Acta histochem. 91, 59-65 (1991) Gustav Fischer Verlag lena
1Anatomische Anstalt, Lehrstuhl n, Universitiit Miinchen, Germany, 2Dept. Environmental and Life Sciences, Murdoch University, Perth, Western Australia
Proteoglycans in the notochord sheath of lampreys*) By ULRICH WELSCH!, RAINER ERLINGER! and IAN C. POTTER2 With 3 Figures
(Received March 1, 1991)
Summary The distribution of proteoglycans in the sheath and epithelial cells of the lamprey notochord had been studied using electron microscopy on material stained with the dye cupromeronic blue (CMB). CMBprecipitates, which indicate the presence of sulphated proteoglycans, were found in the inner collagenous but not the outer "elastica externa" regions of the notochord sheath. Precipitates were also found in the zone of the basement membrane of the notochord epithelial cells, in intercellular spaces within the notochord epithelium, and in intracellular spherical bodies of the epithelial cells. These results indicate that the proteoglycans in the notochord sheath are produced by the notochord cells. The combined presence of sulphated proteoglycans and collagen type II in the notochord sheath parallels the situation found in the cartilage of higher vertebrates.
1. Introduction It has been suggested that the notochord sheath contributes to the vertebral column of elasmobranchs and possibly of dipnoans and some chondrosteans, which have a well developed cell-containing notochord sheath in early developmental stages (see e.g. REMANE 1936). Although the notochord is greatly reduced in most vertebrates, it is still well developed in lampreys and hagfishes, the sole survivors of the agnathan stage in vertebrate evolution (KOELLIKER 1972, KRAUSE 1923, TRETJAKOFF 1926, FLOOD 1973, BORKHYARDT 1972, HARDISTY 1981, POTTER and WELSCH, in press). Cartilagenous elements are not found in association with the notochord of larval lampreys (ammocoetes) and the same generalization is true for that of hagfishes except in the caudal region (GOODRICH 1958, POTTER and WELSCH, in press). However, in each of the body segments of adult lampreys. 2 pairs of rods extend dorsally alongside the spinal cord from their base on the notochord sheath (TRETJAKOFF 1936, GOODRICH 1958, BORKHYARDT 1972, HARDISTY 1981, POTTER and WELSCH, in press). The notochord of lampreys consists of glycogen-rich epithelial cells surrounded by a strong acellular sheath (POTTER and WELSCH, in press). The outer part of the sheath, the socalled elastica extema (Y. EBNER 1895, KRAUSE 1923) contains material which immunohistochemically cross reacts with human elastin (SCIDNKO et al., in press). The collagenous part of the sheath, which is far wider than the elastica extema, comprises closely packed thin collagen fibrils (d = 11 nm) consisting of type II collagen (KIMURA and KAMIMURA 1982). There has been much discussion as to whether the notochord sheath is produced by the outer layer of notochord epithelial cells (e.g. KOELLIKER 1872) or by cells in the mesodermal *) In honour to Professor Dr. Dr. Dr. h. c. I.-H. SCHARF on the occasion of his 70th birthday.
60
U.
WELSCH,
R.
ERLINGER
and l. C.
POTTER
Proteoglycans in the notochord sheath of lampreys
61
connective tissue immediately outside the sheath (HELD 1921) or indeed by cells of both regions (BORKHVARDT 1971, SCHINKO et al., in press). Our recent light microscopical studies have shown that the notochord sheath contains sulphated proteog1ycans (POTTER and WELSCH, in press). However, while alcian blue stains the collagenous region but not the "elastica extema", HID stains both of these layers in the sheath. In order to clarify whether proteog1ycans are present in the entire lamprey notochord sheath, where they are exactly localized, and in which connection to fibrillar elements they occur, we used electron microscopy to study notochord material stained with Cupromeronic Blue (SCOTT 1985). This will increase our knowledge of the composition and arrangement of this important structure and provide information relevant to the question of whether the layers of the sheath are produced by cells within or outside the notochord.
