Arch. histol. jap., Vol. 45, No. 2 (1982) p. 181-189

Formation of Reticular Fibers in the Developing of the Chick Embryo

Katsuhiro FUKUTA and Koshi MOCHIZUKI

Laboratory of Veterinary Anatomy (Prof. K. MOCHIZUKI), Faculty of Agriculture, University of Tokyo, Tokyo, Japan

Received April 17, 1981

Summary. An electron microscope study was made to elucidate the manner in which reticu- lar fibers form in the developing spleen of the chick embryo. On the 7th day of incubation, the splenic reticulum consisted of dendritic reticular cells without reticular fibers. From the 9th day onward, the intercellular gaps between processes of adjacent reticular cells were expanded, becoming occupied by fine flocculent materials. With the growth of the embryos, these materials were aggregated into felt-like bands, exhibiting the characteristics of mature reticular fibers. In the interstices of the reticulum, there appeared microfibrils of 8-30 nm in diameter. The reticular cells possessed numerous free ribosomes and polysomes, well-developed Golgi apparatuses, dilated rough-surfaced endoplasmic reticula, and many coated vesicles. The coated vesicles contained fine flocculent materials as presumable fiber precursors. The vesicles were enlarged as they approached the periphery, where they appeared to discharge their contents by reverse pinocytosis. Some vesicles close to the cell membrane contained ruthenium red-positive materials which were considered to be glycos- aminoglycan in nature and to compose the matrix of fibers. These materials also seemed to be secreted by reverse pinocytosis, being densely accumulated in the intercellular gaps. The differentiation of fibrous proteins into felt-like reticular fibers or striated microfibrils seems, therefore, to be intimately associated with the amount of glycosaminoglycan.

The lymphoid or blood-forming organs have reticular tissues as their basic structure. Three-dimensionally, the reticular tissues are composed of the meshwork of reticular cells and fibers, being filled with numerous blood cells. Reticular fibers, together with and elastic fibers, constitute extracellular connective tissues. Recently, reticu- lar fibers have come to be regarded as a subtype of collagen fibers, based on the fact that the fibrous proteins of both fibers are very similar not only in their chemical com- position but also in their physical arrangement (WINDRUMet al., 1955; ZAIDES et al., 1959; BERRENSand DRIEL, 1962). Nevertheless, little information is available on the exact nature of the reticular fibers themselves. The present study was conducted to clarify the formation process of reticular fibers, using an electron microscope observation of the developing spleen in the chick embryo.

181 182 K. F[JKUTA and K. MocHizuKi:

MATERIALS AND METHODS

Fertilized eggs of the White Leghorn were incubated for use in this study. A total of 33 chick embryos and hatched chicks were obtained. They were killed after 7, 8, 9, 10, 11, 12, 13, 15, 17, 19 and 21 days of incubation, three on each of the days. The spleen of each embryo was cut into small pieces, approximately 1 mm3. The pieces were fixed in a mixture of 2°° paraformaldehyde and 2.5°° glutaraldehyde in 0.08 M cacodylate buffer (pH 7.4) for 2 hrs at 4- C. After being washed with the same buffer, they were postfixed in 2°° osmium tetroxide for 1.5 hrs at 0- C, dehydrated in a series of graded alcohol and embedded in Epon. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed by electron microscope (Hitachi HU-12). In order to demonstrate glycosaminoglycan as the fiber matrix, the splenic tissues from the 11-, 15- and 21-day chick embryos were immersed in a fixative which con- tained 0.05°° ruthenium red, according to LUFT'S (1971) method. The ultrathin sections were not stained, but used directly for electron microscope observation.

RESULTS

In the embryos on the 7th day of incubation, the spleen was already composed of a re- ticular meshwork. Detailed examination, however, revealed that the splenic reticulum consisted only of reticular cells, with no fibrous components. On the 8th day, the reticulum appeared almost the same as that on day 7 (Fig. la, b). Reticular cells were

a b

Fig. 1. Reticular meshwork of a spleen on the 8th day of incubation. a. Reticular meshwork consisting of reticular cells with dendritic processes. There are no blood cells in its interstices. x3,000. b. High magnification of connection sites between cytoplasmic pro- cesses of adjacent cells. The opposing cell membranes are separated by gaps (arrows) about 10 nm in distance. The reticular cells are provided with well-developed Golgi ap- paratuses (G), numerous free rihosomes and polysomes, mitochondria and rough endo- plasmic reticulum. x 18,000 Reticular Fibers in Developing Chick Spleen 183

