The ultrastructure and development of balancers in Ambystoma embryos with special reference to the basement membrane

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6, 36-56 (1962)

J. ULTRASTRUCTURE RESEARCH

The Ultrastructure and Development of Balancers in

Ambystoma

Embryos with Special Reference to the Basement Membrane 1 EVERETT ANDERSON~ and JERRY J. KOLLROS

Department of Zoology, State University of Iowa, Iowa City, lowa Received dune 16, 1961 Balancers from embryos and larvae of Ambystoma at various stages of development have been studied by light and electron microscopy with special reference to the basement membrane. The epithelium covering the balancers consists of a superficial and a basal layer of cells. Beneath the latter at stage 34 the basement membrane makes its appearance and is stainable with toluidine blue and the aniline blue component of Mallory's triple. In subsequent stages (34 + to 45 +) a progressive increase in thickness and staining capacity of the basement membrane is observed. Electron micrographs of the basal epithelial cells at stage 34 reveal evaginations originating from their basal surfaces. Most of the cytoplasmic organelles, e.g., mitochondria and endoplasmic reticulum, are found aggregated above the extensions. At a later stage (36 and 36 +) endoplasmic reticulum, pigment and yolk bodies, and a few mitochondria are found in the larger extensions. The surfaces of the evaginations at stages 34 and 34 + are free of formed elements; however, fine filaments are located at the periphery in close contact with the limiting plasma membrane of stages 36 and 36 + larvae. In stages 37 to 45 + no evaginations are observed; the basement membrane has increased in thickness, being constituted mainly of many fine filaments, some of which show preferred orientation close to the basal epidermal cells. A suggestion is made that these filaments may originate from basal evaginations of these deep epidermal cells. It is well k n o w n that in the course of the development of amphibians, as well as in other organisms, certain structures are formed which regress at a later stage. Some are reduced to vestiges; others disappear completely. Balancers, with which we are herein concerned, are found only during early larval life in certain species of urodeles. They are paired, slender, rod-like appendages which project f r o m the lateral aspects of the head immediately caudal and ventral to the eyes. In addition to providing a sticky surface they function mechanically in holding the head of the larva above the substratum. Notwithstanding the temporary nature of these appendages, their z Supported by grants (RG5479, 8776, and A2202) from the National Institutes of Health, U.S. Public Health Service. 2 Present address: Department of Zoology, University of Massachusetts, Amherst, Massachusetts.

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significance lies in the fact that embryologists have been able to utilize them as analytical structures in attempts to answer some important questions concerning morphogenesis. In Harrison's (4) classical and beautifully illustrated paper, which dealt with the normal structure as well as factors involved in the development of balancers, he called especial attention to the thick basement membrane (2) upon which the basal layer of epithelial cells rests. In 1919 Latta (9) interpreted this membrane to be a dermal bone; however, after using certain staining procedures and also after exposing the membrane to acids, caustic potash, and digestive ferments, Harrison concluded that this membrane was composed of connective tissue of a reticular nature. He stated that the " . . . development of this structure afforded a key to the solution of the problem of the histogenesis of connective tissue, for it is seen to develop out of an intercellular ground-substance without direct participation of mesenchyme cells." Evidence from preliminary observations on the cytoarchitecture of cells composing the epithelium and the structure of the basement membrane (1) supported the view that the constituent filaments of the basement membrane might be derived from the basal epidermal cells, the details of which are hereinafter reported. MATERIALS AND METHODS The animals used in this investigation were larvae of Ambystoma opacum and A. jeffersonianum. The majority of the observations recorded in this study are from A. jeffersonianum. Eggs of this species were collected in local ponds, and kept in finger bowls in the laboratory until they were fixed. The local race of Jefferson's salamander develops only to stage 42 before the onset of feeding, although, like A. maculatum and A. opacum it retains its balancers to stages 45-46. The A. opacum specimens were shipped to us by air express from North Carolina, in stages preceding balancer formation. They were then kept in conditions identical to those of the local species. Larvae were anesthetized with a 2% urethane solution; balancers were removed and fixed for light microscopy in either Bouin's, Helly's, or Carnoy's solution, and stained with either toluidine blue, Mallory's triple, or Heidenhain's hematoxylin. For electron microscopy balancers from larvae similarly anesthetized and staged were fixed in a cold 1% solution of osmium tetroxide buffered at pH 8.0. These were subsequently rapidly dehydrated in a graded series of alcohol solutions. Electron staining with phosphotungstic acid was applied by adding 0.5% phosphototungstic acid to the 70% alcohol used for dehydration. After dehydration the tissue was infiltrated and embedded in methacrylate, Thin sections were cut with a Porter-Blum ultramicrotome and examined in the RCA EMU 3D electron microscope. OBSERVATIONS LIGHT MICROSCOPY

