lattice in the development of feathers and feather patterns. Science 157, 947-948. TRELSTAD, R. L. (1973). The developmental biology of vertebrate collagens. J. Histochem. Cytochem. 21, 521-528. VAN DEN HOOFF, A., LEVENE, C. I., and GROSS, J. (1959). Morphologic evidence for collagen changes in chick embryos treated with B-aminopropionitrile. J. Exp. Med. 110, 1017-1021. VIAL, J., and PORTER, K. R. (1974). The surface topography of cells isolated from tissues by maceration. Anat. Rec. 78, 502.
90
DEVELOPMENTAL BIOLOGY
WEISS, P., and GARBER, B. (1952). Shape and movement of mesenchyme cells as functions of the physical structure of the medium. Proc. Nut. Acad. Sci. 38, 264-280. WESSELLS, N. K. (1965). Morphology and proliferation during early feather development. Develop. Biol. 12, 131-153. WESSELLS, N. K., and COHEN, J. H. (1968). Effects of
VOLUME 48, 1976
collagenase on developing epithelia in vitro lung, ureteric bud, and pancrease. Develop. Biol. 18, 294-309. WESSELLS, N. K., and EVANS, J. (1968). The ultrastructure of oriented cells and extracellular materials between developing feathers. Develop. Biol. l&42-61.