large and dendritic with several extended processes by which the cells were connected with their adjacent fellow cells, thus forming a three-dimensional meshwork. At the connecting site of the cells, the opposing plasma membranes were separated by an intercalated gap of about 10 nm. No fibrous elements were found in the gap (Fig. 1b), but infrequent tight junctions were detectable. Some reticular cells exhibited mitotic figures. Each reticular cell had a large euchromatic nucleus with one or two prominent nucleoli. The cytoplasm contained numerous free ribosomes, most of which composed polysomes. The reticular cells also had more than two Golgi apparatuses each, as well as scant rough-surfaced endoplasmic reticulum with other scattered cytoplasmic or- ganellae such as mitochondria with lamellar cristae, centrioles, fine filaments and microtubules. The interstices of the reticular meshwork were narrow, containing no blood cells but one or two isolated granular leukocytes. There were some primitive blood vessels, which could not be identified as arteries, veins or capillaries in nature. They contained a few erythrocytes and granulocytes. On the 9th day of incubation, the interstices of the reticular meshwork were occupied by a further increased number of leukocytes. On day 10, arteries, veins or capillaries became distinguishable. In the interstices of the meshwork, a few erythro- cytes appeared for the first time, and the blood cells were increased therein. Especial- ly on days 13 and 15 did the number of granular leukocytes markedly increase, as a sign of active granulopoiesis. From days 9 through 15, the intercellular gaps between cytoplasmic processes of adjacent cells were furthermore expanded (Fig. 2, 3a, b, 4a, b). Some gaps had widths of more than 100 nm, and contained fine flocculent materials of moderate-electron density. On day 10 and thereafter, these materials gradually moved away from the cell membrane to be aggregated into felt-like bands (Fig. 4a, h), looking like mature reticular fibers in the adult spleen (FUKUTAet al., 1976). In the interstices of the reticular meshwork, some amount of microfibrils appeared

Fig. 2. Reticular meshwork at 10 days' incubation. A reticular cell (upper side) has three kinds of coated vesicles; located in the cytoplasm (1), attached the cell membrane and having a small aperture (2), and perforated widely with an aperture (3). Tight junctions (ar- rowheads) are seen here and there. In the intercellular gap, some amorphous materials ( a ) are also observed. x 28,000 184 K. FUKUTA and K. MOCHIZUKI:

(Fig. 3a, 5). They varied from 8 to 30 nm in diameter, and tended to be thicker the farther they were apart from the cell surface. In general, they were randomly directed with occasional ramifications. Fine microfibrils appeared to be closely associated with globular granules of 25-30 nm in diameter. The observation coincided with that in the perinotochordal and perivertebral regions in the chick embryo (FREDERICKSONet al.,

a b

Fig. 3. Fine flocculent materials ( * ) accumlated in intercellular gaps. a. 10-day-old chick em- bryo. Some microfibrils are present (arrowhead). Invaginated parts of cell membranes (arrows) are coated with short bristles, suggesting that the vesicles have finished dis- charging their contents into the extracellular spaces. x 19,000. b. Perforated vesicles (arrows) in a reticular cell on day 15. The contents in these vesicles are very similar to the extracellular materials (* ), and they appear to be continuous with each other. Slightly dilated rough endoplasmic reticulum (er) contains fine granular materials. x 37,000

a b

Fig. 4. Formation of extracellular reticular fibers. a. 10-clay-old chick embryo. Fine flocculent materials are aggregated into felt-like bands ( * ) in the intercellular gap. Arrows in- dicate vesicles approaching the cell periphery from the Golgi area (G). x26,000. b. 15- day-old chick embryo. Felt-like bands of flocculent materials (*) exhibit the charac- teristics of mature reticular fibers. x 40,000 Reticular Fibers in Developing Chick Spleen 185