The embryology, anatomy, and histology of balancers have been the subject of extensive study, particularly by Harrison (4), to whom the reader is directed for

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references. It seemed desirable, however, to include p h o t o m i c r o g r a p h s illustrating certain principal microscopical features of balancers f r o m particular stages in order to facilitate the interpretation and correlation of its structure with that seen in the electron micrographs. The balancers first make their appearance at stage 34 as r o u n d e d protuberances u p o n the mandibular arch. Histologically they are composed of a two-layered epithelium which surrounds a core of mesenchymal cells (Figs. 1-4), t h r o u g h which run a blood vessel and a nerve. Fig. 1 is a p h o t o m i c r o g r a p h of a balancer f r o m a stage 36 larva. The inner epithelial layer is c o m p o s e d of low c o l u m n a r cells, while those of the outer layer are cuboidal. In the cells of each layer, in addition to distinct nuclei, are m a n y dense particles which presumably consist of a mixed population of y o l k inclusions and pigment bodies. In sections of balancers f r o m stage 34, stained with Mallory's triple or toluidine blue, is seen a thin moderately stained blue "line," which is the basement membrane. At stages 36 and 36 + the basement m e m b r a n e is slightly thicker and stains more deeply (Fig. 1, BM). There is evidence of a fine cortical striation, perpendiular to the surface of the outer layer of cells, which also stains with the previously mentioned stains (Fig. 1, C) In subsequent stages (37 to 45 + ) the major changes taking place are: (a) a rapid lengthening of the balancer with a change in the shape of cells of both inner and outer epithelial layers f r o m cuboidal to squamous (Figs. 2, 3 and 4); (b) the surface striations become more p r o m inent and stain more deeply (stage 37, Fig. 2, C; stage 45, Fig. 4, C); and (c) a progressive increase in thickness and staining capacity of the basement m e m b r a n e (stage 37, Fig. 2, BM;stage 45, Fig. 4, BM). The virtual absence of mitotic figures in cells composing this epithelium has led to the t h o u g h t that the growth in length of the balancer is accomplished largely by the change in shape of the epithelial cells (4). By stage 40 the basement m e m b r a n e at the base of the balancer appears as an expanded cone or funnel, and remains essentially unchanged in this appearance until stage 45 (Fig. 3, BM~). Soon after this the balancers are broken off at their bases, leaving the cone-shaped portion of the basement m e m b r a n e embedded in the sur-

FIO. 1. A photomicrograph of a longitudinal section of a balancer of a stage 36 larva, showing the two layers of epithelial cells, each showing a distinct nucleus and many dense bodies in its cytoplasm. The basal layer of cells is underlain by the basement membrane (BM). At S are mesenchymal cells (some of which contain dense bodies), which compose a portion of the core of the appendage. A slight indication of the cortical area of the outer epithelial cells is seen at C. Bouin's fixative; Heidenhain's hematoxylin, x 600. Fio. 2. A longitudinal section of a balancer of a stage 38 larva fixed in Helly's solution, and stained with Mallory's triple. Note the heavy staining of the basement membrane (BM), the definite outer cortical area (C), and mesenchymal cell at S. x 500. FIGS. 3 and 4. Sections of balancers from a stage 45 larva fixed in Helly's, and stained with Mallory's triple. Note the distinct cortical area (C, Fig. 4), the shape of the epithelial cells, and a few mesenchymal cells (S, Fig. 4). In Fig. 3 the cone-shaped portion of the basement membrane (BM1) is observed. Fig. 3, x 400; Fig. 4, x 500.

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rounding mesenchymal cells. As stated by Harrison (4) this casting off of the balancer "... is a process of autotomy, and is not due merely to an accidental blow from without."

