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995 Development 113, 995-1005 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 Budding-specific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates KAZUO KAWAMURA*, SHIGEKI FUJIWARA and YASUO M. SUGINO Department of Biology, Faculty of Science, Kochi University, Akebono-cho, Kochi 780, Japan * Author for correspondence Summary We have examined immunocytochemically the expression, localization and in vivo function of a calciumdependent and galactose-binding 14xlO3Afr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner epithelium of a double-walled bud vesicle. Soon after the bud entered the developmental phase, the granules were secreted into the mesenchymal space, where the lectinpositive extracellular matrix (ECM) developed. The lectin was also produced and secreted by granular leucocytes during budding. Hemoblasts, pluripotent stem cells in the blood, were often found in association with the ECM and they aggregated with epithelial cells to form organ rudiments. The lectin showed a high binding affinity for hemoblast precursors. The blockage of epithelial transformation of stem cells by galactose in in vivo bioassay was ineffective in the presence of the lectin. Polyclonal anti-lectin antibody prevented the hemoblasts spreading on the ECM and moving toward the epithelium, but it did not block the cell-cell adhesion of hemoblasts. By three days of bud development, lectin granules and ECM have almost disappeared from the developing bud together with a cessation of hemoblast aggregation. These results show that Polyandrocarpa lectin is a component of the ECM induced specifically in budding and suggest strongly that it plays a role in bud morphogenesis by directing the migration of pluripotent stem cells to the epithelium. Introduction the C-type lectin plays an important role in cell-cell communication in a wide variety of animal species. Recently, we have purified a 14X103 Afr protein from the tunicate, Polyandrocarpa misakiensis. Its amino acid sequencing and biochemical characterization showed that it is a calcium-dependent, galactosebinding lectin (Suzuki et al. 1990), referred to as TC-14 (tunicate-derived C-type lectin of i^xlO 3 MT) tentatively in this work. Unlike P. misakiensis with budding capacity (Watanabe and Tokioka, 1972; Kawamura and Watanabe, 1983), solitary tunicates such as Styela plicata and Ciona intestinalis did not contain TC-14 (Suzuki et al. 1990 and our unpublished data). TC-14 might be involved in asexual reproduction of budding tunicates. In this work, we have purified TC-14, labeled a portion of it with fluorescein or biotin and prepared anti-TC-14 polyclonal antibodies. They were used to examine the expression, localization and possible role of the TC-14 lectin in asexual development of Polyandrocarpa. This is the first report of a budding-specific protein from tunicates. The results are discussed in the context of the manner by which TC-14 plays a role in Animal lectins have recently been classified into two groups (Drickamer, 1988). One, referred to as C-type lectin, requires calcium to exert carbohydrate-binding activity; the other, the S-type lectin, depends on the protection of SH group in polypeptide chains for its activity. The C-type lectins have been extracted from barnacle (Muramoto and Kamiya, 1986), fly (Takahashi et al. 1985), sea urchin (Giga et al. 1987) and many organs of mammals (Drickamer et al. 1984). They have a characteristic carbohydrate-recognition domain (CRD) consisting of 120-130 residues including four invariable half-cysteines that form two intrachain disulfide bridges (Drickamer, 1988). Lectin in the fly, Sarcophaga, is involved in the development of embryos or pupae (Takahashi et al. 1986) as well as defense mechanisms of instars (Komano et al. 1983). In mammals, glycoproteins containing a lectin domain at the N terminus serve as a lymphocytehoming receptor (Lasky et al. 1989) and platelet-vascular endothelium adhesion molecule (Johnston et al. 1989). Thus, increasing evidence strongly suggests that Key words: lectin, extracellular matrix, stem cells, epithelial transformation, budding, tunicate. 