Autoradiographic studies of the origin of the basement lamella in Ambystoma

DEVELOPMENTAL

BIOLOGY,

7, 152-168

Autoradiographic

Studies

Basement

Lamella

ELIZABETH Department

(1963)

of Anatomy,

of the ,Origin

of the

in Ambystoma’

D. HAY AND J. P. REVEL Haruard

Accepted

Medical October

School, Boston, Mussachusetts 6, 1962

INTRODUCTION

In 1954, Weiss and Ferris, using the electron microscope, described an orthogonal system of fibrils in the basement lamella that underlies the epidermis of amphibian larvae. The collagenous nature of this lamella (Porter, 1954; Edds, 1961) and the well-developed endoplasmic reticulum of the associated fibroblasts (Weiss and Ferris, 1956; Weiss, 1956; Kemp, 1959; Hay, 1962), together with the longstanding evidence that fibroblasts can secrete collagen, make it tempting to conclude that the fibrils of the lamella are produced by sublamellar fibroblasts. Edds and Sweeny (1962) have recently suggested that new fibrils are added to the lamella at its epidermal surface, and they have emphasized the possibility that the epidermis also contributes some product to the lamella, a theory which appealed to many earlier workers (see Singer and Andrews, 1956; Kemp, 1959, 1961; Porter and Pappas, 1959; Salpeter and Singer, 1960). In the present autoradiographic study, we chose tritiated proline to study the formation of the lamella because collagen is known to be unusually rich in proline and hydroxyproline. In the past year, work carried out simultaneously in several laboratories has led to the successful application of autoradiographic techniques to electron microscopy (see Revel and Hay, 1961, for partial review). These new techniques have enabled us to study with better resolution than previously possible, the intracellular synthesis of the proteinaceous precursors of the lamella and to trace their subsequent fate as they become incorporated into this amazingly well-ordered epithelial substratum. 1 This 979-Cl-A

research was supported by Grants CA05196-04Sl from the United States Public Health Service. 152

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Larvae of three species of salamanders were used in our preliminary experiments, but the observations to be reported here deal primarily with 25-mm Ambystoma madatum larvae. The animals were injected intraperitoneally with 10 PC of tritiated proline dissolved in 0.01 ml of distilled water (Schwarz Bioresearch, Inc. ); the limbs, which had been regenerating for 12 days (2-3 fingerbud stage), were fixed at appropriate intervals thereafter. Two limbs were fixed at 1, 5, 10, and 15 minutes, and at 1 and 2 hours post-injection, and four limbs were fixed at the 30-minute interval and the 4-hour interval. Limbs were also fixed at 1, 2, 3, 4, 7, 16, and 28 days thereafter. The results agreed with earlier experiments in which larger amounts of isotope were used, but all the illustrations in this report are from the experiment described above. Fixation was carried out in 1% osmium tetroxide for 1 hour at 4”C, and the limbs were then dehydrated in alcohol and embedded in n-butyl methacrylate. Details on amputation level, temperature, em-

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FIG. 1. Diagram summarizing the autoradiographic present study. See text for explanation.

technique

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bedding, and other procedures are given in a previous report (Revel and Hay, 1961). The thin (~0.1 p) sections mounted on grids for electron microscopy were coated with photographic emulsion by the “loop” technique, or they were attached to a glass slide with tape and dipped in melted Ilford L-4 emulsion (diluted 1: 8 with distilled water). Glass slides containing thicker sections (OS-l.0 p) for light microscopy were dipped in the same emulsion (Fig. 1). The autoradiographs were stored 3 weeks at 4°C in light-tight boxes to expose the emulsion to the beta rays emitted by the tritium within the sections. After appropriate development, sections on grids were treated with a lead stain in 0.02 N NaOH, which removes most of the visible gelatin of the emulsion and makes the specimen more transparent. The gelatin of the emulsion over the sections for light microscopy can also be removed with NaOH (Fig. 1). The sections illustrated (Figs. 2-6, 11, and 12) were stained with 1% toluidine blue. The electron micrographs (Figs. 7-10) of the autoradiograms were taken at original magnifications of 2000-5000 x with a Siemens Elmiskop I electron microscope. The autoradiographic resolution achieved is -0.2 p in both the light and electron microscopic preparations. RESULTS

