A reinterpretation of the structure and development of the basement lamella: An ordered array of collagen in fish skin

DEVELOPMENTAL

A

BIOLOGY

20. 304-331

Reinterpretation of the

(1969)

of the

Basement

Lamella:

Collagen JOSEPH Biological

B.

NADOL, Laboratories,

Structure

JR.,

An Ordered

in Fish R.

JOHN

Harvard

Univerxity,

Accepted

April

and

Development Array

of

Skin1

GIBBINS,

AND

Cambridge,

KEITH

R.

Massachusetts

PORTER 021.38

15, 1969

INTRODUCTION

The basement lamella, just beneath the basement membrane of the epidermis of aquatic vertebrates, contains a highly ordered array of collagen fibrils. The arrangement of the fibrils has been described as orthogonal (Porter, 1954a, 1956; Rosin, 1946), that is, in layers or plies with the unit fibrils in adjacent layers oriented approximately at right angles to one another. A similar arrangement of fibrils is also found in the skin of amphibians (Edds, 1964; Kemp, 1959; Weiss, 1957; Weiss and Ferris, 1954), the cornea (Jakus, 1961), the notochord sheath and in encapsulated spherical organs (Porter, 1967). Previous investigators (Edds, 1964; Weiss, 1957; Weiss and Ferris, 1954) have drawn an analogy between the structure of the amphibian basement lamella and plywood, suggesting that each layer of fibrils is continuous around the body of the animal and parallel both to the other layers and to the basement membrane or, as it is now also called, the basement lamina (Fawcett, 1963). Little is understood concerning the mechanism whereby such a highly ordered extracellular array is first established. Proponents of the “plywood” theory (Edds, 1964; Weiss and Ferris, 1954) have suggested that the lamella results from a sequential deposition of layers of collagen just at the lower surface of the basement membrane, which has been implicated as the site of collagen polymerization by several investigators (Edds, 1964; Grobstein, 1964; Hay and Revel, 1963). Some sort of “switching mechanism” at the basement membrane (Edds, 1964) is required by this plywood interpretation to account for the abrupt shift in orientation, if each layer is to be deposited at an angle of approximately 90’ to ’ This investigation was supported carried out at the Marine Biological the Biological Laboratories, Harvard

by USPH Training Laboratories, Woods University, Cambridge, 304

Grant 26;.707-06 and was Hole, Massachusetts, and Massachusetts.

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the one that preceded it. Although several mechanisms have been proposed for this apparent switching (Gross et al., 1952; Weiss, 1957; Edds, 1964), the plywood interpretation does not readily suggest a means by which the basement lamella can expand as the epidermis of the growing animal increases in area. In the hope of shedding further light on this problem, we have examined the skin of the rapidly developing Fund&s heteroclitus. commonly known as the mummichog. Our findings have led us to propose a different interpretation of the structure of the basement lamella and a new theory of its development. the “scindulene” theory (shingle theory). This is based primarily on the observation that collagen fibril layers descend from the basement membrane in a pattern resembling shingles on a roof, rather than being parallel to it as proposed by the “plywood” theory. The “scindulene” theory obviates the necessity of a “switching mechanism,” more easily accounts for the dynamics of growth, and gives to the basement membrane a greater role in the initiation of collagen assembly and the establishment of the lamellar structure. MATERIALS

AND

METHODS

Eggs and sperm, obtainable from the adult Fund&s heteroclitus in June and July at Woods Hole, (and somewhat later in the summer in the salt marshes of Gloucester, Massachusetts), were stripped into a fingerbowl containing a few milliliters of seawater, stirred to mix, and washed with seawater 15 minutes later. Nearly loo’, of the eggs were fertilized. Eight to twelve embryos were maintained in shallow seawater in each fingerbowl at 18OC. At this temperature hatching occurred in 14-17 days. Fish of stages prior to hatching were removed from the egg membrane before fixation. This was accomplished in seawater or fixative in a depression slide under the dissecting microscope by pulling the pierced membrane away from the embryo with two pairs of sharpened forceps. Adult skin was obtained simply by removing a scale from the flank or by removing a larger area of skin, scales, and muscle to retain the original orientation of the scales. Fixation was accomplished by 6’, glutaraldehyde in 0.088 M phosphate buffer with 0.005”r calcium at pH 7.4 for 3 hours at room temperature. The tissue was then washed for 2.5 hours in 0.1 M phosphate buffer with 10”; sucrose at room temperature. It was subsequently postfixed in lc; osmium tetroxide in 0.1 M phosphate buffer

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for 1 hour at room temperature, dehydrated in ethanol, and embedded in epoxy resin. In the cases of embryos 5, 15, and 17 days old as well as the larval Fund&s, cross sections of the entire fish in the mid-body region were cut at approximately 45’ to the long axis of the fish. This was done because collagen fibrils spiral around the body, crossing the lateral midline at approximately 45” in both directions. Thus sections cut at this angle showed the collagen of the basement lamella in longitudinal and transverse aspects. The exposed part of scales of the adult fish were cut in vertical section parallel to the long axis of the fish. Morphological comparisons of the 6ne structure of embryo and larval stages were based on tissue of the mid-body region slightly dorsal to the lateral midline. Sections cut on a Porter-Blum MT-Z microtome were stained with uranyl acetate followed by Reynolds’ lead citrate (Reynolds, 1963). Electron micrographs were taken with the Philips EM 200 and RCA EMU-3D electron microscopes. OBSERVATIONS