996 K. Kawamura and others epithelial-mesenchymal collaboration during bud morphogenesis of Polyandrocarpa. Materials and methods Purification of TC-14 Colonies of Polyandrocarpa (Eusynstyela) misakiensis were attached to glass slides and reared in the inlet near the Usa Marine Biological Institute, Kochi University. A galactosebinding 14xl(r MT lectin (TC-14) was purified, as described previously (Suzuki et al. 1990). In brief, about 100 g of asexually developing animals was homogenized with 300 ml of 7.5 mM phosphate buffer (pH7.2) and extracted for 60min in an ice bath. After centrifugation (12000g, 15min), a portion of the supernatant containing lmM phenylmethylsulfonyl fluoride (PMSF) was stored as crude extracts at —80°C. The supernatant was fractionated with 40-95 % saturated ammonium sulfate, after boiling and centrifugation. The crude lectin fraction was dialyzed against 0.1M ammonium acetate and passed through a gel filtration column (Ultrogel AcA44; 3x100 cm) equilibrated with the same buffer. After dialysis against 20mM Tris-HCl (pH8.0), the lectin fraction was applied to a column (16x40mm) of DE-32 (Whatman Biosystems, Ltd), equilibrated with the same buffer. The column was eluted overnight with a linear gradient of 0-0.5 M NaCl in the same buffer (400 ml). The eluate was monitored for absorbance at 280 nm. The purity of TC-14 was estimated from the elution profile of anion exchange chromatography and SDS-PAGE. Its amino acid composition was determined in an amino acid analyzer (Hitachi 8335-50), as described previously (Suzuki et al. 1990). SDS-PAGE and western blots SDS-PAGE of the crude extracts and purified TC-14 was carried out on 15 % acrylamide gel or 5-20 % gradient gel containing 0.1% SDS in 0.375M Tris-HCl (pH8.8) (Laemmli, 1970). The gels were transferred electrically to nitrocellulose for 1.5 h at 30 volts. Polyclonal antibody Polyclonal anti-TC-14 antibodies were raised in rabbits. The antiserum as well as non-immunized rabbit serum were fractionated with 50% saturated ammonium sulfate. After centrifugation (12000g, lOmin), a crude y-globulin fraction was suspended in 20 ml of 20 mM phosphate buffer (pH7.4) containing 0.15 M NaCl and dialyzed against the same buffer. Total amount of proteins in the y-globulin fraction was determined by the method of Lowry et al. (1951). Immunohisto chemistry Buds of various developmental stages and adult animals were used. These animals were fixed either in Zamboni's fixative (Zamboni and DeMartino, 1967) at 4°C for 30min or in methanol at —20°C for 20 min followed by a rinse with ethanol for the same duration. After dehydration, the specimens were embedded in JB-4 plastic medium (Polysciences, Inc.) and sectioned with glass knives at 2jan. Sections were mounted serially on coverslips. The blocking was carried out in 2 % dry milk or 2 % bovine serum albumin (BSA) suspended in 20 mM Tris-buffered salt solution (TBS containing 0.15 M NaCl, pH7.4) for 30 min. Goat anti-rabbit IgG antibody labeled with horseradish peroxidase was purchased from Zymed Laboratories. The primary and secondary antibodies were diluted 2000-fold with 20 mM TBS containing 0.2 % dry milk or BSA. Sections were reacted with the antibodies for 30min, followed by washing with 0.1 % Tween 20. They were stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Graham and Karnovsky, 1966). As controls, sections were reacted with nonimmunized rabbit y-globulin or anti-TC-14 antibody absorbed by antigen. Electron microscopy Specimens were prefixed in 2.5% glutaraldehyde in phosphate buffer (pH7.4) containing 8% sucrose for 2h in an ice bath. After washing with the buffer, they were postfixed in 1% OsO4 solution for 2h. They were dehydrated and embedded in Spurr low-viscosity resin (Spurr, 1969). Sections were stained with uranyl acetate and Reynolds' lead citrate (Reynolds, 1963), and observed with a JEOL JEM 100U electron microscope. Bioassay Growing buds of 3-4 mm in length were extirpated from the parental