ELECTRONMICROSCOPY

Stage 34, 34 +, and 36 Fig. 5 is a section showing the general distribution of cells composing the epithelium of a balancer from a stage 34 larva. The cells of the basal layer show a morphological polarity, which is established by the evaginations from their basal surfaces (PE). Sometimes, the surfaces of the extensions appear broken in places. Whether or not the discontinuities arise during fixation or are attributable to a particular physiological state of the cells is not clear. These evaginations, whose internal cytoplasm consists of a fine granular material, are similar to those observed in regenerating skin of Triturus by Salpeter and Singer (22). The extensions appear to be naked, i. e., they do not come in contact with mesenchymal cells nor are they seen in contact with a thin, dense osmiophilic line which has been termed the "adepidermal membrane" in the skin of amphibians by Salpeter and Singer (21), and in mammalian skin the "dermal membrane" by Selby (24). Most of the cytoplasmic organelles, i.e., cisternae of the endoplasmic reticulum (Fig. 6, ER), and mitochondfia (Fig. 6, M), appear to be aggregated just above the evaginations. By stages 36 and 36 + the evaginations are larger and contain many profiles of rough-type endoplasmic reticulum (ER), few mitochondria (M), and some pigment and yolk bodies (Fig. 6, YB). It is difficult at this time to relate categorically such an elaboration of the basal surfaces of these cells with a specific function, but it is evident that these evaginations greatly increase the active surface area of the base of these cells. Of special interest is the first indication of the constituent filaments of the basement membrane. These appear as fine filaments upon the external surfaces of the evaginations (Fig. 8, BF). The filaments are randomly oriented with a thickness of approximately 60-70 A, and with no evidence of a periodic axial structure. At this stage the filaments were never seen to extend out into the space between the epidermal cells and mesenchymal cells, nor were they observed in close association with the surfaces of the mesenchymal cells. The position of these filaments would suggest that they have their origin from a precursor that is presumably liberated at the surfaces of the evaginations of the epidermal cells. The nuclei of the basal cells are limited by a double membrane envelope. In addition to the endoplasmic reticulum and mitochondria, the cytoplasm contains dense

FIG. 5. Electron micrograph showing a slightly tangential section of the balancer epithelium of a stage 34 larva. Note the nuclei (N), some of which are indented by yolk bodies (YB). Also illustrated are pigment bodies (PB), the outer cortical area (C), and pseudopodial extensions (PE). ×6000.

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E. ANDERSON AND J. J. KOLLROS

FIG. 6. A section of a portion of a cell from the basal epithelial layer of a stage 34 larva. The nucleus (N), with granular nucleoplasm, is limited by a double membrane envelope (NE), and indented by yolk bodies (YB). Scattered in the cytoplasm are mitochondria (M), cisternae of the endoplasmic reticulum (ER), and many dense granules. Note the pseudopodial extensions (PE). x 16,800.

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FIG. 7. A section showing portions of two adjacent cells of the superficial epithelial layer from a stage 36 larva, illustrating cisternae of the endoplasmic reticulum (ER), mitochondria (M), dense particles, Golgi complexes (GC), tonofilaments (TF), pigment (PB), and secretory (SB) bodies. Note the cortical area (C) showing some vacuoles, a fine filamentous material, and a microvillus (MV). x 20,000.

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particles which appear morphologically like ribonucleoprotein particles and measure 150-250 A in diameter (16). Also observed are pigment (Figs. 5 and 6, PB), and yolk bodies (Figs. 5 and 6, YB), varying in size and density; some of the yolk bodies indent the nuclear envelope (Figs. 5 and 6). The cells composing'the outer epithelial layer are likewise morphologically polarized. This polarity is evidenced by a distinct outer cortical area whose surface is beset with microvilli (Fig. 7, MV) and a few cilia. This cortical area is composed of many compartments oriented perpendicular to the surface. The compartments are presumably related to the striae observed with the light microscope. They appear to be limited by thin membranes and have an interior of a fine granular material. The nucleus, like that found in the basal cells, is limited by a double membrane envelope and frequently indented by yolk bodies. In the cytoplasm are found pigment bodies (Fig. 7, PB), mitochondria (Fig. 7, M), multivesicular bodies (Fig. 7, MVB), typical Golgi complex (Fig. 7, GC), cisternae of the endoplasmic reticulum (Fig. 7, ER), and clusters of dense particles. Other bodies (Fig. 7, SB) are also seen and are similar in their ultrastructure to secretory bodies observed in other cell types. Observed also are bundles of tonofilaments, randomly oriented, which weave among the cytoplasmic constituents (Fig. 7, TF). Such elements were never found in the cytoplasm of cells from the basal layer. The plasma membranes of the outer epithelial cells abut on one another as well as on the membranes of cells of the basal layer. At their points of contact membrane specializations in the form of desmosomes are observed similar to those described for amphibian and mammalian skin (11, 14, 18, 24).