Morphology

of Development’

Embryo, 5 days. This stage of embryonic development is approximately 11-12 days before hatching. The embryo is curled around the large yolk sac, and the trunk has not yet straightened [Fig. 1; stage 28 embryo (Armstrong and Child, 1965) removed from chorion]. The epidermis consists of 1 to 2 cell layers and varies in thickness from 6 to 9 cc. Lying just beneath the epidermis there is a basement membrane (basement lamina) (BM, Fig. 2), a finely fibrillar sheet approximately 500 A thick. A clear zone (Cz) separates the plasma membrane of the epidermal cell from the basement membrane. The embryo of the fifth day possessesno true basement lamella. However, collagen fibrils (Co), about 150 A in diameter, appearing both singly and in bundles and in both cross and longitudinal section, are found closely applied to the basement membrane throughout the subepithelial extracellular space and adjacent to the surface of mesenchymal cells (Mea). Embryo, 12 days. The epidermis, now approximately 8 p thick, is much more uniforn in thickness than in the 5-day embryo, and ’ Embryos of several ages other than those described interest of limiting the length of this report observations lected ages only.

here were studied, but in the are presented on a few se-

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FIG. 1. Drawing of Fundulus embryo 5 days after fertilization. removed from its chorion. Stage 28, Armstrong and Child (1965). mission of the editors of the Biological Bulletin.

The embryo Reproduced

307

has been with per-

FIGS. 2, 3, and 5-13. Unless otherwise stated all electron micrographs represent sections taken in the mid body region of the fish at approximately 45O to its long axis. FIG. 2. Skin of embryo 5 days after fertilization. Although no true basement lamella is present, collagen fibrils (Co) are found closely applied to the basement membrane (EM) in the subepithelial extracellular space and adjacent to mesenchymal cells (Mes). Collagen fibrils are found both singly and in bundles and both in cross and longitudinal sections. A clear zone (Cz) separates the epithelial cell membrane from the basement membrane. x 17,500: ‘Unless

otherwise

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1 micron.

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possesses a regularly scalloped external surface. Both cell layers of the epidermis are replete with finely fibrillar material, probably tonofilaments (To, Fig. 3). Dense regions (HD) similar in appearance to half-desmosomes, called “bobbins” by Weiss and Ferris (1954), are common at this stage along the cytoplasmic side of the cell membrane where it faces the basement membrane. The basement membrane (BM) is more condensed and much thicker (now 950 A) than that of the 5-day-old embryo. Basement granules (BG), about 350 A in diameter, an extracellular component not present at all on day 5, have appeared in the clear zone (CZ) below the basal surface of the epithelium and are closely applied to the basement membrane, forming a sheet of densely staining spherical granules. A basement lamella (BL), approximately 0.7 ~1 thick, and made up of collagen fibrils (ca. 350 8, in diameter) arrayed in 8-10 seemingly orthogonal layers, is deposited beneath the basement membrane between days 5 and 12 of life. The number of rows of collagen fibrils per layer is uniformly 2 to 3 throughout the lamella except in the layer adjacent to the basement membrane and in the deepest layers, the latter of which are often interrupted by intruding dermal cells. In these latter layers there is often only one row of fibrils per layer. Although the number of rows of fibrils per layer changes with the stage of development, the number of layers (8-10) established by day 12 is maintained through posthatching stages. Hut&Zing, 17 days. The skin of the hatchling (Fig. 4) is still without scales. The epidermis is similar to that of the 12-day-old embryo. It is about 6-7 p thick and stratified into two layers, the basal cells staining more densely than the superficial ones. Occasionally the continuity of the stratum of densely staining basal cells is interrupted by what appear to be chloride cells, also found in the gill arch of Fund&s (Philpott and Copeland, 1963). Just beneath the basal cells of the epidermis there is a planar array of basement granules (BG, Fig. 5), which appear between the fifth and the twelfth day. In addition, the basement membrane (BM) is thicker (now 1500 A) and more finely textured than that of the earlier stage. The basement lamella is also thicker (now 0.9 M) than at the twelfth day (0.7 ,.L). This0 is due to an increase in fibril diameter (now approximately 400 A) and in the number of rows of collagen fibrils per layer rather than to an increase in the number of layers.

FIG. 3. Embryo 12 days after fertilization. The epidermis consists of two cell layers, both of which contain tonofilaments (To). The lower, or basal, layer stains more densely than the upper, or superficial layer. Densities called half-desmosomes (HD) or “bobbins” by previous investigators extend into the epithelial cytoplasm from the cell membrane adjacent to the basement membrane (BM). Beneath the clear zone (Cz) and at the upper surface of the basement membrane there is a sheet of densely staining basement granules (BG). The basement lamella (BL) has been deposited by day 12 with S-10 “orthogonally” arrayed layers of collagen fibrils; the number of layers maintained until posthatching stages. Each layer with the exception of that adjacent to the basement membrane and the deepest layer of the lamella, has 2 to 3 rows of fibrils. X25,000. 309

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FIG. 4. Drawings of Fundulus hatchling. large yolk sac. Stage 35 of Armstrong and of the Biological Bulletin.

AND

The Child

newly (1965).