animal. They were incubated in Millipore-filtered sea water (MFSW) containing 0.1 ^jg, 0.3 ^g, 1/igor lO^gml" 1 of TC-14 and/or 5mM galactose, and were allowed to develop for two days in the presence of streptomycin (0.5xlO- 4 gUmr') and kanamycin (lxlO^gUmF 1 ). Bioassays were also done, using 5/zgml"1 or 10/igml"1 of antiTC-14 y-globulin fraction in the presence of antibiotics. As a control, non-immunized rabbit y-globulin was used. Buds treated were incubated in 1 mM colchicine for the last 12 h before fixation in order to augment mitotic figures (Kawamura and Nakauchi, 1986a). They were fixed in Bouin's fixative, dehydrated and embedded in paraffin. The specimens were sectioned at 5^m and stained with hematoxylin and eosin. Labeling of TC-14 In order to determine target cells of the lectin, lyophylized powder of TC-14 (about 0.5 mg) was labeled with fluorescein isothiocyanate (FITC, F-7250 Sigma) in an ice bath for 60 min in 2 ml of a reaction mixture containing 50 mM sodium bicarbonate buffer (pH9.0), 0.5 mg FITC, 0.1 M galactose and 0.5 mM CaCl2. The products were dialyzed against distilled water. The lectin (0.5 mg) was also labeled with biotin. It was dissolved in 0.5 ml of 0.1M sodium bicarbonate buffer (pH8.5) and incubated with 0.1 mg of N-hydroxysuccinimidyl-6-(biotinamido)-hexanoate (biotin-NHS, Vector Laboratories) for 4h at room temperature. The reaction products were dialyzed against 20mM Tris-HCl (pH7.4). Sections were stained for 30 min with TC-14-FTTC or biotinyl TC-14 diluted 500-fold with 20 HIM Tris-HCl (pH 7.2) containing 10 mM CaCl2. In the case of biotinyl TC-14, they were reacted secondarily with avidin-peroxidase (Vector Laboratories). They were colored with DAB, as described above. Blood smears were fixed in methanol at —20°C and stained as above. As controls, specimens were stained in the presence of either 10mM EDTA or 50 mM lactose. Results Appearance and localization of TC-14 during budding A monomeric, 14X103 MT lectin is a major component of Polyandrocarpa's 95 % ammonium acetate fraction (PAM95). It shows the electrophoretic mobility equivalent to a 17 x 103 Mr protein on SDS-PAGE (Lane a of Fig. 1A) (see Suzuki et al. 1990). After gel filtration (Fig. IB), it was found in the fraction of PAM95-5 Budding-specific lectin in tunicates 997 kDa 0.2- o oo 0.1- B 50 100 200 150 FRACTION NUMBER Fig. 1. Purification of TC-14 from colonies of P. misakiensis (see Materials and methods). (A) Coomassie brilliant blue staining after SDS-PAGE (a,b,d), and anti-TC-14 polyclonal antibody staining after western blotting to nitrocellulose membrane (c,e). (a) Crude extracts; (b,c) PAM95-3 fraction after gel nitration; (d,e) TC-14 purified from PAM95-5 by anion exchange chromatography (DE-32). (B) Gel filtration profile of PAM-95. G D Fig. 2. Schematic illustration of bud formation and development in P. misakiensis. Each square corresponds to photographs in Figs 3 and 4. (A) Bud primordium and adjacent parental mantle wall. (B) Growing bud. Parental epidermis and atrial epithelium form the outer and inner epithelia of the bud. (C) Developing bud, one day after isolating from the parent. Square shows the morphogenesis domain. (D) 2-day developing bud. Organ rudiments form at the proximal end (square at the bottom). Another square shows a non-morphogenetic domain, a, atrial epithelium; b, blood cell; e, epidermis; g, gut rudiment; i, inner epithelium; p, pharyngeal rudiment. {Lane d of Fig. 1A). A polypeptide with the same electrophoretic mobility was also found in PAM95-3 {Lane b of Fig. 1A). Rabbit anti-TC-14 polyclonal antibody reacted with the respective bands from PAM95-3 and PAM95-5 {Lanes c and e of Fig. 1A). So, it appears that in living animals there are some polymeric forms of TC-14 as well as the monomeric one. To provide the context for considering results of immunohistochemistry, we describe briefly bud formation and development of P. misakiensis. The bud primordium forms as an evagination of the parental mantle wall, which consists of the epidermis and atrial epithelium (Fig. 2A). It grows to form a