Stage 38 + The evaginations of the basal cells are lost at this stage and the limiting plasma membrane shows no hemi-desmosomes or "bobbins" (29) like those observed in other skin cells (7, 14). The electron micrograph (Fig. 9) shows that the basement membrane (BF) has increased in thickness by this stage. The constituent filaments of the basement membrane are approximately 70-90 A in thickness and show some preferential orientation close to the plasma membrane of the epidermal cells; however, farther from the basal portion of the cell (to the left in Fig. 9) these filaments become randomly oriented. At higher magnification these filaments are seen to have an axial periodicity of approximately 100-120 A. Yolk bodies, some of which are concentrically laminated, are frequently observed embedded in the network of filaments. The cytoplasm of the basal cells becomes filled with many dense particles and cisternae of the endoplasmic reticulum (Fig. 9, ER). Also found in the cytoplasm are yolk bodies, Golgi complexes, and many mitochondria (Fig. 9, M). In a few cells,

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FIG. 8. A relatively small field of a portion of a pseudopodial extension of a cell from the basal epithelial layer of a stage 36 larva, showing cisternae of the endoplasmic reticulum (ER), and a mitochondrion (M). Note the fine filaments (BF) at the surface of the extensions (PE). x 28,000.

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E. ANDERSON AND J. J. KOLLROS

FIG. 9. A section showing portions of two adjacent cells of the basal epithelial layer from a stage 36 larva. Illustrated here are mitochondria (M), cisternae of the endoplasmic reticulum (ER), dense particles, nucleus (N), and tonofilaments (TF). The l~asement membrane (BE) is composed of fine filaments. These filaments show some preferred orientation close to the basal plasma membrane. x 20,000.

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FIG. 10. A small field of a portion of a cell from the superficial layer of a balancer from a stage 38 larva, showing elements of the endoplasmic reticulum (ER), mitochondria (M), Golgi complex (GC), a few tonofilaments (TF), pigment (PB), and secretory (SB) bodies. Note: in the insert is a tangential section of a superficial cell illustrating the nucleus (N), and the outer compartments with homogeneous interiors (C). × 56,000; insert, x 8000.

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E. ANDERSON AND J. J. KOLLROS

concentrically arranged, smooth-type membrane systems were observed; their function and identification alike remain obscure. Unlike the cytoplasm of the basal cells of stages 34 and 34 +, the cytoplasm of these cells contains many tonofilaments, some of which come into close contact with the outer nuclear envelope (Fig. 9, TF). In the cytoplasm of cells of the outer epithelial layer is observed more endoplasmic reticulum, which is interconnected (Fig. 10, ER). Among the pigment bodies (Fig. 10, PB), mitochondria (M), and Golgi complex (GC), there is a host of secretory bodies (Fig. 10, SB); many are seen in thin section as oval or elliptical profiles of varying sizes and densities, of which the outer surface may or may not be limited by a thin membrane. The outer striated or cortical area of these cells is more distinct than that observed in stage 34 (Fig. 10, insert, C), and is filled with many secretion globules. The latter may consist of a mucoid substance which is presumably released onto the surface of the balancer. The features of the outer cortical area of these cells are similar to those depicted by Olsson (15) for the cortical area of epidermal cells of Amphioxus. The mesenchymal cell illustrated in Fig. 11 is typical of stage 34 + larvae as well as those of stage 38. These cells are rather elongate, and contain an abundance of endoplasmic reticulum (ER), a number of Golgi complexes (GC), and yolk bodies (YB). The heavily granulated nucleoplasm with its nucleolus (NCL) is limited by a double nuclear envelope. No filamentous material was observed in either the inner cytoplasm of these cells or at their surfaces.