PORTER

hatched fry Reproduced

still possesses a with permission

As before the number of layers stands at B-10. Here the greatest number of rows in any layer is three, and this maximum is achieved in the middle layers of the lamella. The average diameter of the collagen fibrils on the other hand decreases with increasing distance from the basement membrane. Occasionally a fibril which stays within one layer over several microns of the image suddenly descends and merges with the next layer of fibrils having the same orientation (CF, Fig. 5). These have been named “crossover fibrils.” The basement lamella is not continuous around the body of the fish. Although extending into the dorsal fin, it rapidly decreases in layer and row number toward the tip of the fin. The basement granules (BG, Fig. 6) become progressively less closely packed in the same zone. The lamella of the fin near the junction of the fin to the body has only about 6 to 8 layers (BL, Fig. 61, and these are less regularly organized than those in the lamella of the flank region. Near the tip of the fin the basement lamella is reduced to one or two layers of single fibril rows. At the very tip of the fin, the basement granules and basement lamella are completely absent. Between the lamella of the fin and the sublamellar dermal cells lies a rod (R, Fig. $), which in thin section shows the periodicity of collagen (640-650 A). It is the developing supportive major ray of the fin. A similar structure has been described in the caudal fin, where it has been called “elastoidin” (Fitton-Jackson, 1964). Dermal cells are closely applied to the lower surface of the ray. The collagenous supportive ray, like the basement lamella is completely absent at the very tip of the fin.

FIG. 5. Skin of hatchling. Basement granules (BG), first found at day 12 are still prominent below the clear zone (CZ). The basement membrane (BM) is thicker than at earlier stages. The basement lamella (BL) is thicker than at day 12 due to an increase in the average number of fibril rows per layer and in the average fibril diameter. The number of layers, however, has remained constant at 8-10. Collagen fibril layers are not parallel to the basement membrane, but descend toward the underlying connective tissues at a slight angle from it. Thus layer N descends beneath a longitudinal fibril beginning near $. In the same zone, layer (Y increases from 1 to 2 rows of collagen fibrils. Other examples of collagen fibril descent from the basement membrane are found in Figs. 7 and 10. The average diameter of the fibril decreases with increasing distance from the basement membrane. ~23,500. 311

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FIG. 6. The basement lamella (BL) of the hatchling slowly tapers or thins in the dorsal fin. This decrease in thickness is the result of a decrease in the number of layers and in the number of rows of collagen fibrils per layer. A supportive ray (R) exhibiting the banding period of collagen is present beneath the basement lamella. At the very tip of the fin the basement lamella, basement granules (BG), and collagenous rays (as seen in this micrograph) are entirely absent. X 16,500.

Larval Fundulus. At this stage, several days after hatching, the larva has completely absorbed its yolk and has achieved a length of approximately 7-10 mm. However, the skin still does not possess scales, and the epidermis is in fact quite similar to that of the two previous stages, being 5-10 P thick.

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The basement membrane has not thickened since day 17. The basement granules (BG, Fig. 7) are less densely staining, less closely packed, and of smaller diameter (now 200 A) than those in earlier stages. The basement lamella has thickened considerably, now being approximately 1.7 P. Nevertheless the number of fibril layers remains at 8-10; this, it will be recalled, is the number found as early as day 12 after fertilization. However, the number of fibril rows per layer has increased to a maximum of 4-5 rows. Although the deepest layer may be interrupted by dermal cell extensions, the number of fibril rows per layer generally increases with increasing distance from the basement membrane. In contrast the average fibril diameter, at a maximum of 400 A in the layers adjacent to the basement membrane, decreases in the same direction. Adult Fundulus. Dramatic changes occur in the skin between the hatching and adult stages. Over the exposed region of the scale there is a stratified epithelium about 12 cell layers for a total thickness of 650 ,.L This replaces the two-layered, 10 ,J thick epithelium of the l7-day-old hatchling. The cell shape changes gradually from columnar in the deepest layers to cuboidal in the middle layers and finally to squamous at the most superficial surface. Basement granules, so prominent in the earlier stages, have completely disappeared, and only a clear zone (CZ, Fig. 8) lies between the 1900 A-thick basement membrane and the basal cell membrane. The basement lamella (BL, Fig. 8) is no longer the planar structure of the earlier stages but is corrugated and thickened to approximately 3.6 H. However, the 8-10 layers observed in previous stages are still present. The increase in thickness of the lamella is due, as before, to an increase in the number of fibril rows per layer, reaching a maximum of approximately 10 in the deepest layer, where not interrupted by intruding dermal cells. The maximum collagen fibril diameter has also increased to 500 A. The cells beneath the lamella have become less tightly packed than the mesenchymal cells of earlier stages, and the collagen fibrils extend considerably below the lamella proper (CO, Fig. 8). There they intermingle with long, thin processes (Pr) of dermal cells. Cell bodies of the latter are rich in rough endoplasmic reticulum, while their surfaces possess a number of pits, especially on the cell surface facing the lamella. The structure of adult skin is complicated by scales, which develop in the dermis after hatching. The epidermis continues uninter-

FIG. 7. Larval stage. The basement lamella (BL) is considerably thicker than at earlier stages. This increase in thickness is due to an increase in the number of rows of collagen fibrils per layer. The number of layers, however, remains at 8-10, the number observed as early as day 12. The layers of collagen fibrils are not parallel to the basement membrane (BM), but descend at a slight angle from it. Thus layer (Y passes beneath 0, 0 passes beneath y, and y beneath 6. Basement granules (BG), so prominent in the embryo of 12 days and in the hatchling, are much less densely staining in the larva. The number of collagen fibril rows per layer increases with increasing distance from the basement membrane, except in the deepest layers, which are often interrupted by intruding processes of fibroblasts (F). X 18,500. 314