double-walled vesicle (Fig. 2B). The inner epithelial cells take a squamous shape (Fig. 3A) and have a long Gj (Go) 998 K. Kawamura and others Fig. 3. Cellular behaviors during the earliest stage of bud development. (A) Growing stage, the proximal end. Inner epithelial cells are squamous. Hemoblasts cannot be recognized. Bar, 25/mi. (B) 36 h after isolation, the proximal end. Inner epithelial cells are cuboidal and the nucleus becomes swollen. Hemoblasts are associated with the inner epithelium. Bar, 25 fan. (C) 48 h after isolation. Arrowheads show mitotic figures. Note that the inner epithelium becomes temporarily multilayered probably owing to hemoblast adhesion. Bar, 25 fan. (D) The proximal end of a 2-day developing bud. The gut rudiment is established. Arrow shows hemoblast aggregation. Bar, 100/an. e, epidermis; g, gut rudiment; i, inner epithelium; t, tunic phase of cell cycle (Kawamura et al. 1988). After isolation from the parent, the bud enters the developmental phase. In this work, all specimens were encouraged to develop by extirpating them from the parent with razor blades. The wound heals after about 10 h, the inner epithelial cells at the cut end become thickened by 30 h (Figs 2C, 3B), and then the cells enter the cell division cycle (Fig. 3C, Kawamura and Nakauchi, 1986a; Kawamura et al. 1988). Together with hemoblasts (about 6 fxm in diameter) with a prominent nucleolus (Wright's nomenclature, 1981), they form first the gut rudiment and then the pharyngeal rudiment (Figs 2D, 3D, Kawamura and Nakauchi, 1991a). There is experimental evidence showing that the DNA replication of epithelial cells and epithelial transformation of hemoblasts are essential for establishing those primary organ rudiments (Kawamura and Nakauchi, 1991&). Anti-TC-14 antibody reacted with granules of the inner epithelial cells at the primordial bud stage (Figs 2A, 4A, 4B). Parental tissues around the bud primordium did not have such granules (Fig. 4C). The basal lamina was stained, but it was a non-specific reaction, as shown later. As the bud grew, lectin- Fig. 4. Expression of TC-14 in Polyandrocarpa buds visualized by peroxydase-DAB (see also Fig. 1). A-D, H and K were fixed in Zamboni's fixative and others in alcohol. (A) Bud primordium. (B) Higher magnification of the primordium. Arrows show positive granules in the inner epithelium. (C) Parental tissues adjacent to the primordium. There were no granules in the atrial (inner) epithelium. (D) 1-day developing bud, the proximal end (phase-contrast microscopy). Note that the granules are. secreted in the mesenchymal space (arrows) and that the ECM forms in situ (arrowhead). (E) 2-day developing bud, morphogenesis domain. The ECM developed fully. Blood cells and their periphery became lectin-positive (arrowhead). (F) Higher magnification of blood cells, granular leukocytes, which are characterized by large granules in the cytoplasm. (G) An aggregate of hemoblasts (arrow) and single hemoblasts associated with the ECM. (H) Aggregating hemoblasts, hematoxylin-eosin staining. (I) Non-morphogenetic domain of a 2Aiay-old bud. Only the basal lamina was positive (arrowhead). No ECM was observable. (J) Control staining of the morphogenesis domain of a 2-day-old bud. The primary antibody was absorbed by TC-14 before staining. The basal lamina still stained (arrowhead). (K) Three-day-old bud. The granules (arrows) were situated on the apical surface of the gut rudiment and atrial epithelium. The ECM disappeared, a, atrial epithelium; e, epidermis; g, gut rudiment; gr, granular leukocyte; h, hemoblast; i, inner epithelium, mo, morula cell; mu, muscle cell; t, tunic. Bars in A and E, 100/en. Others bars, 75 fan. Budding-specific lectin in tunicates inner epithelial cells began to secrete lectin granules into the mesenchymal space at the proximal end of a bud (Figs 2C, 4D arrows), and the extracellular matrix (ECM) appeared (Fig. 4D arrowhead). The ECM developed fully within two days of bud development and extended its dendritic extremities to the epidermis (Fig. 4E). Granular leukocytes were also stained positively (Fig. 4E,F). This type of cell is characterized positive granules were observed throughout the proximodistally elongated inner vesicle (not shown). Morula cells, a kind of .vacuolated blood cell in tunicates (Fig. 5A), reactedpositively after fixation in Zamboni's fixative (Fig. 4B); but, not after alcohol fixation (cf., Fig. 4F). This was owing to endogenous peroxidase activity (detailed account will be published elsewhere). About one day after the onset of bud development, • v,.