Stages 45 and 45 + At these stages the two layers of epithelial cells show variable stages of degeneration (Fig. 12). The cells of the outer layer begin to lose contact with adjacent ones, and many protoplasmic projections are observed at their surfaces (PJ). The endoplasmic reticulum has lost most of its preferential orientation and many dense particles are scattered in the cytoplasm. The nucleoli are irregular in shape and are composed of many dense granules and a few clear zones (Fig. 12, NCL). The outer layer of epidermal cells is strikingly characterized by its large complement of homogeneous bodies (Fig. 12, HB), and large vacuoles (Fig. 13, V1). Frequently one sees smaller vacuolated bodies which may be degenerating mitochondria (Fig. 13, V2); the Golgi complex is not recognizable; however, a number of multivesicular bodies are present (Fig. 13, MVB). The distinct cortical area is usually present, but the discrete globular units observed in stage 38 and 38 + larvae are no longer seen. The cells of the basal layer remain closely applied to the basement membrane and their extended lateral cytoplasmic edges become thin, much like the attenuated cytoplasmic area of endothelial cells (Figs. 13 and 14, BC). The basal plasma membranes

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do not show hemi-desmosomes. More tonofilaments are discernible at this time than at any other stage of development (Figs. 12 and 14, TF). The constituent filaments of the basement membrane, beneath the epidermal cells, as well as in its cone-shaped portion embedded in mesenchymal cells, appear thicker (130-140 A) than do those of previous stages. The axial periodicity is quite clear and measures approximately 220 A, with no evidence of an intraperiod banding (see insert, Fig. 14). A few of these filaments still show some preferential orientation near the base of the epidermal cells; however, farther from the epidermis (downward on the micrograph) a tendency to random orientation is observed. A similar differential ordering has been described by Kemp (7) in the basement lamella in skin of a stage 20 frog embryo. A comparable orientation of filaments of the basement lamella has also been described by Weiss (26, 27, 30) in both anurans and urodeles during the reconstruction of the basement lamella after wounding. Such a comparison seems to support Pease's (17) contention that "... the healing process can be expected to recapitulate embryological development ..." DISCUSSION Bell (2) was the first to note the peculiar basement membrane which is characteristic of balancers in certain salamanders. From his meticulous studies he suggested that it was of a collagenous nature. Since that time relatively few studies have been made on the cytology of this organ; however, more studies are available on embryonic (7, 11, 28) and adult skin (12, 14, 24). The early concern in utilizing the electron microscope on larval amphibian skin was to visualize more clearly the structure of the basement lamella, with the hope of explaining the peculiar geometrical pattern of orthogonality made by the underlying collagenous fibers. This architectural pattern was originally pointed out in the elegant phase-microscopical studies of Rosin (20). In 1954 Weiss and Ferris (29), with the aid of the electron microscope, first showed the finer details of such a complicated design in the basement lamella of skin from the larvae of A. opacum and Rana pipiens. They found that the lamella consisted of about 20 layers of ground substance, in which cylindrical collagenous fibers, approximately 500 ~ in diameter, are embedded. These are parallel to one another, but with the fiber direction alternating by 90 ° from layer to layer. Later (1956) these same authors (30) studied the reconstruction of the lamella after wounding and found that "... (1) epidermal cells cover the wound exudate by migration. (2) Rather uniform fibers of small size ( < 200 A) appear in the space between the -pidermal underside and subjacent fibroblast; these fibers are sparse and oriented t random. (3) Proceeding from the epidermal surface downward, a wave of organization spreads over this primitive fiber tangle, resulting in the fiber becoming (a) 4 - 62173313 J . Ultrastructure Research

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E. ANDERSON AND J. J. KOLLROS

FIG. 11. A section showing a portion of a mesenchymal cell of a stage 45 larva, illustrating the nucleus with its nucleolus (NCL), all of which is surrounded by a double membrane envelope. Also observed are mitochondria (M), Golgi complex (GC), endoplasmic reticulum (ER), and a yolk body (YB). x 39,000.

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FIG. 12. A section showing the epithelium of a stage 45 larva. Note the heavily folded nuclei (N), the large homogeneous bodies (HB) in the superficial layer, and the degenerated cortical area (C). Protoplasmic projections are labeled PJ. The basal cell, closely applied to the basement membrane (BF), contains many tonofilaments (TF). The filaments of the basement membrane show some preferential orientation close to the basal cell's plasma membrane, x 12,000.