2. Material and Methods Artificially reared 3 week and 6 month old ammocoetes of Lampetra japonica were kindly provided by Prof. Dr. Y. HONMA, Niigata, Japan. Geotria australis, representing stage 6 in metamorphosis, were collected from rivers in south-western Australia. N.B. By stage 6 in metamorphosis, most of the morphological characteristics of the young adult lamprey have been developed. Small pieces of notochord were fixed in glutaraldehyde, washed in 0.2 molll acetate buffer pH = 5.6 and then stained overnight in block in 1 % Cupromeronic Blue (CMB, Aldrich) in the same buffer but now containing 0.3 molll MgClz. The material was rinsed in the same solution but without dye, after which 0.5% NaW04 was added to the buffer. After 1 h the material was transferred to 30% ethanol, containing 0.5% NaW04 , for overnight storage. Following dehydration in ethanol, the material was embedded in Araldite (Merck). Thin sections, stained with uranyl acetate in 70% methanol or without any heavy metal ions, were examined in a Zeiss EM 10 or a Philips CM 10 electron microscope.
3. Results While CMB-precipitates were abundant in the collagenous inner region of the notochord sheath of metamorphosing Geotria australis, they were observed neither in the outer "elastica" region of that sheath, nor in the patches of similarly electron dense material found in the midline of the collagenous region of the sheath (Fig. la, b). The precipitates in the collagenous region have the appearance of thin unbranches needles. Although most of the precipitates run parallel with the densely-packed collagen fibrils (Fig. 1d), some run at either oblique or almost perpendicular angles to these fibrils (Fig. Ie). Since the banding pattern on these thin fibrils (d = 11 nm) is very indistinct, it was not possible to determine the specific binding sites of the CMB precipitates to the fibrils.
Fig. 1. Cross section through a metamorphosing (Stage VI) representative of the southern hemisphere lamprey Geotria australis showing sulphated proteoglycans in the notochord sheath.
a. Positive reaction shown by the presence of needle-shaped Cupromeronic blue precipitates in the collagenous part of the sheath (CP) and in association with collagen fibrils (CF) outside the sheath. N.B. No precipitates are present in the "elastica externa" (EE) of the sheath. With uranyl acetate. X 46,690. b. Collagen fibrils (CF) outside the sheath are in direct contact with the "elastica externa" (EE). CP collagenous part of the sheath. Arrows denote CMB-precipitates. Without uranyl acetate. X 46,690. c. Notochord epithelial cells (NEC) underlain by a zone representating the basement membrane (BM) in which there are aggregates of precipitates (arrows). Asterisk homogenous material opposite hemidesmosomes. Arrowhead spherical body with CMB precipitates. CP collagenous part of the sheath. With uranyl acetate. x46,690. d and e. Collagenous part of the sheath (CP) with most of the precipitates orientated parallel to the collagen fibrils (d) or also in an irregular manner (e). Without uranyl acetate. (d) x92,OOO; (e) X 46,690.
62
U. WELSCH, R. ERLINGER and I. C. POTTER
,
I
I
I
Fig. 2. Sulphated proteoglycans in the notochord epithelium. a. precipitates inside variously sized vesicles (arrows), probably in the Golgi area, of a 6 month old ammocoete of Lampetra japonica. With uranyl acetate. x 46,690. b. Reaction product in an intracellular vesicle (large arrow) and in the intercellular space (small arrows) of a metamorphosing Geotria australis. Without uranyl acetate. x 37,720.
Precipitates were also found in the zone of the basement membrane of the notochord epithelial cells, which is about 0.25 !!m thick. These precipitates tend to be thicker than in the collagenous part of the notochord sheath and sometimes form small local accumulations (Fig. lc). CMB precipitates were rarely found in amorphous masses of homogenous dense material opposite hernidesmosomes in the notochord epithelial cells (Fig. I c). Single and sometimes relatively long CMB precipitates were observed in the intercellular spaces between the notochord cells and in the variously sized spherical bodies which are found in the cytoplasm of these cells close to the Golgi field and close to the basal plasma membrane (Figs. lc, 2a, b). Precipitates were observed in association with the collagen fibrils outside the sheath (Fig. I a). Collagen fibrils were also frequently in contact with the "elastica extema" (Fig. lb). As was the situation with metamorphosing Geotria australis, CMB-precipitates were confined to the collagenous region of the notochord sheath of young ammocoetes of Lampetra japonica (Fig. 3a, b). While CMB-precipitates were also found in the spherical bodies in notochord epithelial cells, those that are likewise present in the zone of the basement membrane do not form the local accumulations which characterised their presence of this region in metamorphosing Geotria australis.
