Monoclonal Antibody Specific to Urochordate ...

Mar. Biotechnol. 5, 388–394, 2003 DOI: 10.1007/s10126-002-0087-9

 2003 Springer-Verlag New York Inc.

Monoclonal Antibody Specific to Urochordate Botryllus schlosseri Pyloric Gland Ziva Lapidot, Guy Paz, and Baruch Rinkevich* Israel Oceanographic & Limnological Research, Tel Shikmona, P.O.Box 8030, Haifa 31080, Israel

Abstract: We describe here the development of a new hybridoma cell line, CF12F6, that produces a specific antibody to Botryllus schlosseri (a colonial tunicate). The monoclonal antibody was isotyped as IgG1 (by enzyme-linked immunosorbent assay), and the cellular localization of the antigenic epitope that reacts specifically with CF12F6 was confined to the cells of the pyloric gland of the zooid (by immunohistochemistry). The pyloric gland participates in osmoregulation, digestion, glycogen storage, and various other secretion functions that will be studied further by the use of monoclonal antibody CF12F6, the first in botryllid ascidians that recognizes cells of a whole, single organ. Key words: ascidians, Botryllinae, monoclonal, pyloric gland, zooid.

INTRODUCTION The colonial tunicate Botryllus schlosseri, a cosmopolitan invertebrate of the subfamily Botryllinae, inhabits shallow waters abundantly throughout the world, especially in harbors (Berrill, 1950; Abbott and Newberry, 1980; BenShlomo et al., 2001). Adult colonies are made of few to several hundred genetically identical units (each one is called a zooid), which are grouped in typical star-shaped structures (systems; each 3 to 10 zooids), and are embedded within a translucent, gelatinous matrix, the tunic. All systems, as well as zooids within a single system, are interconnected to one another by a network of blood vessels, which bear spherical to elongate termini (called ampullae)

Received May 1, 2002; accepted September 17, 2002. *Corresponding author: telephone 972-4-8565275; fax 972-4-8511911; e-mail buki@ ocean.org.il

near the surface of the tunic, between the systems and around the peripheral border of the colony (Milkman, 1967). Sexual reproduction is commenced by a tadpole larva with a chordate body plan. Upon releasing from the maternal colony, the larva swims to a nearby subtidal surface and undergoes metamorphosis to a sessile juvenile individual (oozooid). The oozooid starts then with cycles of asexual budding processes (each called blastogenesis) that eventually give rise to a multi-individual colony. The blastogenic cycles (at 18
to 20
C) are weekly synchronized developmental processes that terminate at a stage in which all functional zooids degenerate and are replaced by a new generation of zooids developed from paleal buds. The developing buds produce secondary buds, so 3 sequential generations of zooids are simultaneously present in normal colonies (Berrill, 1951; Milkman, 1967; Sabbadin, 1969).

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Botryllus has become increasingly important as a model experimental species for a variety of scientific disciplines such as cell biology, aging, developmental biology, immunology, genetics, physiology, evolutionary ecology, and more (Milkman, 1967; Boyd et al., 1986; Lauzon et al., 1992; Rinkevich and Weissman, 1992; Rinkevich et al., 1992, 1995; De Tomaso et al., 1998; Stoner et al., 1999). For the purpose of these studies, several cellular tools such as cell cultures, (Rinkevich and Rabinowitz, 1993, 1994) and molecular markers such as microsatellites (Pancer et al., 1994; Stoner et al., 1997) were developed. Monoclonal antibodies (MAbs), one of the primary types of markers in biological sciences, have also been used in studies with B. schlosseri. Three different sets of MAbs were initiated. The first set of MAbs was developed against Botryllus blood cell surface antigens (Schlumpberger et al., 1984a). Some of these monoclonal antibodies recognized embryonic cells as well (Schlumpberger et al., 1984b). Lauzon et al. (1992) developed a monoclonal antibody that labeled all B. schlosseri blood cells and the perivisceral epithelium of mid-cycle zooids. More recently, another monoclonal antibody binding to an epitope localized at the border of the atrial siphon and on the inner surface of blood vessels and ampullae (Fagan and Weissman, 1998) was developed. Most of the above-mentioned antiBotryllus MAbs are either lost or not available (B. Rinkevich; 1 personal, communication). It is of great importance therefore to add additional MAbs to the above panel. Here we report the developing of a new MAb that specifically detects the pyloric gland in B. schlosseri zooids.