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E. ANDERSON AND J. J. KOLLROS

FIG. 13. A section of a portion of the superficial and basal epithelial cells (BC), and the filamentous basement membrane (BF) of a stage 45 larva. In the superficial cell may be observed the nucleus (N), large (V:) and small (VD vaeuolated structures, multivesicular bodies (MVB), and a pigment body (PB). x 48,000.

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FIG. 14. A relatively small field of the basal area from a stage 45 larva, illustrating the attenuated edge of a basal cell (BC), consisting of many tonofilaments (TF), and the basement membrane (BF), whose filaments show a preferential orientation near the basal cell and some axial periodicity (see insert). ×72,000; insert, x140,000.

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E. ANDERSON AND J. J. KOLLROS

straightened; (b) oriented; (c) packed into the characteristic layered structure; and (d) brought up into the 500 A diameter class." Kemp (7) studied the submicroscopic development of the basement lamella in anurans from the tailbud stage onward, and was able to show that the early steps in the embryonic differentiation of the basement lamella essentially parallel those described by Weiss (26, 27) and by Weiss and Ferris (28, 29). Such a precise geometrical pattern, as previously mentioned, is characteristic not only of amphibians, but has also been found in the fiber layer which is located immediately below the single-layered epidermis of Amphioxus (15), as well as in the corneas of the dogfish, rabbit, and human by Jakus (5; see also 10). It is evident that the term basement lamellae would be inadequate when applied to the structure which lines the underside of the basal epithelial cells of balancers. It is also evident that the term basement membrane as used by recent investigators is equally inadequate (see Pease for discussion). We will, however, retain the term basement membrane as used by Bell (2), for want of a better one. Our present use of the term is subject to modification as more data becomes available to stabilize or modify the morphological as well as the physiological concepts with respect to this structure. The present studies demonstrate that the filaments composing the basement membrane of balancers do not possess the high degree of orientation which is observed in larval skin from other regions of the body. However, as previously pointed out, there is some preferential orientation of these filaments, particularly immediately adjacent to the epidermis, and especially in older embryos (e.g., stage 45). In the early stages of balancer growth, however, filament orientation appears to be random, just as it is at first during the reconstruction of the membrane system during wound healing (26, 27, 30). Weiss (30) stated that after this stage of organization "... a wave of organization spreads over the primitive fiber tangle .... " which eventually results in the characteristic orthogonality. This "wave" may be thought of as one of chemical alteration in the environment, for it has been pointed out by Schmitt (23) that "the manner in which the macromolecules will behave under particular conditions, i.e., whether they will orient themselves in parallel or in antiparalM array, in register or staggered with respect to macromolecular ends, depends upon the chemical environment at the moment." One wonders if the filaments of the basement membrane of balancers might orient themselves in an orthogonal pattern if these appendages could be maintained in a healthy state and their life span extended. Certain experiments may be helpful in testing such a speculative hypothesis. For example, experiments dealing with transplanting balancers, like those performed by Bell (2), Nakamura (13), and Kollros (8), in their studies of the factors controlling the balancer's life span, may be made.

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The question dealing with the origin of the filaments composing the basement membrane of balancers, like similar ones found in the basement lamella in other regions of the body of amphibians, has been considered by many investigators. No efforts will be made in this paper to review the extensive literature dealing with fibrillogenesis. Gross (3), Schmitt (23), Wassermann (25), Porter and Pappas (19), and Karrer (6) have given excellent treatments to this subject, and the reader is referred to these papers for critical analysis and references to the literature of fibrillogenesis. It is only necessary to point out here that some authors think that the basement membrane of balancers is of mesenchymal origin (2), while others suggest that it is of ectodermal origin (4). We think that the present observations give evidence that the constituents of the basement membrane have their origin, at least in part, from epidermal cells. In the electron micrographs illustrating this paper it can be seen that during the early phases of development the basal epidermal cells showed many evaginations from their inner surfaces. It was also seen that the basement membrane could be stained as early as stage 34. At this stage no filaments were observed; however, at stages 36 and 36 + fine filaments, with no evidence of an axial period, could be seen at the periphery of the evaginations, and at still later stages the filaments of the basement membrane had increased in both number and size and showed a definite axial periodicity. Morphologically the filaments of the basement membrane appear to be collagen; however, an unequivocal statement cannot be made as to their precise nature until they can be isolated and characterized physicochemically. picture such as presented above invites one to speculate concerning a possible on~!n of the precursor or precursors of the fibrils. One possibility is that such precursors may be elaborated at the surfaces of the basal evaginations as afibrillar materials from which the fibrils may be synthesized. Such a hypothesis concerning the origin of these fibrils is similar to that of Gross (3) in his studies dealing with fibrogenesis of collagen. Gross stated that "the fibroblast secretes tropocollagen particles and these are spontaneously precipitated in the form of collagen type fibrils because of the characteristics of the ionic environment." If, indeed, the filaments of the basement membrane of the balancer are synthesized from an afibrillar material, one would assume that such a material would pass across the plasma membrane. In this connection it has been suggested by Karrer (6), from a study on the fine structure of embryonic chick aorta, that the precursors of the collagen fibers are synthesized by the endoplasmic reticulum of fibroblasts. In considering mechanisms whereby synthesized fiber precursors leave the fibroblast, he suggested two possibilities. The first was that a continuity is established between the lumen of the endoplasmic reticulum and the extracellular space. He envisions that once the fusion has been accomplished there is a subsequent rupture of the fused membranes. The second mechanism suggested by Karrer was the release into the extracellular space of whole dilated cisternae, or cysts,