Proteoglycans in the notochord sheath of lampreys
63
Fig. 3. Sulphated proteoglycans in the notochord sheath of (a) 3weeks old and (b) 6 months old ammocoetes of Lampetra japonica. NEC notochord epithelial cell. CP collagenous part of notochord sheath. EE "elastica extema". (a) without uranyl acetate, X46,690. (b) with uranyl acetate, X144,900.
4. Discussion The present EM study using material stained with Cupromeronic Blue has shown that sulphated proteoglycans are abundant in the collagenous part of the lamprey notochord sheath, but are present not in the "elastica extema". These results were obtained with Scott's critical electrolyte concentration technique, which is highly specific for sulphated glycosaminoglycans with the corresponding salt concentrations (SCOTT 1989) and are in agreement with the light microscopical results using Alcian Blue (POTTER and WELSCH, in press). However, the "elastica extema" does react positively with HID. Since HID also reacts with elastin (SANNES 1986), as well as with other sulphated glycoconjugates (SPICER et aI. 1981), it is assumed that the positive IDD reaction in the "elastica extema" reflects the presence in this location of an elastin-like material rather than glycosaminoglycans (see also SCHINKO et aI., in press). The combined presence of collagen type II (KIMuRA and KAMIMURA 1982) and sulphated proteogIycans in the collagenous part of the lamprey notochord sheath parallels the situation in the cartilage of higher vertebrates (NARDINGHAM 1981). Although this close agreement suggests that the glycosaminoglycan chain in the lamprey notochord sheath may likewise be condroitin sulphate, further
64
U. WELSCH, R. ERLINGER and I. C. POTTER
work is required to verify that this is the case. However, the above parallels would be consistent with the view that the material of the notochord sheath contributes to the vertebral column (see REMANE 1936). It seems to be relevant that the morphology of the precipitates inside the spherical bodies close to the basal plasma membrane of the notochord epithelial cells is similar to the aggregated precipitates in the zone of the basement membrane. Although these intracellular precipitates could represent lysosomal degradation, they could indicate that the glycosarninoglycans in this zone are produced by the notochord epithelial cells. Such a view would be consistent with the fact that proteoglycans are assembled within the cell (ALBERTS et a!. 1989). It also seems likely that the epithelial cells supply the glycosarninoglycans of the collagenous part of the sheath. This view is based on the fact that, since the "elastica extema" is dense and does not contain glycosarninoglycans, it is very unlikely that this layer acts as a transport route for glycosarninoglycans from production sites outside the notochord to the collagenous region of the sheath.
Acknowledgement We are grateful to Prof. Dr. Y. HONMA, Marine Biological Sciences, University of Niigata, Japan, for the gift of the young ammocoetes of Lampetra japonca. Financial support was provided by the Australian Research Grant Scheme.
References ALBERTS, B., LEWIS, D., RAFF, I., ROBERTS, K., and WATSON, D., Molecular Biology of the Cell. Garland, New York 1989. BORKHVARDT, V. G., Chordal membranes of cyclostomes and fishes. Vop. Ikhtio!. 11, 1061-1070 (1972). V. EBNER, J., Uber den feineren Bau der Chorda dorsalis der Cyclostomen (Vorl. Mitt.) Sitzgsber. Akad. Wiss. Wien, Math.-naturwiss. Kl. III, 104, 7-16 (1895). FLOOD, P. R., The notochord of Myxine glutinosa L. related to that of other chordates. Acta Roy. Soc. Scient. Litt. (Zoo!.), 14, 14-16 (1973). GOODRICH, E. S., Studies on the Structure and Development of Vertebrates. New York: Dover 1958. HARDINGHAM, T., Proteoglycans: Their structure, interactions and molecular organization in cartilage. Biochem. Soc. Trans. 9,489-497 (1981). HARDISTY, M. W., The skeleton. In: HARDISTY, M. W., and POTTER, I. C. (eds.), The Biology of Lampreys. Academic Press, London 1981. HELD, H., Uber die Entwicklung des Achsenskeletts der Wirbeltiere. Abh. math. phys. K!. sikhs. Akad. Wiss. 3, 5, 1-2 (1921). KIMURA, S., and KAMIMURA, T., The characterisation of lamprey notochord collagen with special reference to its skin collagen. Compo Biochem. Physio!. 