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ruptedly around the exposed part of the scale, although it is thicker on the dorsal or exposed surface. Between the adepithelial basement lamella (BL, Fig. 8) and the scale there is loose connective tissue. The scale itself has at least two distinct layers. One is composed of densely staining fine fibrils. This layer, called the “bony plate” (Van

FIG. 8. Skin of the adult. The adepithelial basement lamella (IX) above the scale possesses B-10 layers of collagen fibrils, the number also found in the embryo of day 12. The number of rows of collagen fibrils per layer increases with increasing distance from the basement membrane (B&f). Basement granules, found in the embryo, hatchling, and less prominently in the larva, are now completely absent, and only a clear zone (CZ) separates the epithelial cell membrane from the basement membrane. Collagen fibrils (Co) descend for a considerable distance beneath the lamella proper, and long fibroblast processes (Pr) intrude into the deepest layers of the lamella. ~21,500.

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Oosten, 1957) overlies the second layer, which consists of large bundles of collagen fibrils arranged in strata. This second layer has been called the “fibrillar plate” (Van Oosten, 1957) (FP, Fig. 9). These collagen fibrils, 600 A in diameter, are very closely packed into a hexagonal array, and in some regions their arrangement resembles that of the basement lamella. Beneath the fibrillar plate there is a thin epithelial sheet (EP, Fig. 9), recognizable by the presence of large desmosomes (Des). This overlies an extremely thick admuscular lamella (AL) composed of an orthogonal array of collagen fibrils of smaller diameter (350 A) and less closely packed than those of the fibrillar plate of the scale. Like the adepithelial basement lamella the number of fibril rows per layer of the admuscular lamella increases with the distance from the superficial surface of the fish. However, in contrast to the adepithelial lamella, fibroblast cell bodies and processes (PR, Fig. 9) are present in the admuscular lamella. Collagen Orientation and Morphology ment Lamella Junction

of Basement Membrane-Base-

Cross section. Previous investigators (Edds, 1964; Weiss, 1957; Weiss and Ferris, 1954) have described the basement lamella of amphibian skin as being composed of alternate layers or plies of collagen fibrils, parallel to the overlying basement membrane. However, in every stage of the development of the basement lamella of Fundulus that we have examined, the collagen fibrils were observed to descend at a very small angle from the basement membrane rather than lying parallel to it. For example, in a hatchling (Fig. 10) the uppermost layer cut in cross section can be followed beginning at the top of the field (CY)as it descends beneath a longitudinally sectioned fibril layer that begins at p, which in turn, passes beneath a third layer seen in cross section ( y). Thus the fibril layer ((Y) that is adjacent to the basement membrane at the top of the field is the third removed from it at the bottom. This descent of collagen fibrils is also shown to advantage in Figs. 5 and 7. In Fig. 5, also taken from a hatchling, as the layer CYdescends beneath the fibrils viewed in longitudinal aspect (B), it increases in thickness from one to two fibril rows. In Fig. 7, taken from a larva, layer (Y passes beneath a longitudinal fibril layer 8, which increases from one to two fibril rows in thickness as it itself passes beneath another layer 7. This layer also passes beneath the layer 6, observed in longitudinal aspect. Thus layer a, which is adjacent to the basement membrane at

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FIG. 9. The scales, present in the adult fish, lie beneath the epidermis and adepithelial basement lamella (Fig. 8). The scale is composed of two layers the “bony plate” and the “fibrillar plate.” Only the fibrillar plate is shown here (FP). Beneath the scale there is an epithelial extension (Ep), recognizable by the presence of desmosomes (Des). Adjacent to the muscle there is an admuscular lamella (AL), composed of orthogonally arrayed layers of collagen fibrils. In contrast to the adepithelial lamella, long fibroblast processes (Pr) are found throughout the admuscular lamella. X33,000.

the all 3’. the

bottom of the field, is the fourth removed from it at the top. In these cases, the angle of descent of fibril layers is approximately It should be noted that obliquity of sectioning angle cannot alter apparent orientation of the layers. For example, no matter how a

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FIG. 10. Fundulus hatchling. The collagen layers of the basement lamr 4la al -e not parallel to 1the basement membrane (BM), but descend at a slight angle frc pm it. Thus layer (Y des tends beneath layer p, which in turn descends beneath layer y Xl’ 9,000.

piece Of plywood is cut, the anothc 2r 2md none tapers off at Obl iqu :e section. In order collag en fibrils of adjacent

plies always appear paralle 1 to one the surface. beti ween to observe the relationship layers and between fibrils and the

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we studied sections tangential overlying basement membrane, to or slightly oblique with respect to the superficial surface of the skin. Sections including the upper surface of the basement membrane revealed the densely staining basement granules in a closely packed array. Deeper penetration revealed the fabric of collagen fibrils with adjacent layers crossing each other at angles of 105-110” (Fig. 11) rather than at right angles as described by Weiss (1957). Layers of fibrils cut nearly tangenin Ambystona tially appear in sheets or bundles (Bu), whereas layers cut more obliquely are disposed in a “herring-bone” pattern (HB), in which collagen fibrils alternately enter and leave the plane of section. At the sublamellar surface (SS, Fig. 11) and at the surface of the basement membrane (BM, Fig. 12) the fibrils are also arranged in bundles crossing each other at 105-110’.