-, -\.,.. i mm -iiv ;/'»A'>•*-: -r-pT ^ . l l - V o l - Q I ^ 999 ...... , f 1000 K. Kawamura and others by several acidophilic granules in the cytoplasm (Fig. 5B, see Kawamura et al. 1988). Positive reaction around the cell body was considered as coelomic lectin secreted by granular leukocytes (Fig. 4F). Hemoblasts were observed associated with the lectinpositive ECM at the morphogenesis domain (Fig. 4G). They were identified by a prominent nucleolus in the large nucleus (Fig. 4H). Cell boundaries of aggregated hemoblasts could not be stained with anti-TC-14 antibody (Fig. 4G arrow), suggesting that TC-14 does not play a role in cell-cell adhesion at this stage. Neither the ECM nor hemoblast aggregation could be seen at the non-morphogenesis domain of a 2-day developing bud (Figs 2D, 41). As controls, sections of 2-day-old buds were stained with the antibody absorbed by TC-14 or with nonimmunized rabbit y-globulin. In both cases, no staining could be observed in the inner epithelium, ECM and granular leukocytes. However, the basal lamina was still stained (Fig. 4J), showing that it was a non-specific reaction. From two days of bud development onward, the granules of the inner epithelium and gut rudiment became located at the apical surface of the cells (Fig. 4K arrows). They were secreted into the lumen and hardly observed in further developing buds and zooids (see Fig. 4C). At the same time, the ECM disappeared from the mesenchymal space (Fig. 4K). Binding affinity of TC-14 for blood cells Bud sections were stained with TC-14 labeled with FTTC or biotin. Only undifferentiated blood cells (precursors of hemoblasts, see Discussion) with diameters of 4-5 pan were stained heavily (Fig. 6A,B arrowheads). These cells had a high nucleus: cytoplasm ratio and had no vacuoles in the cytoplasm (Figs 5C, 6C). The plasma membrane and, especially, pseudopods showed a high affinity for TC-14 (Fig. 6C,D). Unlike the hemoblast precursors, single or aggregated mature hemoblasts did not bind TC-14 (Fig. 6E,F). Galactose-binding plant lectin, peanut agglutinin (PNA), showed a similar pattern of staining (not shown). The binding was also found in biotinyl TC-14 (Fig. 6G). It was blocked completely by EDTA (Fig. 6H). Biotinyl TC-14 also stained the outline of morula cells (Fig. 6G). This type of cell was sometimes observed in association with the ECM or inner epithelium at the morphogenesis domain (not shown), but their function was unclear in the process of bud development. Fig. 5. Electron micrographs of Polyandrocarpa blood cells. (A) Morula cell. Vacuoles include electron-dense materials. (B) Granular leukocyte. The cell is characterized by several acidophilic granules and myelin figures. (C) Hemoblast precursor. The nucleus is relatively large. The cytoplasm is not specialized, g, granule, mi, mitochondrion; my, myelin figure; n, nucleus; v, vacuole. Bar, I/an. Effect of TC-14 and anti-TC-14 antibody on bud development The gut rudiment is thefirstorgan to be established (cf., Fig. 3D). TC-14 did not accelerate the formation of this organ rudiment (Table 1). At a concentration of lO^gml" 1 , it reduced the average number of mitotic figures in the morphogenesis domain by half compared with non-treated controls (Table 1). It may result from a non-specific effect of the protein. 