73B, 335-339 (1982). KOELLIKER, A., Kritische Bemerkungen zur Geschichte der Untersuchungen tiber die Scheiden der Chorda dorsalis. Wiirzb. Verh. N. F. 3, 335-345 (1872). KRAUSE, R., Mikroskopische Anatomie der Wirbeltiere in Einzeldarstellungen, IV. Teleostier, Plagiostomen, Zyklostomen und Leptokardier. W. de Gruyter, Berlin/Leipzig 1923. POTTER, I. C., and WELSCH, U., Arrangement, histochemistry and fine structure of the connective tissue architecture of lampreys. J. Zoo!., London, in press. REMANE, A., Skelettsystem. I. Wirbelsiiule und ihre Abkiimmlinge. In: Handbuch der vergleichenden Anatomie der Wirbeltiere (BOLK, L., GOPPERT, E., KALLIUS, E., und LUBOSCH, W. K., EDS.), Urban & Schwarzenberg BerlinlWien 1936, pp. 1-206. SANNES, P. L., Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains. Histochemistry 84,49-56 (1986). SCHINKO, I., POTTER, I. C., WELSCH, U., and DEBBAGE, P., Structure and development of the notochord "elastica extema" and nearby components of the elastic fiber system of agnathans. Acta zoologica (in press). SCOTT, J. E., Proteoglycan histochemistry - a valuable tool for connective tissue biochemists. Collagen Relat. Res. 5, 765-774 (1985). - In praise of dyestuff histochemistry. Bas. App!. Histochem. 33, 9-15 (1989).
Proteoglycans in the notochord sheath of lampreys
65
SPICER, S. S., BARON, D. A., SATO, A., and SCHULTE, B. A., Variability of cell surface glycoconjugates Relation to differences in cell function. J. Histochem. Cytochem. 29, 994-1002 (1981). TRETJAKOFF, D., Die Wirbelsaule des Neunauges. Anal. Anz. 61, 387-396 (1926). Author's address: Prof. Dr. Dr. U. WELSCH, Anatomische Anstalt, Ludwig-Maximilians-Universitat, D-W-800D MUnchen 2, BRD.
5 Acta histochem., Bd. 91, 1
1Anatomische Anstalt, Lehrstuhl n, Universitiit Miinchen, Germany, 2Dept. Environmental and Life Sciences, Murdoch University, Perth, Western Australia
Proteoglycans in the notochord sheath of lampreys*) By ULRICH WELSCH!, RAINER ERLINGER! and IAN C. POTTER2 With 3 Figures
(Received March 1, 1991)
Summary The distribution of proteoglycans in the sheath and epithelial cells of the lamprey notochord had been studied using electron microscopy on material stained with the dye cupromeronic blue (CMB). CMBprecipitates, which indicate the presence of sulphated proteoglycans, were found in the inner collagenous but not the outer "elastica externa" regions of the notochord sheath. Precipitates were also found in the zone of the basement membrane of the notochord epithelial cells, in intercellular spaces within the notochord epithelium, and in intracellular spherical bodies of the epithelial cells. These results indicate that the proteoglycans in the notochord sheath are produced by the notochord cells. The combined presence of sulphated proteoglycans and collagen type II in the notochord sheath parallels the situation found in the cartilage of higher vertebrates.
1. Introduction It has been suggested that the notochord sheath contributes to the vertebral column of elasmobranchs and possibly of dipnoans and some chondrosteans, which have a well developed cell-containing notochord sheath in early developmental stages (see e.g. REMANE 1936). Although the notochord is greatly reduced in most vertebrates, it is still well developed in lampreys and hagfishes, the sole survivors of the agnathan stage in vertebrate evolution (KOELLIKER 1972, KRAUSE 1923, TRETJAKOFF 1926, FLOOD 1973, BORKHYARDT 1972, HARDISTY 1981, POTTER and WELSCH, in press). Cartilagenous elements are not found in association with the notochord of larval lampreys (ammocoetes) and the same generalization is true for that of hagfishes except in the caudal region (GOODRICH 1958, POTTER and WELSCH, in press). However, in each of the body segments of adult lampreys. 2 pairs of rods extend dorsally alongside the spinal cord from their base on the notochord sheath (TRETJAKOFF 1936, GOODRICH 1958, BORKHYARDT 1972, HARDISTY 1981, POTTER and WELSCH, in press). The notochord of lampreys consists of glycogen-rich epithelial cells surrounded by a strong acellular sheath (POTTER and WELSCH, in press). The outer part of the sheath, the socalled elastica extema (Y. EBNER 1895, KRAUSE 1923) contains material which immunohistochemically cross reacts with human elastin (SCIDNKO et al., in press). The collagenous part of the sheath, which is far wider than the elastica extema, comprises closely packed thin collagen fibrils (d = 11 nm) consisting of type II collagen (KIMURA and KAMIMURA 1982). There has been much discussion as to whether the notochord sheath is produced by the outer layer of notochord epithelial cells (e.g. KOELLIKER 1872) or by cells in the mesodermal *) In honour to Professor Dr. Dr. Dr. h. c. I.-H. SCHARF on the occasion of his 70th birthday.