General Features of the Regenerating

Basement Lame&

The forelimb of the Ambystoma larva regenerates at a very rapid rate. A blastema of mesenchyme-like cells derived from the stump tissues appears 3-6 days after amputation and grows into a paddleshaped bud which redifferentiates to form a new limb 10-15 days post amputation. At 12 days, the cartilaginous skeleton is clearly delineated, muscle is redifferentiating, and a new basement lamella is forming under the growing epidermis of the regenerate. In the distal portion of the regenerate the collagenous components of this lamella are not yet organized into the orthogonal layers described by Weiss and Ferris, and the fibroblasts believed to be involved in its production are fairly close apposition to the epidermis (Fig. 2). In the is more proximal region of the regenerate, however, differentiation advanced and the fibroblasts are separated from the epidermis by a lamella of collagen fibers -4 p in thickness, which, when viewed with the light microscope, has the appearance of a basement “membrane” (BL, Fig. 3). Tl re electron microscope reveals that the lamella (BL,

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Fig. 7) is composed of bundles of collagen fibrils arranged in very regular layers. The long axes of the fibrils in each layer are parallel to each other, but are perpendicular to the long axes of the fibrils of adjacent layers (Weiss and Ferris, 1954; Porter, 1954). At this time in development, the layers of fibrils adjacent to the epidermis may be narrower and less organized than the deeper layers (Kemp, 1961). The outermost component at this stage appears as a single line or “adepidermal membrane” ( Salpeter and Singer, 1960). In the less differentiated, distal portion of the regenerate, new collagen in the basement lamella is not laid down in orthogonal layers but is subsequently reorganized (see Weiss and Ferris, 1956). In the present report, we shall concern ourselves with a description of the manner in which new protein is deposited in the formed basement lamella of the proximal portion of the regenerate (Fig. 3). Location of the Isotope 15-30 Minutes after Injection At 12 days post amputation, all the experimental animals were injected intraperitoneally with tritiated proline and the limbs were fixed at various intervals thereafter. In the l-10 minute period after the injection we were unable to detect any isotope in the limb tissues by autoradiographic techniques. In the limbs of animals fixed 15 minutes after injection of the labeled amino acid, however, radioactivity could be detected in the cytoplasm of the fibroblasts underlying the basement lamella. Inasmuch as the more soluble molecules, such as the circulating amino acid, seem to be removed from the tissue during fixation and dehydration, it seems likely that the H”-proline revealed in the autoradiographs has been incorporated into protein molecules or their immediate precursors which are bound in the cytoplasm. There is no detectable radioactivity in the vessels or extracellular space. Protein synthesis is known to be very rapid, and the I5-minute lag here may simply reflect limitations of the present technique in detecting the first molecules formed. In animals fixed 30 minutes after the injection of the isotope, radioactivity has increased in the differentiating cells of the blastema. At this time, tritiated proline is localized in the cytoplasm of the fibroblasts under the basement lamella (Fib, Figs. 2 and 3). In electron micrographs of thin sections of the same material (Fig. 7)) it would appear that the tritiated proline at this time is preferentially localized in the Golgi apparatus and endoplasmic reticulum of the fibroblasts.