FIG. 11. An oblique section through the epithelium (Ep), basement membrane (BM), basement lamella (BL), and sublamellar surface (SS) of the hatchling. The collagen fib& are arranged in bundles (Bu) crossing at 105-llO”. Where the angle of sectioning becomes less tangential to the surface of the skin, collagen fibrils appear to be disposed in a herringbone pattern (HB). 9,500.

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FIG. 12. Hatchling. Oblique section. At the basement membrane (BM)-basement lamella (BL) junction, collagen fibrils seem to be arranged in bundles (Bu). Fibrils of adjacent layers cross each other at 105-1100. Basement granules (BG) are also visible. X 22,500.

Higher resolution microscopy revealed the presence within the basement membrane of another extracellular fibrillar component, which we have called “microfilaments.” These vary in diameter from 10 to 40 A and are found at the junction of basement membrane and basement lamella and throughout the lamella itself. Occasionally, these filaments appear in two orientations, crossing at approximately 95O, to form a microgrid within the basement membrane (arrows, Fig. 13). DISCUSSION

Scindulene Model: Lamella Structure On the basis of the observations presented above, we conclude that the collagen layers, or “scindulae”, are not parallel to the basement membrane, but rather depart from it at a small angle (which

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FIG. 13. Hatch@. Oblique section. At the basement membrane (BM)-basement lamella (BL) junction, microfilaments (arrows), lo-40 A in diameter, are occasionally found in two orientations crossing each other at approximately 95O to form a microgrid. X 284,000.

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is exaggerated in the model in Fig. 14 A) and furthermore that collagen fibrils of the scindulae are inserted at end points into the basement membrane and into the sublamellar region. In the limited field imposed by the electron microscope, this extension toward the sublamellar region does indeed look like an addition of plies, just as the observation of a limited region of a myelin sheath of a nerve cut in cross section suggests a parallel ply arrangement rather than the actual continuous spiral of membranes (Robertson, 1955). This descent of fibril layers toward the underlying connective tissue has been observed in every stage of developing Fund&s studied, whenever a basement lamella was present. The fibrils of adjacent layers, or scindulae, cross each other at 105-110” (Fig. 14B), as observed in tangential sections (Fig. 12). If the long axis of the fish is established along the line in Fig. 14B, the angles (Y and p, both 50-55”, correspond well with the observations that sections must be cut at an angle of approximately 50” to the long axis of the fish to obtain longitudinal and cross section of fibrils in the same plane of section. The overall configuration of the scindulene structure thus resembles “shingles” of collagen fibril bundles with each succeeding “shingle” crossing the next at an angle of 105” to llO” and all extending from the basement membrane. Furthermore, like shingles, the scindulae grow thicker with increasing distance from their area of insertion into the basement membrane toward their sublamellar extent. The progressive increase in average number of fibril rows per layer with increasing distance from the basement membrane accounts for this thickness. It is interesting that scales, which are apparently dermal in origin (Van Oosten, 1957) and form in posthatching stages,

FIG. 14A. Model of basement lamella as proposed in “scindulene” or shingle theory. Lamella as seen in transverse sections. Collagen fibril layers, or scindulae (Co) descend at a slight angle (here exaggerated) from the basement membrane (BM).

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FIG. 14B. Model of basement lamella as proposed in “scindulene,” or shingle theory. Lamella as seen in oblique section. Collagen fibril layers in both orientations are inserted into the basement membrane (B&f). Adjacent layers, or scindulae, cross each other at 105-IlOo. The line represents the long axis of the body. The angle between the long axis of the body and the long axis of fibril layers (01 and 0) is 50-55”. This explains why sections must be taken at approximately 50” to the long axis of the fish to obtain fibril layers in both cross and longitudinal section.

are also composed of collagen fibrils and that these too are arranged like shingles on a relatively macroscopic level. It is not necessary to suppose, however, that all the fibrils of a given layer observed in stages of development after day 12 are inserted into the basement membrane, but only that during the initial establishment of the lamellar array were the first fibrils so inserted. Thus in the 12-day-old embryo there is little difference in the number of rows of fibrils per layer, whereas in later stages of development the number of rows of fibrils substantially increases with increasing distance

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from the basement membrane. We therefore suggest that the first fibrils that establish the apparently orthogonal array prior to day 12 are all inserted into the basement membrane and that with growth more fibrils are inserted into the membrane in each layer (Fig. 15). However, the progressive increase in row number in deeper layers suggests that other fibrils may be polymerized adjacent to the original fibrils without insertion into the basement membrane. The insertion of collagen fibrils of the basement lamella into the basement membrane proposed by the scindulene theory agrees well

Basement

membrane

TRANSVERSE

/

TANGENTIAL

SECTION

SECTION

FIG. 15. Growth of the basement lamella as Thickening of the basement lamella with growth posthatching stages is the result of an increase per layer rather than in the number of layers. with radial and longitudinal growth of the fish, basement membrane are produced for insertion fibrils in adjacent “areas of initiation” are oriented

interpreted by the scindulene theory. from day 12 after fertilization until in the number of collagen fibril rows As the basement membrane expands additional “rows of initiation” in the of more fibril rows per layer. Collagen at 105-110’ to each other.