1 /igml" 1 of TC-14 often enhanced the mitotic activity, although the Budding-specific lectin in tunicates 1001 H Fig. 6. Identification of lectin-binding cells. (A-F) Specimens were stained with TC-14 labeled with FITC. (G,H) Biotinyl TC-14 staining followed by avidin-peroxidase and DAB. (A) Section of a growing bud, phase-contrast microscopy. Arrowheads show precursors of hemoblast. (B) The same section as A, fluorescent microscopy. Only hemoblast precursors emitted the fluorescence (arrowheads). (C) Hemoblast precursor smeared on glass slide, phase-contrast microscopy. It has the large nucleus. (D) Fluorescent microscopy of the same specimen. Pseudopods were stained. (E) Morphogenesis domain of a 2-day developing bud. (F) Fluorescent microscopy of the same section. Neither aggregated hemoblasts nor inner epithelium emitted fluorescence. (G) Blood smear. The outline of hemoblast precursor was stained heavily (arrowhead). (H) Blood smear stained in the presence of 10 mM EDTA. Hemoblast precursors were stained negatively (arrowheads), a, aggregate of hemoblast; e, epidermis; i, inner epithelium; m, morula cell; n, nucleus. Bar, 25/an. difference was not statistically significant. In most specimens examined, the epithelial transformation of hemoblasts was observed in the mesenchymal space. Galactose, an inhibition sugar of the lectin, did not have so drastic an effect on organogenesis (Table 1). The mitotic activity was reduced, but in some cases high activity was maintained (see 3.6±6.6 in Table 1). Galactose blocked the epithelial transformation of hemoblasts to large extent (Table 1, Fig. 7A). It should be noted that the inner epithelium was single-layered, in contrast to the untreated controls which have transiently multilayered epithelium owing, partly, to hemoblast adhesion (cf., Figs3B, 3C). This inhibition became ineffective by the addition of TC-14 (Table 1, Fig. 7B). Buds were allowed to develop in the presence of lO^gml" 1 of anti-TC-14 antibody (10 cases). In all cases, small aggregates of hemoblasts were scattered in the mesenchymal space (Fig. 7C arrows). They had no pseudopods and were never associated with the inner epithelium. There were no cases in which organogenesis took place. At a concentration of 5/igmF 1 , the 1002 K. Kawamura and others Table 1. Effects of TC-14 and galactose on bud development in P. misakiensis Treatment Control 10/igml"1 TC-14 l/zgrnr 1 TC-14 0.3/igmP 1 TC-14 0.1/igmr 1 TC-14 5mM Gal 5mM Gal + 1/igml"1 TC-14 5mM Gal+OJ/igmP 1 TC-14 No. of cases % Formation of gut rudiment No. of mitotic figures/section 16 10 11 10 6 10 7 5 62.5 60.0 63.6 40.0 50.0 40.0 42.9 40.0 9.1±2.8* 4.3±2.3 10.7±6.3 9.5±13.1 6.8±2.9 3.6±6.6 4.7±2.6 N.D. % Formation of aggregated hemoblasts 93.8 100 100 80.0 83.3 20.0 100 80.0 2-day-old buds were examined. * The ranges show the limit of 95 %confidence. Fig. 7. Results of bioassays in 2-day developing buds. (A) 5mM of galactose. This photograph shows an exceptional case in which hemoblasts have aggregated. Note that the inner epithelium remains single-layered. Arrowhead shows a mitotic figure. Bar, 25 /an. (B) 5ITLM of galactose and 0.3/igml"1 of TC-14. Some hemoblasts extend pseudopods to the inner epithelium. Arrowheads show mitotic figures. Bar, 25 /an. (C) 10/tgmF 1 of anti-TC-14 antibody, differential interference microscopy. Arrows show small aggregates without pseudopods. Bar, 50/an. (D) 5/igml"1 of antibody, differential interference microscopy. Thick arrows show large aggregates with pseudopods. Thin arrow shows an aggregate without pseudopods. Bar, 50 fim. h, hemoblast; i, inner epithelium. antibody blocked the organogenesis in 10 cases out of 15. In the mesenchymal space of most cases, some aggregates of hemoblasts extended pseudopods toward the thickened epithelium at the morphogenesis domain (Fig. 7D thick arrows), as observed in intact buds. But, there existed another kind of aggregate, which was far distant from the inner epithelium and had a smooth outline without any pseudopods (Fig. 7D thin arrow). Non-immunized rabbit y-globulin had no such deteriorative effect on bud development (not shown). Discussion Monomeric and polymeric forms of TC-14 Recently, a monomeric, 14xlO3A/r protein has been purified from the hemolymph of the tunicate, Poly an- Budding-specific lectin in tunicates 1003 drocarpa misakiensis (Suzuki et