MATERIALS

AND

METHODS

Animals We used wild and laboratory-bred colonies of B. schlosseri. Wild colonies were collected from floating docks and submerged artificial objects in Monterey, Moss Landing, and Half Moon Bay, California in the United States, or from the lower surfaces of stones lying atop the substrate in the Istra Peninsula in Croatia, and Tel Shikmona, Caesarea, and Michmoret in Israel. In the laboratory, colonies were attached to 5 · 7.5-cm glass slides, 1 colony per slide, held in slots of glass staining racks within 17-L glass tanks, and maintained as described (Rinkevich and Shapira, 1988).

Production of Hybridoma Cells Botryllus colonies were first kept in sterile (0.22-lm) seawater, without feeding for 2 days. The colonial systems were then carefully separated from the tunic matrix by needles (27G), cut into small pieces with a surgical blade on ice, and suspended in Dulbecco’s–phosphate-buffered saline (PBS). Female mice were immunized intraperitoneally with 0.1 to 0.2 ml of Botryllus cells and with small pieces of torn tissues on days 0, 14, 32, and 94. Four days after the last immunization, mice spleens were removed, placed in Dulbecco’s Modified Medium (DMEM), and forced through wire mesh using a syringe plunger. Cells were washed twice in cold DMEM without fetal calf serum (FCS) by centrifuging for 7 minutes at 1200 rpm. NSO myeloma cells were washed in the same way. Spleen cells (108 cells) were mixed with the myeloma cells (107 cells). The cells were spinned at 1200 rpm for 7 minutes. The medium was aspirated completely, and the pellets were loosened by flicking on the tube. While agitating the tube, 1 ml of 50% polyethylene glycol (PEG; Boehringer Mannheim GmbH) at 37
C was added drop-by-drop over 1 minute. This procedure was followed by 1 minute of swirling at 37
C and by adding: 1 ml of DMEM medium without FCS 37
C over 1 minute, 3 ml of medium over 3 minutes, and as the last step, 10 ml of medium over 5 minutes. The cells were spinned at 900 rpm for 7 minutes and resuspended in 40 ml of DMEM containing 100 lm hypoxantine, 4 lm aminopterin, 16 lm thymidine, and 20% FCS, 10% DCCM-1 (vol/vol), 1 mM sodium pyruvate, 2 mM glutamine solution, 10 U/ml penicillin sodium salt, and 100 lg/ml streptomycin (all the above materials were supplied by Biological Industries, Kibbutz Beit HaEmek, Israel). Two drops per well of medium containing cells were carefully added. Cells were cultured in 96-well tissue culture plates and incubated in 10% CO2 at 37
C. Supernatants from wells showing colony growth were tested for the presence of mouse immunoglobulins and for specific antibodies to Botryllus schlosseri, using enzyme-linked immunosorbent assays (ELISA). Cells from positive wells were expanded and subcloned 3 times by limiting dilution (Fazekas et al., 1980; Eshhar, 1985).

Elisa Wells of ELISA plates (U-shaped microtiter NUNC, Denmark) were coated with 0.1 ml of Botryllus schlosseri cell homogenates (1 colonial system in 0.5 ml of 0.1 M carbonate

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buffer, pH 9.8) containing 1 mM phenylmethylsolfonyl flouride (PMSF), incubated overnight at 4
C, fixed with 10% formaldehyde for 10 minutes at 4
C, and then washed with PBS containing 0.05% Tween 20 (PBS-T) 4 times for 5 minutes each. Wells were post coated with 0.15 ml of 4% bovine serum albumin (BSA) in PBS-T. Following overnight incubation at 4
C and washing, aliquots of 0.1 ml of culture supernatants, as well as positive controls (mouse anti–B. schlosseri serum) and negative controls (normal mouse serum, myeloma culture fluid), were each added to wells and incubated for 2 hours at 37
C. After washing, goat antimouse conjugated to alkaline phosphatase (No. 115055068 Jackson Research Laboratories) was added to each well at a predetermined dilution and incubated for 1 hour at 37
C. Following washing, 0.1 ml of substrate for alkaline phosphatase, p-nitrophenylphosphates (pNPP; Cat. No. 104–105, Sigma) were added at 1 mg/ml to each well and incubated at 37
C. The optical density (OD) was determined using a micro ELISA reader at 405 nm. Each ELISA result was expressed as the ratio between the mean absorbance of tested sample in positive (P) and the mean absorbance in negative (N) wells (Naot and Remington, 1981).