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with the fact that in the collagen systems so far observed, the ends of collagen fibrils are usually found in conjunction with ground substance or basement membrane material. Indeed, where basement membrane and collagen fibrils are associated, the fibrils merge with or are embedded in the membrane material. For example, Karrer (1956) found collagen fibrils embedded in the aveolar basement membrane of mouse lung, and Yamada (1960) observed an intimate association of collagen fibrils and the basement membrane of the frog renal glomerulus. Porter (1966) has also observed that collagen fibrils are inserted into basement membrane material of the tracheal epithelium of the rat and that collagen fibrils of frog tendon appear to be anchored at the myotendon junction (Porter, 1954b) in the same manner. Therefore, in the case of the basement lamella, the claim that collagen fibrils insert into the basement membrane not only brings the basement lamella into accord with other collagen-basement membrane systems, but also offers a means by which the dermis firmly supports and binds the epidermis, just as one would expect a connective tissue to do. Scindulene

Model:

Deposition

of the

Lamella

Rather than relying on a “switching mechanism” to ensure that succeeding layers of collagen be laid down at right angles to each other, as proposed by the plywood theory (Edds, 1964), the scindulene model suggests that the lamella is formed by a polymerization of the fibrils of every layer over a limited area of the skin at the same time, beginning at the basement membrane and proceeding toward the sublamellar region. We postulate that there are areas of initiation and insertion for the fibrils in the basement membrane, with each area directing deposition of collagen in only one orientation and doing so continuously in direction if not in time (see Fig. 15). Adjacent areas of the basement membrane direct collagen deposition at 105-110” to each other to form the “orthogonal” array. Microfilaments observed throughout the basement membrane-lamellar regions and especially at the margin of the basement membrane with the lamella may mark the origins of this nearly orthogonal array. The plywood theory claims that the deepest layers of the lamella were first laid down at the basement membrane, and that later layers were placed atop them. Each layer is therefore different in time of origin, the deepest being the oldest. In contrast, we suggest that the deepest layer observed in a limited field is actually the deepest extent

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of a layer of collagen fibrils still in large part anchored at the basement membrane. The deepest layer may in fact be the last portion of the scindula to be polymerized if collagen fibril extension occurs at the fibril end not attached to the basement membrane. Conversely, it may have been the first portion to have been polymerized if collagen fibril extension occurs at the end attached to the basement membrane. The second interpretation is supported by the autoradiographic findings of Hay and Revel (1963), which indicate that in a regenerating system in an amphibian presumed collagen precursors are first incorporated into the upper layers of fibrils and that the label subsequently moves down through the lamella. Nevertheless, abundant evidence suggests that fibroblasts (found only below the basement lamella in early stages of Fundulus) synthesize collagen (Gaines, 1960; Grobstein, 1964; Porter and Pappas, 1959) and therefore it is conceivable that the polymerization of collagen fibrils occurs at sites other than the basement membrane and closer to the source of monomeric protein. Furthermore, if we consider collagen fibril polymerization in time as a rapid accretion of precursors onto a central core resulting in a fibril of increasing diameter, the obvious decrease in average fibril diameter with increasing distance from the basement membrane (Fig. 5) suggests that fibril extension occurs at the fibril end not inserted into the basement membrane; that is, a later time of polymerization is indicated for the deeper layers than for the uppermost layers. Fibrils of very small diameter found throughout the lamella are probably the result of intussusceptive addition of fibrils once the lamella has been established. Implications

of the Theory

Crossover fibrils. Since in the scindulene theory all layers of the basement lamella could polymerize at approximately the same time, at least over limited areas of the skin, it would be conceptually a simple matter for regions of two layers having the same orientation to fibril” during the period the join together and form a “crossover lamella is being established between the fifth and twelfth days. Such a crossover fibril is much more difficult to explain on the basis of the plywood theory, which claims that fibrils in separate layers differ in time of deposition and hence by definition, are discontinuous. Growth. The plywood theory becomes difficult to support when one considers events in the development of the lamella after the twelfth day. The number of layers established by the twelfth day is

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maintained without increase until well after hatching. The increase in thickness is due, then, to an increase in the number of fibril rows per layer rather than the the number of layers. The plywood theory proposes either (a) that the switching mechanism is programmed to allow increasingly more fibril rows per layer before the next layer in an orthogonal orientation is begun, or (b) that a totally different mechanism intervenes to allow addition of fibril rows to layers that are completely separated from the basement membrane. The scindulene theory, on the other hand, explains the increase in the number of rows of fibrils with age as a natural extension of the proposed mechanism of original deposition. Expansion of the basement membrane with radial and longitudinal growth of the fish would also expand the areas of initiation of collagen fibrils in both directions, making possible an increase in the number of sites of initiation and consequently the number of fibril rows per layer (Fig. 15). Then also the scindulene theory much more easily allows for radial and longitudinal growth. It is inferred from the plywood theory that concentric layers of fibrils surround the animal. This would seem to require some sort of breaking and joining of fibrils (Edds, 1964) to account for the necessary increase in fibril length as the epidermis grows. The scindulene theory more easily allows for growth by a sliding of adjacent scindulae or layers relative to each other and/or the increase in collagen fibril length at the end points, either in the basement membrane or in the sublamellar region. Variation in lamella thickness. The basement lamella is not of equal thickness around the entire fish. Indeed it tapers out in the dorsal fin area and disappears completely near the tip of the fin. Edds (1964) has also described a decrease in the number of layers toward the ventral region of Rana pipiens larva. Since the plywood theory implies concentric layers of fibrils around the animal, the only way this theory could account for tapering of the lamella would be a decrease in the speed of the “switching mechanism” relative to reabsorption in those areas with fewer layers or plies. This would mean, however, that adjacent areas would be out of phase. Such different rates of “switching” could never result in a continuous lamellar structure. The scindulene theory can account for lamellar tapering more easily by proposing that the scindulae, or layers of fibrils, grow less rapidly at the sublamellar end in the tapered regions, resulting in fewer observed layers in a limited field. Role of the basement membrane. The basement membrane is a