Explanation

of

Micrographs

Figures 2-6 are photographs of autoradiographs viewed in the light micromicrographs of autoradiographs of thinner scope. Figures 7-10 are electron sections of the same material. The arrows in some of the figures point to silver grains in the autoracliographic emulsion overlying the section. Figures 11 and 12 are light micrographs of autoradiographs of later stages. BL, basement lamella hl, muscle cytoplasm Ep, epidermis N, nucleus ER, cndoplasmic reticulum cess, blood vessel Fib, fibroblast u, small vesicle in cytoplasm CA, Golgi apparatus FIG. 2. Light micrograph showing labeled fibroblasts and epidermal cells 30 minutes after administration of H”-proline (arrows). The diffuse grains over nuclei inclicate incorporation of the amino acid into nucleoprotein. There is very little background fog in these preparations. Magnification: x900. FIG. 3. Light micrograph showing a labeled fibroblast in the proximal region of the limb 30 minutes after H3-proline administration. The area in the rectangle is shown in an electron micrograph of an adjacent section in Fig. 7. Note absence of label over blood vessel lumen. Magnification: x900. FIG. 4. Light micrograph showing labeled fibroblasts and epidermal cells 1 hour after tritiated thymidine administration. There is little, if any, label in the basement membrane as yet. Magnification: X 900. FIG. 5. Light micrograph showing accumulation of label at the basal surface of the epidermal cells (arrows) 2 hours after administration of H3-proline. Electron micrographs (Fig. 9) confirm the impression that some of the label is now located extracellularly at the epidermal-lamellar junction. Magnification: x 900. FIG. 6. Light micrograph showing further accumulation of labeled protein at the epidermal-lamellar junction (arrows) 4 hours after administration of H”proline. The juxtaepidermal label is now predominantly extracellular (Fig. lo), and there are a few grains over the deeper portions of the lamella. Magnification: x 900. 157

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The epidermal cells have also incorporated the labeled amino acid. The reticulum and Golgi apparatus tend to be perinuclear in location in epidermal cells and most of the label is associated with this membrane-rich region of the cytoplasm (Ep, Fig. 2; ER, Fig. 8). There is less incorporation of isotope into nucleoproteins, muscle proteins, and other cytoplasmic proteins low in proline content. Little, if any, bound isotope can be detected in the extracellular compartment. These observations are interpreted as indicative of the active synthesis of a protein, rich in proline, by both the fibroblasts and the epidermal cells adjacent to the basement lamella. Location

of the Isotope 1-4 hours after Injection

If we now examine autoradiographs of the limbs of animals injected as above with tritiated proline but fixed at hourly intervals after the injection, we can learn the subsequent fate of the cytoplasmic materials labeled by tritiated proline in the 30-minute period postinjection. One hour after the injection (Fig. 4), the location of the isotope in the fibroblasts is similar to that at 30 minutes. We have the impression, however, that the label in the epidermal cells in the proximal region of the regenerate is already preferentially located in the epidermal cytoplasm adjacent to the forming basement lamella. By 2 hours, it is clear that a significant amount of tritiated proline in these cells has moved to the epidermal-lamellar border (Fig. S). Electron micrographs of autoradiographs suggest that the radioactive material at the junction of epidermis and connective tissue is now located in the extracellular basement lamella adjacent to the epidermis (Fig. 9), as well as in the cytoplasm of the cells. Thus, it would seem that the first labeled extracellular protein containing proline appeared within 2 hours after the initial exposure of the cells to the tritiated precursor. If smaller amounts were secreted earlier, however, they might not be detected by the present technique. There are small vesicles (500-1000 A in diameter) in the basal region of the epidermal cells (inset, Fig. 9) and near the cell membrane of the FIG. 7. Electron micrograph of portions of a fibroblast and epidermal cell in a limb fixed 30 minutes after HZ-proline administration. The section is thinner than the adjacent section that was processed for light microscopy (Fig. 3). The localization of the silver grains over the fibroblast, however, is similar in the two autoradiographs. The long arrows in Fig. 7 indicate grains over the Golgi apparatus, and the short arrows, endoplasmic reticulum. Magnification: X7000.