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E. ANDERSON AND J. J. KOLLROS

of endoplasmic reticulum. He views this to be achieved by a rupture of the plasma membrane over a superficially located cisterna. We have observed no morphological evidence that would indicate that the filaments of the basement membrane in our studies are derived in the interesting manner presented by Karrer. In this study no evidence was found that would implicate the mesenchymal cell in the formation of fibrils. We are not, however, ruling out the possibility that the mesenchymal cells m a y be functioning as fibroblasts and contributing in some way to fibril formation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

ANDERSON,E. and KOLLROS,J. J., Anat. Record 138, 330 (1960). BELL, E. T., Anat. Anz. 31, 283 (1907). GROSS, J., J. Biophys. Biochem. Cytol. 2, No. 4, Suppl., 261 (1956). HARRISON,R. G., J. Exptl. Zool. 41, 349 (1925). JAKUS,M. A., Proc. Intern. Conf. Electron Microscopy, Berlin, 1958, p. 344. KARRER,H. E., J. Ultrastructure Research 4, 420 (1960). KEMP, N. E., Developmental Biol. 1, 459 (1959). KOLLROS,J. J., d. Exptl. Zool. 85, 33 (1940). LATTA,J. S., Anat. Record 17, 62 (1919). MAXIMOW,A. A. and BLOOM, W., A Textbook of Histology, 7th ed., p. 557. Saunders, Philadelphia, 1957. MENEFEE,M. G., 3". Ultrastructure Research 1, 49 (1957). MONTAGNA,W., The Structure and Function of Skin. Academic Press, Inc., New York, 1956. NAKAMURA,O., Rot. and Zool. 6, 1051 (1938). ODLAND, G. F., 3. Biophys. Biochem. CytoI. 4, 529 (1958). OLSSON,R., Z. ZeIlforsch. u. mikroscop. Anat. 54, 90 (1961). PALADE, G. E., in PALAY, S. L. (Ed.), Frontiers in Cytology, p. 283. Yale University Press, New Haven, 1958. PEASE, D. C., Proc. Intern. Conf. Electron Microscopy, Berlin, 1958, p. 139. PORTER, K. E., Proc. Intern. Conf. Electron Microscopy, London, 1954, p. 539. PORTER, K. E. and PAP1'AS, G., J. Biophys. Biochem. Cytol. 5, 153 (1959). RosiN, S., Rev. Suisse Zool. 51, 376 (1944). SALPETER,M. M. and SINGER,M., J. Biophys. Biochem. Cytol. 6, 35 (1959). -Anat. Record 136, 27 (1960). SCHMITT, F. O., Proe. Intern. Conf. Electron Microscopy, Berlin, 1958, p. 1. SELBY, C. C., J. Biophys. Bioehem. Cytol. 1,429 (1955). WASSERMANN,F., Ergeb. Anat. Entwicklungsgeschichte 35, 240 (1956). WEISS,P. A., J. Cellular Comp. Physiol. 49, 105 (1957). -Proc. Natl. Acad. Sci. 42, 819 (1956). -ibid. 40, 528 (1954). WEISS, P. A. and FERRIS, W., Exptl. Cell Research 6, 546 (1954) - - - - J. Biophys. Bioehem. Cytol. 2, 275 (1956).