Determination of Mouse Immunoglobulin Isotypes Wells of ELISA plates were coated with 50 ll (in carbonate buffer pH 9.8) of goat antimouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, antimouse k, or K (Southern Biotechnology Associates, Birmingham, Ala.). Following overnight incubation at 4
C and washing, wells were postcoated with 1% BSA in PBS-T. Aliquots of samples being tested were added to the wells for a 2-hour incubation period at room temperature. Biotin conjugated to antimouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, k, or K were added, respectively, for an additional 2-hour incubation period at room temperature. Streptavidin conjugated to alkaline phosphatase was added for a 1-hour incubation period at room temperature. Each well was rinsed 3 times with PBS-T between each incubation period. Substrate for alkaline phosphatase, p-nitrophenylphosphates was added to each well. After incubation, the optical density was determined using an ELISA reader.

Immunohistochemistry Colonial systems were fixed in Bouin’s solution for 1 hour, dehydrated through a series of graded concentrations of

Figure l. SDS-PAGE protein profile of B. schlosseri exposed to: lane 1, mouse anti–B. schlosseri; lane 2, normal mouse serum; lane 3, dilution buffer (3% dry milk in TBS); lane 4, NSO myeloma culture fluid; lane 5, MAb CF12F6. The molecular weight 50 kDa is indicated on the right (an arrow).

ethanol, and embedded in paraffin wax. Serial sections were cut at a thickness of 5 lm using a hand microtome, and then attached to a microscope slide that was covered with poly-L lysine (100 lg/ml). Paraffin sections were dewaxed and hydrated by decreasing concentrations of ethanol. The sections were permeabilized in 0.01% BSA, 0.01% Triton X-100, and 0.02% horse serum in PBS for 3 hours at 25
C (Miller et al., 2000). Nonspecific binding sites were blocked by incubation in 4% BSA in 0.15 M PBS-T (pH 7.2). The sections were then incubated with MAbs (growing medium concentrated to ·3) and controls for 18 hours at room temperature, washed (·3) in PBS, and were incubated with alkalinephosphatase-conjugated purified goat antimouse IgG + IgM (Jackson Research Laboratories) for 1 hour at 37
C. The slides were then washed in PBS (·3), and the reactions

Monoclonal Antibody for Pyloric Gland 391

Figure 2. Botryllus schlosseri histologic sections immunostained with: (a) mouse anti–B. schlosseri serum (·100); (b) normal (control) mouse serum (·100); (c) PBS (control) ( · 100); (d–f) Mab CF12F6 (·100, ·400, ·1000; respectively), concentrating on the pyloric gland. Abbreviations: b, primary bud; e, endostyle; g, gonad; i, intestine; p, pyloric gland; t, tunic matrix; tu, tubules of the pyloric gland; z, zooid. Scale bars = 10 lm.

were visualized by using a 5-bromo-4-chloro 3-indolyl phosphate nitroblue tetrazolium (BCIP-NBT; Sigma).

Electrophoresis and Immunoblotting Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in accordance with the method of Laemmli (1970) using 7.5% resolving gels. Systems of B. schlosseri were cut by a surgical blade in Tris buffer containing 1 mM PMSF, 10 lg chymostatin, 10 lg/ml antipain, 10 lg/ml leupeptin, and 10 lg/ml pepstatin A. After sonication, the B. schlosseri homogenate was dissolved in a dissociation buffer containing 2 mercaptoethanol and boiled for 5 minutes. The samples were separated in the gels, and the proteins were transferred to nitrocellulose sheets (Towbin et al., 1979). For immunodetection of antigens, nitrocellulose sheets were first incubated in a blotting buffer

containing 10 mM TBS-T, 0.04% NaN3, and 3% (wt/vol) nonfat dry milk at pH 7.6. Incubations with MAbs or control sera diluted in a blotting buffer were carried out for 18 hours at room temperature. The strips were washed 4 times, 10 minutes each, with TBS-T. Alkaline-phosphatase-conjugated, affinity-purified goat antimouse IgG + IgM were diluted in a blotting buffer and added for 1 hour at 37
C. The strips were washed again and exposed to the alkaline phosphatase substrate BCIP-NBT. The reaction was ended by washing the strips with PBS containing 2 mM EDTA (pH 8.0).