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fibroblasts. It may be that some of the labeled protein is contained within them and is carried to the cell surface by such intracellular membrane-limited vesicles. It is tempting to think that vesicles of this type originate in the Golgi zone of the cell. At 4 hours after the injection of tritiated proline, the outer layer of the basement lamella is even more heavily labeled (Fig. 6) than at 2 hours. The label is now predominantly extracellular (Fig. 10). The cytoplasm of the fibroblasts is not as heavily labeled (Fig, 6) at 4 hours as at 3 hours (Fig. 5), and we have the impression that the fibroblasts begin to lose radioactivity at about the same time as detectable labeled extracellular protein appears (2 hours). The same is true for epithelial cells. The preferential label in the base of the epidermal cells disappears 4 hours post-injection (Fig. 6). There is a general increase in nuclear labeling (N, Fig. 6) during the 14 hour period post-injection and the background cytoplasm of epidermal cells and fibroblasts and the myofibrils of muscle cells (hl, Fig. 6) are also more labeled at 2 and 4 hours than at 1 hour. There would appear to be a slow accumulation, then, of labeled intracellular as well as extracellular protein for several hours as more and more of the circulating proline is incorporated into the cells. A shorter exposure to the isotope (“pulse” type label) would be helpful in analyzing the events that take place after 4 hours, and we have initiated such a series of experiments. It is interesting to note, however, that in the present experiments we found that the label at the epidermal-basement lamellar border did not increase after 1 day. That is to say, we have accomplished in the present experiment a “pulse” type label to the extent that the limb was exposed to radioactive proline for less than a 24-hour period. After 1 day, all the tritiated proline has been absorbed from the intraperitoneal cavity. If we examine limbs at subsequent intervals, it would appear that the labeled juxtaepidermal band of collagen is displaced inward 4-7 days later (Fig. 11) and finally (2-4 weeks later) reaches the fibroblast layer where it is apparently reabsorbed (Fig. 12). This inward dis-

FIG. 8. Electron micrograph showing localization of the label over the endoplasmic reticulum (short arrows) and Golgi apparatus (long arrows) in an epidermal cell 30 minutes after administration of H”-proline. A small portion OF the basement lamella is included in the lower right-hand corner of the figure. Magnification: X 6500.

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placement of old collagen is predictable, for our experiments show that the new protein of the lamella is laid down in the region adjacent to the epidermis. The “youngest” or smallest collagen fibrils are to be found next to the epidermis; as more fibrils form, these fibrils are left behind or, to speak loosely, they “move” from the epidermal to the inner surface of the lamella. DISCUSSION

The present autoradiographic study has demonstrated that most, if not all, of the new proline-rich protein of the basement lamella in the regenerating Ambystoma limb is deposited at the epidermal-lamellar junction. Evidence pertinent to the participation of adjoining cells in the synthesis of these components has also been obtained and, although space does not permit a full discussion of these data in relation to previous literature (see introduction), it seems worthwhile to examine the data briefly as they bear on the following three questions. 1. Does the epidermal basement lamella?

cell contribute

a proteinaceous

product

to the

Several points revealed in the present autoradiographic investigation favor an affirmative answer to this question. A proline-rich material, probably a protein or protein precursor, appears in the cytoplasm of the epidermal cells 15 minutes after the injection of the tritiated amino acid into the animal and accumulates in the endoplasmic reticulum-Golgi regions of the cell. At 30 minutes, some of the label is located in the basal cytoplasm. At 1 hour, the basal cytoplasm is more heavily labeled, and at 2 hours the first detectable extracellular label appears immediately adjacent to the epidermal cell membrane; the label accumulates at the epidermal-lamellar junction. The label in the basal regions of the epidermal cells decreases after 4 hours. These facts are compatible with the hypothesis that the epidermis secretes a protein into the underlying connective tissue. Indeed, it is difficult to suggest an alternative explanation for these observations. FIG. 9. Higher magnification electron micrograph of the epidermal-lamellar junction in a limb fixed 2 hours after administration of H3-proline. Extracellular material adjacent to the epidermal cell membrane is labeled. The area enclosed in the rectangle is shown at higher magnification in the inset, We are not certain whether label is actually localized in the small vesicles depicted. Magnification: X 19,000; inset, X 50,000.