al. 1990, and our unpublished observation). Its characterization and amino acid sequencing have shown that it belongs to a calcium-dependent (C-type), galactose-binding lectin that has four conservative Cys residues forming two intrachain disulfide bridges (cf. Drickamer, 1988). Galactose-binding lectins of hemolymph origin have been extracted previously from tunicates, Halocynthia roretzi (Yokosawa et al. 1982), Phallusia mamillata (Parrinello and Canicatti, 1983), Didemnum candidum (Vasta et al. 1986), Ascidia malaca (Parrinello and Arizza, 1988) and Botrylloides leachii (Schluter and Ey, 1989). Although some of them require Ca 2+ to exert carbohydrate-binding activity, it is uncertain whether they belong to a C-type lectin family, because the information is lacking about the primary structure of proteins except D. candidum lectin (DCL-1) (Vasta et al. 1986). No reports are available for the expression, localization and possible role of tunicate lectins in relation to embryonic or postembryonic development. The present work has shown for the first time that, in P. misakiensis, the tunicate-derived C-type lectin (TC-14) is induced specifically during budding, although there might be a trace of lectin in the hemolymph of adult animals (not shown). Rabbit anti-TC-14 polyclonal antibody reacted with granules in the bud's inner epithelium from the earliest stage of bud formation until two days of bud development. It was during this stage of bud development that the granules were secreted into the mesenchymal space where the ECM is formed. The antibody also reacted with granular leukocytes, a kind of blood cell in the hemolymph (see Wright, 1981) of a developing bud, suggesting that the lectin is of both epithelial and blood cell origins. Immunoblot analyses suggested that TC-14 existed in both monomeric and polymeric forms. It seems likely that the humoral antigen secreted by granular leukocytes is identical to the monomeric lectin eluted as PAM95-5 fraction in gel filtration chromatography. It is possible that the antigen remains around the blood cells, because there is no blood flow in a bud until it develops into a functional animal. However, the ECM secreted by the inner epithelium may include molecular complexes consisting of TC-14 and other components, as seen in the PAM95-3 fraction. Alternatively, the ECM may consist of high molecular weight proteins(s) with lectin domains. For example, the lymphocytehoming receptor or platelet-endothelium adhesion molecule (GMP-140) in mammals has a C-type lectin domain at the N terminus (Lasky et al. 1989; Johnston et al. 1989). Fibroblast proteoglycan core protein is also known to contain the lectin domain (Krusius et al. 1987). Now, we are trying to purify the functional ECM from developing buds. Cytochemically, inner epithelial cells of a bud are different from the parental atrial epithelium, as they carry lectin granules at the very start of budding. Interestingly, lectin granules were also induced in the process of regeneration (Kawamura, in preparation). They were found only in regenerating organ rudiments that originated from the inner (atrial) epithelium. So, the lectin granules may be essential for both budding and regeneration of Polyandrocarpa. The role of TC-14 in budding As already discussed, both monomeric and polymeric forms of TC-14 seem to exist in Polyandrocarpa buds and it is possible that they might exert different physiological activities. A monomeric TC-14 bound to small undifferentiated blood cells with high affinity in a calcium-dependent manner. The staining pattern was similar to that of PNA (a galactose-binding phytohemagglutinin). However, single or aggregated hemoblasts did not show such an affinity, suggesting that they might lose galactosyl conjugate(s) from the plasma membrane in the process of epithelial transformation. In some budding tunicates, many kinds of somatic tissues and germ cells differentiate from blood cells with a prominent nucleolus, suggesting pluripotent stem cells in the hemolymph (for review, Kawamura and Nakauchi, 1991a). In