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protein-polysaccharide complex (Bennett, 1963). All connective and supportive tissue, including the ground substance of the lamella itself (Edds and Sweeny, 1961) contain such mucoproteins (Meyer, 1957). Furthermore, the interface of basement membrane and lamella is not abrupt, but rather the basement membrane material seems to merge with both the collagen fibrils and the collagen ground substance. Increasing evidence suggests that the basement membrane is synthesized at least in part by the overlying epithelium. Pierce et al. (1964) found ferritin-labeled antibodies to a basement membrane antigen localized in the endoplasmic reticulum of parietal yolk epithelial cells. Mukerjee et al. (1965) have demonstrated by histochemical, immunochemical, and electron microscopic studies that this basement membrane material, secreted by an epithelial tumor, is the counterpart of normal basement membrane. What little is known presently about the basement membrane and associated connective tissues suggests a positive interaction between basement membrane and basement lamella. For example, there is a chemical similarity between components of basement membrane and collagen. Goodman et al. (1955) have shown that the basement membrane from glomerular capillaries has antigens in common with collagen fibrils. Similarly, Rothbard and Watson (1961) have demonstrated that rabbit antibody to rat collagen localizes in the basement membrane of rat glomeruli. Kefalides and Winzler (1966) have found that the amino acid composition of basement membrane isolated from dog glomeruli resembles that of mammalian collagen and that X-ray diffraction patterns of basement membrane and denatured collagen are similar. They concluded that the basement membrane is composed of a collagen-like protein and a glycoprotein. Furthermore, Wood (1960) and Wood and Keech (1960) have demonstrated that a low concentration of polyanions, such as those commonly found in the basement membrane, accelerate the nucleation phase and rate of collagen fiber formation and induce the development of thin fibrils. Thus the concentration of mucopolysaccharides in the extracellular phase may influence the rate of polymerization and average diameter in uiuo. Thus the basement membrane shows biochemical and antigenic properties similar to collagen and collagen ground substance. Furthermore, common chemical components of basement membrane affect collagen polymerization. One might therefore expect an interaction

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between the basement membrane and the lamella. In fact, it may not be excessive to speculate that collagen fibrils represent a transformation of basement membrane material; that is, the collagen fibril is composed at least in part of basememt membrane components polymerized in a more highly ordered array. Moreover, at the next level of order, the basement membrane could play a role in establishing the orthogonality of the collagen fibrils of the basement lamella. We believe that the scindulene theory bestows new importance upon the basement membrane as an area of initiation and insertion of collagen fibrils, by proposing the insertion of collagen fibrils into the basement membrane and hence the possible continuity and identity of at least certain components of the basement membrane and collagen. The theory also offers a new approach to understanding the involvement of basement membrane and underlying connective tissue in the establishment of highly ordered extracellular arrays of collagen fibres, such as the basement lamella. SUMMARY

An adepithelial basement lamella of 8-10 layers of apparently orthogonally arrayed collagen fibrils develops in the skin of Fundulus heteroclitus between the fifth and twelfth days after fertilization. Subsequent thickening of the lamella with growth until posthatching stages is the result of an increase in the number of rows of fibrils per layer, not in the number of layers. In contrast to the admuscular lamella, which forms in posthatching stages, the adepithelial lamella is acellular except in the sublamellar region. A morphological study of the skin of developing stages of Fundulus places the present “plywood” theory of basement lamella structure and development in doubt and supports a new “scindulene (shingle)” theory. Collagen fibril layers are observed to descend at a slight angle from the basement membrane, rather than to lie parallel to it as proposed by the plywood theory. Deposition of collagen in the basement lamella appears not to result from sequential switching and layering of collagen fibrils parallel to the basement membrane, as suggested by the plywood theory. Rather it appears that the collagen fibrils are inserted at end points into the basement membrane and are polymerized in one orientation only in any given area of the basement membrane and that adjacent areas are oriented at 105-llO” to each other. Morphological changes with growth are accounted for by expan-