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2. Does the epidermis secrete collagen? Collagen contains ~25% proline and hydroxyproline. Most other proteins contain <3% proline, except keratin (~10%). The keratin of the epidermal cell is an intracellular product, but most of the protein labeled 15-30 minutes post-injection in the epidermal cells seems to be secreted 2-4 hours later and in earlier periods is located over the endoplasmic reticulum-Golgi region of the cell instead of over its “keratin” filaments (tonofilaments). Although the idea that epidermal cells might make both keratin and collagen seems heretical to a vertebrate morphologist, it is widely accepted in invertebrate zoology (Rudall, 1955). The most reasonable alternatives to this are that the epidermis is contributing some other material (such as mucopolysaccharide) to the lamella or that the epidermal product is some kind of mucus secreted to the exterior of the limb. However, mucoproteins are not very high in proline content. Moreover, the basement lamella is composed predominantly of collagen known to be rich in proline and hydroxyproline (Edds, 1961). It would not be unreasonable, then, to interpret the present data as compatible with the theory that epidermal cells secrete collagen. Some mention might be made of the marked incorporation of proline into nuclei. “Nucleoproteins” are high in proline content, and protein turnover in the nucleus is known to be extensive. The label in the nuclei does not decrease 24 hours post-injection. It reaches a maximum at this time and slowly decreases over the next 2-3 weeks. It seems unlikely that the nucleus has contributed a significant portion of the secreted proline-rich protein, for this pattern of amino acid incorporation is typical of “intracellular” protein synthesis. 3. What is the role of the fibroblast.? Numerous fibroblasts form a single-layered sheath immediately under the basement lamella, and these fibroblasts have an abundant granular endoplasmic reticulum and Golgi complex similar to that seen in gland cells, a morphology now commonly believed to be characteristic of cells synthesizing large amounts of protein destined FIG. 10. High magnification electron micrograph of the epidermal-lamellar junction in a limb fixed 4 hours after administration of H’-proline. The labeled protein is now predominantly extracellular in location. The plane of the section is oblique to the juxtaepidermal surface of the lamella. Magnification: x 16,000.

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FIG. 11. Light micrograph of an autoradiograph from a limb exposed to tritiated proline 4 days prior to fixation. The labeled “new” collagen that accumulated next to the epidermis is now “old” collagen and has been displaced inward due to the juxtaepidermal deposition of unlabeled collagen after the trix 900. tiated proline “pulse” was exhausted. Magnification: FIG. 12. Light micrograph of an autoradiograph of a limb 4 weeks after the initial exposure to tritiated proline. The labeled collagen is now located next to the fibroblast layer, and we have the impression that some of the label is re-

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for the extracellular compartment. Proline incorporation into the cytoplasm of fibroblasts is impressive, and most of the labeled product leaves the cell 24 hours after injection of the precursor. There is no extracellular accumulation of labeled protein in the immediate vicinity of the fibroblasts. Instead, most of the labeled product seems to accumulate next to the epidermis. In tissue cultures, fibroblasts are known to secrete collagen molecules (“tropocollagen”) which probably polymerize into collagen fibrils extracellularly (see Gross, 1956; Porter and Pappas, 1959). The marked incorporation of tritiated proline by the sublamellar fibroblasts most likely reflects their synthesis of the proline-rich collagen precursors. It is, perhaps, ,difficult to believe that these protein molecules might diffuse through the basement lamella to accumulate on its epidermal surface. The basement lamella, with its well-ordered collagen fibrils, seems to constitute an imposing structural barrier, but certainly all nutrient materials that reach the epidermis must pass through the lamella, and larger unitseven cells (macrophages and white blood cells) and cell processes (nerves, fibroblasts)-seem to enter it at will. In developing cartilage, proteins labeled by tritiated proline can diffuse through an even more imposing matrix (Revel and Hay, unpublished observations). It would seem that collagen and possibly other proteins synthesized by the fibroblasts and epidermal cells are in some way attracted to the epidermal surface of the lamella and that here they polymerize into the layers of fibrils visualized with the electron microscope as an extracellular collagenous framework. SUMMARY