60
U.
WELSCH,
R.
ERLINGER
and l. C.
POTTER
Proteoglycans in the notochord sheath of lampreys
61
connective tissue immediately outside the sheath (HELD 1921) or indeed by cells of both regions (BORKHVARDT 1971, SCHINKO et al., in press). Our recent light microscopical studies have shown that the notochord sheath contains sulphated proteog1ycans (POTTER and WELSCH, in press). However, while alcian blue stains the collagenous region but not the "elastica extema", HID stains both of these layers in the sheath. In order to clarify whether proteog1ycans are present in the entire lamprey notochord sheath, where they are exactly localized, and in which connection to fibrillar elements they occur, we used electron microscopy to study notochord material stained with Cupromeronic Blue (SCOTT 1985). This will increase our knowledge of the composition and arrangement of this important structure and provide information relevant to the question of whether the layers of the sheath are produced by cells within or outside the notochord.
2. Material and Methods Artificially reared 3 week and 6 month old ammocoetes of Lampetra japonica were kindly provided by Prof. Dr. Y. HONMA, Niigata, Japan. Geotria australis, representing stage 6 in metamorphosis, were collected from rivers in south-western Australia. N.B. By stage 6 in metamorphosis, most of the morphological characteristics of the young adult lamprey have been developed. Small pieces of notochord were fixed in glutaraldehyde, washed in 0.2 molll acetate buffer pH = 5.6 and then stained overnight in block in 1 % Cupromeronic Blue (CMB, Aldrich) in the same buffer but now containing 0.3 molll MgClz. The material was rinsed in the same solution but without dye, after which 0.5% NaW04 was added to the buffer. After 1 h the material was transferred to 30% ethanol, containing 0.5% NaW04 , for overnight storage. Following dehydration in ethanol, the material was embedded in Araldite (Merck). Thin sections, stained with uranyl acetate in 70% methanol or without any heavy metal ions, were examined in a Zeiss EM 10 or a Philips CM 10 electron microscope.
3. Results While CMB-precipitates were abundant in the collagenous inner region of the notochord sheath of metamorphosing Geotria australis, they were observed neither in the outer "elastica" region of that sheath, nor in the patches of similarly electron dense material found in the midline of the collagenous region of the sheath (Fig. la, b). The precipitates in the collagenous region have the appearance of thin unbranches needles. Although most of the precipitates run parallel with the densely-packed collagen fibrils (Fig. 1d), some run at either oblique or almost perpendicular angles to these fibrils (Fig. Ie). Since the banding pattern on these thin fibrils (d = 11 nm) is very indistinct, it was not possible to determine the specific binding sites of the CMB precipitates to the fibrils.
Fig. 1. Cross section through a metamorphosing (Stage VI) representative of the southern hemisphere lamprey Geotria australis showing sulphated proteoglycans in the notochord sheath.
a. Positive reaction shown by the presence of needle-shaped Cupromeronic blue precipitates in the collagenous part of the sheath (CP) and in association with collagen fibrils (CF) outside the sheath. N.B. No precipitates are present in the "elastica externa" (EE) of the sheath. With uranyl acetate. X 46,690. b. Collagen fibrils (CF) outside the sheath are in direct contact with the "elastica externa" (EE). CP collagenous part of the sheath. Arrows denote CMB-precipitates. Without uranyl acetate. X 46,690. c. Notochord epithelial cells (NEC) underlain by a zone representating the basement membrane (BM) in which there are aggregates of precipitates (arrows). Asterisk homogenous material opposite hemidesmosomes. Arrowhead spherical body with CMB precipitates. CP collagenous part of the sheath. With uranyl acetate. x46,690. d and e. Collagenous part of the sheath (CP) with most of the precipitates orientated parallel to the collagen fibrils (d) or also in an irregular manner (e). Without uranyl acetate. (d) x92,OOO; (e) X 46,690.