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sion of these “areas of insertion” in the basement membrane. Problems of variation in lamella thickness, crossover fibrils between different collagen layers, and the possible role of the basement membrane in the deposition of collagen into the lamellar structure are discussed. REFERENCES ARMSTRONG, P. B., and CHILD, J. S. (1965). Stages in the normal development ofFun&1u.s heteroclitus. Biol. Bull. 128, 143-168. BENNI~IT, H. S. (1963). Morphological aspects of extracellular polysaccharides. J. Histo&em. Cytochem. 11,14-23. EDDS, M. V., JR. (1964). The basement lamella of developing amphibian skin. In “Small Blood Vessel Involvement in Diabetes Mellitus” (M. Siperstein, A. R. Colwell, and K. Meyer, eds.), pp. 245-250. Am. Inst. Biol. Sci., Washington, D.C. EDDS, M. V., JR., and SWEENY, P. R. (1961). Chemical and morphological differentiation of the basement lamella. Zn “Synthesis of Molecular and Cellular Structure” (D. Rudnick, ed.), pp. 111-138. (Society for the Study of Development and Growth, Symposium 19, 1960. Ronald Press, New York. FAWCE?T, D. W. (1963). Comparative observations on the fine structure of blood capillaries. In “The Peripheral Vessels,” Monograph No. 4, International Academy of Pathology, pp. 17-44. Williams & Wilkins, Baltimore, Maryland. FIITON-JACKSON, S. (1964). Connective tissue cells. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. VI, pp. 483-520. Academic Press, New York. GAINES, L. M., JR. (1960). Synthesis of acid mucopolysaccharides and collagen in tissue cultures of fibroblasts. Bull. Johns Hopkins Hosp. 106,195-204. GOODMAN, M., GREENSPON, S. A., and KRAKOWER, C. A. (1955). The antigenic composition of the various anatomic structures of the canine kidney. J. Zmmunol. 75, 96-104. GROBSTEIN, C. (1964). Role of connective tissue in embryonic development. Znternational Conf. Congenital Malformations. 2nd Conf., pp. 224-232. International Medical Congress Ltd., New York. GROSS, J., HIGHBERGER, J. H., and SCHMITT, F. 0. (1952). Some factors involved in the fibrogenesis of collagen in vitro. Proc. Sot. Exptl. Biol. Med. 80,462-465. HAY, E. D., and REVEL, J. P. (1963). Autoradiographic studies of the origin of the basement lamella in Ambystoma. Deoelop. Biol. 7, 152-168. JAKUS, M. A. (1961). The fine structure of the human cornea. In “The Structure of the Eye” (G. K. Smelser, ed.), pp. 343-366. Academic Press, New York. KARRER, H. E. (1956). The ultrastructure of mouse lung. J. Biophys. Biochem. Cytol. 2, Suppl.. 287-292. KEFALIDES, N. A., and WINZLER, R. J. (1966). The chemistry of glomerular basement membrane and its relation to collagen. Biochemistry 5, 702-713. KEMP, N. E. (1959). Development of the basement lamella of larval anuran skin. Develop. Biol. 1,459-476. MEYER, K. (1957). The chemistry of the mesodermal ground substances. Harvey Lettures Ser. 51, (19551956), 88-112. Academic Press, New York. MUKERJEE, J., SRI RAM, J., and PIERCE, G. B., JR. (1965). Basement membranes. V. Chemical Composition of neoplastic basement membrane mucoprotein. Am. J. Pathol. 46.49-57.

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OF

THE

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PHILPOTT, C. W., and COPELAND, D. E. (19631. Fine structure of chloride cells from three species of Fund&s. J. CelE Rio/. l&389-404. PIERCE, G. B., JR., BEALS. T. F., SRI RAM, J., and MIDGLEY, A. R., JR. (1964). Basement membranes. IV. Epithelial origin and immunologic cross reactions. Am. J. Puthol. 45,929-961. PORTER, K. R. (1954a). Observations on the submicroscopic structure of animal epidermis. Anat. Record 118, 433. PORTER, K. R. (1954bl. The myo-tendon junction in larval forms of Am&stoma punctatum. Anat Record 118,342. PORTER, K. R. (1956). Observations on the fine structure of animal epidermis. Proc. Intern. Conf. Electron Microscopy 3rd, London, 1954, pp. 539-546. Royal Microscopical Society. London. PORTER, K. R. (1966). Morphogenesis of connective tissue. In ‘Cellular Concepts in Rheumatoid Arthritis (C. A. L. Stephens, Jr., and A. B. Stanfield, eds.1, pp. 6-40. Thomas, Springfield, Illinois. PORTER, K. R. (1967). Personal communication. PORTER, K. R., and PAPPAS, G. D. (1959). Collagen formation by fibroblasts of the chick embryo dermis. J. Biophys. Biochem. Cytol. 5, 153-166. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212. ROBERTSON, J. D. (19551. The ultrastructure of adult vertebrate peripheral myelinated nerve fibers in relation to myelinogenesis. J. Biophys. Biochem. Cytol. 1,271-278. ROSIN, S. (1946). iiber Bau und Wachstum der Grenzlamelle der Epidermis bei Amphibienlarven; Analyse einer orthogonalen Fibrillamtruktur. Reu. Suisse Zool. 53, 133201. ROTHBARD, S., and WATSON, R. F. (1961). Antigenicity of rat collagen. J. Enptl. Med. 113,1041-1051. VAN OOSTEN, J. (1957). The skin and scales. In “The Physiology of Fishes” (M. Brown, ed.), Vol. I, pp. 207-244, Academic Press, New York. WEISS, P. (1957), Macromolecular fabrics and patterns. J. Cellular Comp. Physiol. 49, Suppl. 1. 105-112. WEISS, P., and FERRIS, W. (1954). Electronmicroscopic study of the texture of the basement membrane of larval amphibian skin. Proc. N&l. Ad. Sci. U.S. 40, 528-540. WOOD, G. C. (1960). The formation of fibrils from collagen solutions. A mechanism of collagen fibril formation. Biochem. J. 75, 598-605. WOOD, G. C., and KEECH, M. K. (1960). The formation of fibrils from collagen solutions. The effect of experimental conditions; kinetic and electronmicroscope studies. Biohem. J. 75,588-598. YAMADA, E. (1960). Collagen fibrils within the renal glomerulus. J. Biophys. Biochem. Cytol. 7,407.