AND CONCLUSIOii

Tritiated proline was used to follow the synthesis and fate of proteins of the basement lamella of regenerating salamander limbs. Autoradiographs were analyzed in both the electron and light microscopes. Our data are most reasonably interpreted as indicating that the collagen of the basement lamella, and/or other proline-rich proteins, are secreted by associated fibroblasts and by the epidermis and accumulate at the epidermal-lamellar junction. The factors that might influence the protein precursors of this collagenous lamellar “fabric” to polymerize immediately next to the epidermis are unknown, but entering the fibroblast cytoplasm (arrow on the right). The outer juxtaepidermal layers of collagen are new. They have been deposited there subsequent to the exposure of the limb to tritiated proline. Magnification: x900.

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there can be little doubt, as Weiss so clearly recognized, that it is “the epidermal underside which serves as foundation for the architectural development of the basement lamella” (Weiss and Ferris, 1956, p. 279). We are pleased to be able to contribute this paper mental Biology dedicated to Dr. Paul Weiss.

to a volume

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L)fXdO/J-

REFERENCES M. V., JR. ( 1961). Chemical and morphological differentiation of the basement lamella. In “Synthesis of Molecular and Cellular Structure” (D. Rurnick, ed.), pp. 111-138. Ronald, New York. EDDS, M. V., JR., and SWEENY, P. R. (1962). Development of the basement lamella. In “Electron Microscopy” (S. S. Breese, Jr., ed.), Vol. 2, pp. QQ-2 Academic Press, New York. GROSS, J. (1956). The behavior of collagen units as a model in morphogenesis. J. Biophys. Biochem. Cytol. Suppl. 2, 261-274. HAY, E. D. (1962). Cytological studies of dedifferentiation and differentiation in regenerating amphibian limbs. In “Regeneration” (D. Rudnick, ed. ), pp. 177-210. Ronald, New York. KEMP, N. E. (1959). Development of the basement lamella of larval anuran skin. Develop. Biol. 1, 459476. KEMP, N. E. (1961). Replacement of the larval basement lamella by adult-type basement membrane in anuran skin during metamorphosis. Develop. Biol. 3, 391410. PORTER, K. R. ( 1954). Observations on the fine structure of animal epidermis, Proc. Intern. Conf. on Electron Microscopy, London, p. 539. PORTER, K. R., and PAPPAS, G. D. (1959). Collagen formation by fibroblasts of the chick embryo dermis. J. Biophys. Biochem. Cytol. 5, 153-166. REVEL, J. P., and HAY, E. D. ( 1961). Autoradiographic localization of DNA synthesis in a specific ultrastructural component of the interphase nucleus. Exptl. Cell Research 25, 474480. RUDALL, K. M. ( 1955). The distribution of collagen and chitin. Symposia SOC. Exptl. Biol. No. 9, 49-71. SALPETER, M. M., and SINGER, M. (1960). Differentiation of the submicroscopic adepidermal membrane during limb regeneration in adult Triturus, including a note on the use of the term basement membrane. Anat. Record 136, 2740. SINGER, M., and ANDREWS, J. S. (1956). The adepidermal network in the skin of the newt, Triturus ciridescens. Acta Anat. 28, 313-330. WEISS, P. ( 1956). The compounding of complex macromolecular and cellular units into tissue fabrics. Proc. Natl. Acad. Sci. U. S. 42, 819830. WEISS, P., and FERRIS, W. (1954). Electron-microscopic study of the texture of the basement membrane of larval amphibian skin. Proc. Natl. Acad. Sci. U. S. 40, 528-540. WEISS, P., and FEXRIS, W. (1956). The b asement lamella of amphibian skin. Its reconstruction after wounding. J. Biophys. Biochem. Cytol. 2, 275-282. EDDS,