62
U. WELSCH, R. ERLINGER and I. C. POTTER
,
I
I
I
Fig. 2. Sulphated proteoglycans in the notochord epithelium. a. precipitates inside variously sized vesicles (arrows), probably in the Golgi area, of a 6 month old ammocoete of Lampetra japonica. With uranyl acetate. x 46,690. b. Reaction product in an intracellular vesicle (large arrow) and in the intercellular space (small arrows) of a metamorphosing Geotria australis. Without uranyl acetate. x 37,720.
Precipitates were also found in the zone of the basement membrane of the notochord epithelial cells, which is about 0.25 !!m thick. These precipitates tend to be thicker than in the collagenous part of the notochord sheath and sometimes form small local accumulations (Fig. lc). CMB precipitates were rarely found in amorphous masses of homogenous dense material opposite hernidesmosomes in the notochord epithelial cells (Fig. I c). Single and sometimes relatively long CMB precipitates were observed in the intercellular spaces between the notochord cells and in the variously sized spherical bodies which are found in the cytoplasm of these cells close to the Golgi field and close to the basal plasma membrane (Figs. lc, 2a, b). Precipitates were observed in association with the collagen fibrils outside the sheath (Fig. I a). Collagen fibrils were also frequently in contact with the "elastica extema" (Fig. lb). As was the situation with metamorphosing Geotria australis, CMB-precipitates were confined to the collagenous region of the notochord sheath of young ammocoetes of Lampetra japonica (Fig. 3a, b). While CMB-precipitates were also found in the spherical bodies in notochord epithelial cells, those that are likewise present in the zone of the basement membrane do not form the local accumulations which characterised their presence of this region in metamorphosing Geotria australis.
Proteoglycans in the notochord sheath of lampreys
63
Fig. 3. Sulphated proteoglycans in the notochord sheath of (a) 3weeks old and (b) 6 months old ammocoetes of Lampetra japonica. NEC notochord epithelial cell. CP collagenous part of notochord sheath. EE "elastica extema". (a) without uranyl acetate, X46,690. (b) with uranyl acetate, X144,900.
4. Discussion The present EM study using material stained with Cupromeronic Blue has shown that sulphated proteoglycans are abundant in the collagenous part of the lamprey notochord sheath, but are present not in the "elastica extema". These results were obtained with Scott's critical electrolyte concentration technique, which is highly specific for sulphated glycosaminoglycans with the corresponding salt concentrations (SCOTT 1989) and are in agreement with the light microscopical results using Alcian Blue (POTTER and WELSCH, in press). However, the "elastica extema" does react positively with HID. Since HID also reacts with elastin (SANNES 1986), as well as with other sulphated glycoconjugates (SPICER et aI. 1981), it is assumed that the positive IDD reaction in the "elastica extema" reflects the presence in this location of an elastin-like material rather than glycosaminoglycans (see also SCHINKO et aI., in press). The combined presence of collagen type II (KIMuRA and KAMIMURA 1982) and sulphated proteogIycans in the collagenous part of the lamprey notochord sheath parallels the situation in the cartilage of higher vertebrates (NARDINGHAM 1981). Although this close agreement suggests that the glycosaminoglycan chain in the lamprey notochord sheath may likewise be condroitin sulphate, further
64
U. WELSCH, R. ERLINGER and I. C. POTTER
work is required to verify that this is the case. However, the above parallels would be consistent with the view that the material of the notochord sheath contributes to the vertebral column (see REMANE 1936). It seems to be relevant that the morphology of the precipitates inside the spherical bodies close to the basal plasma membrane of the notochord epithelial cells is similar to the aggregated precipitates in the zone of the basement membrane. Although these intracellular precipitates could represent lysosomal degradation, they could indicate that the glycosarninoglycans in this zone are produced by the notochord epithelial cells. Such a view would be consistent with the fact that proteoglycans are assembled within the cell (ALBERTS et a!. 1989). It also seems likely that the epithelial cells supply the glycosarninoglycans of the collagenous part of the sheath. This view is based on the fact that, since the "elastica extema" is dense and does not contain glycosarninoglycans, it is very unlikely that this layer acts as a transport route for glycosarninoglycans from production sites outside the notochord to the collagenous region of the sheath.