Fig. 5. 9-day-old chick embryo. Various sized microfibrils (8-30 nm in diameter) in the inter- stice of a reticular meshwork. Some fibrils diverged (large arrows), and globular granules (small arrows) are present in association with the fine fibrils. Thick fibrils more than 25 nm in diameter have cross striations (arrowheads). x 36,000

b

a C

Fig. 6. 11-day-old chick embryo. Showing ruthenium red-positive materials in a splenic reticu- lum. a. Ruthenium red-positive materials are faintly present on the overall cell surface. However, they are strongly concentrated in invaginated parts of the cell membrane (arrowheads), a cytoplasmic vesicle (arrow) and an intercellular gap ( * ). x 28,000. b. An invagination of cell membrane. It contains ruthenium red-positive materials. x53,000. c. A perforated vesicle with an aperture on its free surface to the extracellu- lar space. Ruthenium red-positive materials, strongly stained, are seen on the cell sur- face around the aperture, suggesting secretion from the vesicles through the aperture (arrow). x 88,000 186 K. FUKUTA and K. MocHIZUKI ;

1977). The microfibrils thicker than 25 nm were provided with obscure but electron- dense cross striations, the intervals of which varied from 27 to 37 nm (Fig. 5). The reticular cells, on days 10 to 15, had well-developed Golgi apparatuses consist- ing of lamellae and vesicles with no vacuoles. The rough endoplasmic reticulum was dilated, containing fine granular materials (Fig. 3b). Many coated vesicles were dis- tributed throughout the cytoplasm. Although those vesicles located near Golgi areas were as small as 40-60 nm in diameter, those vesicles located close to the cell periphery were as large as 130-150 nm (Fig. 2, 3b, 4a). Generally, the coated vesicles contained fine flocculent materials similar to the extracellular materials found in the intercellular gaps. The large vesicles attaching to the cell membrane were each perforated with an aperture. Through these apertures, the vesicles linked together the extracellular spaces. Frequently, a continuation of the contents in the perforated vesicles with the extracellular flocculent materials (Fig. 3b) was encountered. On the cell membrane, some invaginations were observed scattered about and they were also coated with short bristles (Fig. 3a). These invaginations seemed to show the remnants of vesicles, which had just finished releasing their contents, as a sign of reverse pinocytosis. In addition, a few non-coated, electron-lucent vesicles were also observed. From day 17 to the day of hatching, active granulopoiesis was observed in the reticular meshwork. The gaps between reticular cell processes were widened, includ- ing flocculent materials like felt bands. In the interstices of the reticular meshwork, microfibrils ran parallel to the cell surface without making bundles. They were about 30 nm in diameter. The cross striations of microfibrils became more obscure, but their intervals were relatively constant (50-60 nm). The reticular cells showed cytologic features similar to those seen at earlier stages, but contained extremely dilated cisterns of rough endoplasmic reticulum. Many coated vesicles at various growing stages were observed as well. Ruthenium red-positive materials suggested the presence of glycosaminoglycan as a fiber matrix. These materials were observed in the reticular cells, particularly in close association with the cell surfaces, cytoplasmic vesicles and invaginated cell membranes (Fig. 6a-c). Although the overall cell surfaces were very weakly positive, the invaginated cell membranes and the intercellular gaps were very strongly positive. The perforated vesicles at cell periphery were also very strongly positive (Fig. 6c).