P. misakiensis, as shown in this work, hemoblasts (Wright's nomenclature, 1981) take part in histogenesis at the earliest stage of bud development. We considered them to be derived from smaller undifferentiated blood cells. One of the grounds for supporting this is that hemoblasts with typical morphology were rarely observed before the epithelial transformation stage (cf., Figs 3A, 3C). There is a great possibility that hemoblasts may come from other type(s) of cell through, for example, dedifferentiation. Our autoradiographic study (Kawamura et al. 1988) has shown that, in the blood, small undifferentiated cells incorporate [3H]thymidine actively, but we have never observed mitotic figures in those smaller cells. We suggested that smaller and larger undifferentiated cells might merely represent different phases of their cell cycle (Kawamura et al. 1988). This is another reason for considering the smaller cell as a hemoblast precursor. Of course, our grounds are inconclusive because the classification of blood cells is based only on morphological differences, as in other tunicates (Wright, 1981). Molecular markers that recognize particular subpopulations of undifferentiated cells are required. In developing buds, hemoblasts and their precursors were often observed in contact with the lectin-positive ECM. The ECM disappeared at the same stage as the termination of hemoblast aggregation. TC-14 competed with galactose, an inhibition sugar of the lectin, for hemoblast aggregation. Anti-TC-14 antibody prevented hemoblasts from forming a large aggregate, extending pseudopods and being associated with the inner epithelium. These results have strongly suggested that TC-14 serves as a component of ECM to direct hemoblast precursors to the inner epithelium. However, mature hemoblasts should interact with other component(s) of the ECM as they had no affinity for monomeric TC-14. The antibody did not stain cell boundaries of aggregated hemoblasts, nor did it affect cell aggregation as such in the bioassay, indicating that TC-14 plays no part in cell-cell adhesion at this stage. 1004 K. Kawamura and others The Polyandrocarpa ECM is similar to tenascin in its pattern of expression and its possible role. Tenascin is expressed at very limited areas during fetal development of mammals (Chiquet-Ehrismann et al. 1986). It appears to be involved in epithelial-mesenchymal interactions during embryogenesis (Crossin et al. 1986; Aufderheide et al. 1987; Vainio et al. 1989) and regeneration (Mackie et al. 1988; Arsanto et al. 1990). We do not know whether the Polyandrocarpa ECM contains tenascin and/or other components such as fibronectin, laminin and vitronectin. Some of these components have a unique amino acid sequence essential for cell-ECM adhesion; for example, ArgGly-Asp-Ser in fibronectin (Pierschbacher and Ruoslahti, 1984) and Arg-Gly-Asp-Val in vitronectin (Suzuki et al. 1985). TC-14 does not have such a RGDX structure. Among C-type lectins, only echinoidin from sea urchin has been shown to possess a single RGD domain (Giga et al. 1987). Mature hemoblasts show a high mitotic activity (Kawamura and Nakauchi, 1986a, 1991a; Kawamura et al. 1988). This activity should not be attributed to a monomeric lectin, as the extrinsic lectin did not act as mitogen in spite of enough time to work through the cut surface. Of course, it is possible that polymeric TC-14 and/or other components of the ECM may trigger cell cycling. For example, fibroblast proteoglycan core protein contains epidermal growth factor-like repeats, following the C-type lectin domain (Krusius et al. 1987). The fly lectin from Sarcophaga peregrina can be induced in instars by surgical injury (Komano et al. 1980). It plays a role in defense mechanisms (Komano et al. 1983). Monomeric TC-14 may serve as opsonin, as suggested in other tunicate lectins (see Vasta et al. 1986). This function is important, because Polyandrocarpa buds may possibly be attacked by foreign materials during early development following extirpation from the parent. 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