Acknowledgement We are grateful to Prof. Dr. Y. HONMA, Marine Biological Sciences, University of Niigata, Japan, for the gift of the young ammocoetes of Lampetra japonca. Financial support was provided by the Australian Research Grant Scheme.
References ALBERTS, B., LEWIS, D., RAFF, I., ROBERTS, K., and WATSON, D., Molecular Biology of the Cell. Garland, New York 1989. BORKHVARDT, V. G., Chordal membranes of cyclostomes and fishes. Vop. Ikhtio!. 11, 1061-1070 (1972). V. EBNER, J., Uber den feineren Bau der Chorda dorsalis der Cyclostomen (Vorl. Mitt.) Sitzgsber. Akad. Wiss. Wien, Math.-naturwiss. Kl. III, 104, 7-16 (1895). FLOOD, P. R., The notochord of Myxine glutinosa L. related to that of other chordates. Acta Roy. Soc. Scient. Litt. (Zoo!.), 14, 14-16 (1973). GOODRICH, E. S., Studies on the Structure and Development of Vertebrates. New York: Dover 1958. HARDINGHAM, T., Proteoglycans: Their structure, interactions and molecular organization in cartilage. Biochem. Soc. Trans. 9,489-497 (1981). HARDISTY, M. W., The skeleton. In: HARDISTY, M. W., and POTTER, I. C. (eds.), The Biology of Lampreys. Academic Press, London 1981. HELD, H., Uber die Entwicklung des Achsenskeletts der Wirbeltiere. Abh. math. phys. K!. sikhs. Akad. Wiss. 3, 5, 1-2 (1921). KIMURA, S., and KAMIMURA, T., The characterisation of lamprey notochord collagen with special reference to its skin collagen. Compo Biochem. Physio!. 73B, 335-339 (1982). KOELLIKER, A., Kritische Bemerkungen zur Geschichte der Untersuchungen tiber die Scheiden der Chorda dorsalis. Wiirzb. Verh. N. F. 3, 335-345 (1872). KRAUSE, R., Mikroskopische Anatomie der Wirbeltiere in Einzeldarstellungen, IV. Teleostier, Plagiostomen, Zyklostomen und Leptokardier. W. de Gruyter, Berlin/Leipzig 1923. POTTER, I. C., and WELSCH, U., Arrangement, histochemistry and fine structure of the connective tissue architecture of lampreys. J. Zoo!., London, in press. REMANE, A., Skelettsystem. I. Wirbelsiiule und ihre Abkiimmlinge. In: Handbuch der vergleichenden Anatomie der Wirbeltiere (BOLK, L., GOPPERT, E., KALLIUS, E., und LUBOSCH, W. K., EDS.), Urban & Schwarzenberg BerlinlWien 1936, pp. 1-206. SANNES, P. L., Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains. Histochemistry 84,49-56 (1986). SCHINKO, I., POTTER, I. C., WELSCH, U., and DEBBAGE, P., Structure and development of the notochord "elastica extema" and nearby components of the elastic fiber system of agnathans. Acta zoologica (in press). SCOTT, J. E., Proteoglycan histochemistry - a valuable tool for connective tissue biochemists. Collagen Relat. Res. 5, 765-774 (1985). - In praise of dyestuff histochemistry. Bas. App!. Histochem. 33, 9-15 (1989).
Proteoglycans in the notochord sheath of lampreys
65
SPICER, S. S., BARON, D. A., SATO, A., and SCHULTE, B. A., Variability of cell surface glycoconjugates Relation to differences in cell function. J. Histochem. Cytochem. 29, 994-1002 (1981). TRETJAKOFF, D., Die Wirbelsaule des Neunauges. Anal. Anz. 61, 387-396 (1926). Author's address: Prof. Dr. Dr. U. WELSCH, Anatomische Anstalt, Ludwig-Maximilians-Universitat, D-W-800D MUnchen 2, BRD.
5 Acta histochem., Bd. 91, 1