DISCUSSION

Reticular fibers can be histologically distinguished from other fibers when they are stained with periodic acid Schiff and alkali silver methods. Reticular fibers are thought to consist of a unit fibril protein or tropocollagen embedded in an abundant matrix which gives certain histochemical characteristics to the fibers by a large amount of glycosaminoglycan (GLEGGet al., 1953; ISHII, 1968 a, b; BLOOMand FAWCETT,1975). Our previous works (FUKUTA et al., 1975, 1976) demonstrated that in the chicken spleen, the morphology of reticular fibers was similar to that of the basement membrane, and that the reticular fibers were continuous with the basement membrane. The felt- like bands and the electron-lucent bands of the reticular fibers may be equivalent to the lamina densa and the lamina lucida of the basement membrane, respectively (KAJI- KAWA,1975; KATSUYAMAet al., 1977). In the present study, the spleen of 7-day-old chick embryos had a reticular mesh- work consisting only of reticular cells. This finding is very similar to that of the primary vascular reticulum in the human fetal spleen (WEIss,1973). With the growth Reticular Fibers in Developing Chick Spleen 187 of chick embryos, the intercellular gaps between the cytoplasmic processes of two adjacent cells were gradually widened with an increasing accumulation of fine floc- culent materials which, in turn, made up the felt-like bands of reticular fibers. The reticular cells themselves were characterized by having numerous free ribosomes (most of which formed polysomes), well-developed Golgi apparatuses, dilated cisterns of rough endoplasmic reticulum and many vesicles of various sizes. As regards , it has been noticed that they contain numerous aggregated ribosomes in proportion to the degree of collagen-forming activity (LESSONand LEESON, 1964). Moreover, there is vesicular-typed endoplasmic reticulum in the active fibro- blasts (PEACHet al., 1961). In accordance with this similarity of cellular features to fibroblasts, the reticular cells in the developing spleen are thought to have an ability to synthesize fibrous proteins. In contrast, there is a paper reporting that reticular fibers in the lymph nodes of rats and mice are not formed by reticular cells (KAJIKAWA, 1964). The vesicles observed in the reticular cells were coated with short bristles and they contained fine flocculent materials. Usually, coated vesicles are considered to be the results of endocytosis. However, the coated vesicles in the reticular cells observed in this study should be considered to play an exocytotic role, on the basis of the follow- ing findings: 1) the vesicles grow up as they approach the cell periphery; 2) the vesi- cular contents are very similar to the materials accumulated in the intercellular gaps; 3) the contents of perforated vesicles attaching to the cell membrane are continuous with the exracellular materials; 4) the extracellular materials formed arrangements as the incubation time progressed. For the dilated endoplasmic reticu- lum, its contents were found to be fine granular materials, seemingly different in appearance from the precursor materials included in the vesicles. They possibly con- tribute to the formation of fibrous proteins. The present study revealed the presence of variously sized microfibrils mostly in the interstices of the reticular meshwork. These microfibrils seem to have no relation- ship with the fine filaments located in the reticular cells themselves. The extracellular microfibrils more than 25 nm in diameter had cross striations. This observation con- flicts with the reported finding that striaitons appear in 16 nm or more thick collagen fibrils cultured in vitro (GOLDBERGand GREEN,1964). It is unknown where these micro- fibrils in the interstices are transported. Glycosaminoglycan is generally considered to be formed in fibrous protein- producing cells as fiber matrix (BALAZS and HOLMGREN,1950; NORMANand SCHMIDT, 1967; KAJIKAWA,1975), though there is a supposition that it can be produced from the blood in the case of healing wounds (BENTLEY,1967). The present study also showed evidence of the secretion of glycosaminoglycan from the reticular cell together with fibrous proteins. MERKER and STRUWE (1971) suggested different routes for glyco- saminoglycan and fibrous proteins. The present study failed to make clear whether both substances can be transported and discharged by the same vesicles. In lymph nodes, reticular fibers are generally enclosed in reticular cell bodies with- out being exposed to tissue spaces (HAN, 1961, 1962; MOE, 1963). Also in the spleen, the reticular fibers were formed in the closed spaces, i.e., in the intercellular gaps. On the other hand, the microfibrils with cross striations appeared in the reticular mesh- work interstices which were considered to be communicated with blood vessels as previously mentioned (FUKUTAet al., 1976). Therefore, it is presumed that the differ- entiation of fibrous proteins depends on the amount of glycosaminoglycan; the fibrous proteins aggregate to form reticular fibers in the closed spaces which contain a high 188 K. FUKUTA and K. MocHIzuKI: concentration of glycosaminoglycan. It is also presumed that the fibrous proteins are further polymerized to make up microfibrils with cross striations in the open spaces where glycosaminoglycan is diluted and neutralized. It is well known that if collagen fibers are dissolved in acid, reconstituted fibers will automatically appear when the acid is neutralized (GROSS,1956). Our presumption on fiber differentiation in vivo well agrees with the results in vitro, because glycos- aminoglycan is acidic in nature (MYERSet al., 1969; KAJIKAWA,1975).

Acknowledgement. The authors wish to thank Prof. Y. EGUCHI,Azabu University, for his critical reading of the manuscript.

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福 田 勝 洋 Dr. Katsuhiro FUKUTA 〒113東 京 都 文 京 区 弥 生1-1-1 Laboratory of Veterinary Anatomy 東 京 大 学 農 学 部 Faculty of Agriculture 家 畜 解 剖 学 教 室 University of Tokyo Bunkyo-ku, Tokyo, 113 Japan