RESULTS

AND

DISCUSSION

Here we describe a hybridoma cell line (CF12F6) that produces a specific anti–B. schlosseri antibody, recognizing cells of the pyloric gland. This monoclonal is a subculture

392 Ziva Lapidot et al.

of clone CF12 and is highly specific to a B. schlosseri epitope as determined by ELISA (p/N values: clone CF12F6, 3.14; clone CF12, 2.05; mouse anti–B. schlosseri 1:5000 dilution, 7.14; normal mouse serum 1:5000 dilution, 1.13; control, 1.0). Monoclonal CF12F6 was isotyped as IgG1. To further identify antigen specificity by MAb CF12F6, extracts of B. schlosseri were electrophoresed under reducing condition on 7.5% SDS polyacrylamide gel, transferred to nitrocellulose membranes, and exposed to mouse anti–B. schlosseri serum, normal serum, myeloma culture fluid, dilution buffer, and MAb CF12F6 (Figure 1). The mouse anti–B. schlosseri serum, as expected, reacted with multiple antigenic bands (lane 1, Figure 1). The 3 controls (lanes 2, 3, and 4) reacted nonspecifically with a single low molecular weight band, approximately 32 kDa, and only MAb CF12F6 reacted specifically with a single band of molecular weight of 50 kDa. The cellular localization of the antigenic epitope that reacts with MAb CF12F6 was evaluated by immunostaining of B. schlosseri histologic sections. Zooids were sectioned parallel to their anteroposterior axis such that the sections span through the whole dorsoventral plane. Staining with mouse anti–B. schlosseri serum revealed positive recognition of most of the animal’s organs’ cellular milieu (Figure 2, a). The mouse serum stained blood cells located in the spaces between the epidermis and the perivesceral epithelium, blood cells within lacunae around visceral organs, and blood cells in the ampullae. The tunic and cells in the tunic, the stomach, intestine, branchial basket, pyloric gland, endostyle, nerve complex, siphons, ampulla, epidermis, testis, ovaries, and embryos, all were immunostained. Normal mouse serum (Figure 2, b) or PBS reacted weakly and unspecifically with only the tunic matrix. Conversely, MAb CF12F6 reacted specifically with cells in the pyloric gland and weakly with the tunic (Figure 2, d, e, f). All of the pyloric gland cells are stained (Figure 2, d, e, f) including the cells lining the tubules, while the nearby intestine cells are not stained The pyloric gland that is found in all tunicates (Berrill, 1950) has been studied by various researchers (Fouque, 1954; Burighel and Milanesi, 1977; Gaill, 1977; Mirre and Thouveny, 1977). It grows from the basal region of the stomach over the outer wall of the zooid’s intestine. This gland consists of a network of tubules and blind ampullae that differentiate early in development from evagination of the gastric epithelium. It is further connected, on the wall of the intestine, with large blood sinuses and is supported

by a connective tissue. The ampullae and tubules join a collecting duct that opens into the posterior end of the stomach near its junction with the intestine. On the cellular level, the epithelium of the pyloric gland comprises a single cell type that may undergo different phases of activity (Fouque, 1954; Gaill, 1977; Mirre and Thouveny, 1977). The cells are cuboidal and carry long cilia and numerous microvilli. In Botryllus schlosseri the gland cells uniquely develop a labyrinthine network of extracellular canals (Mirre and Thouveny, 1977), whereas the basal and lateral parts of adjacent cells are interdigitated. Participation of the pyloric gland in sugar metabolism was documented as well as its function as a major repository for glycogen (Gaill, 1977; Burighel and Cloney, 1997). The glandular cells store glycogen and resemble in function hepatocytes of the vertebrate liver (Mugnaini and Harboe, 1967). Osmoregulation, digestion and excretory functions have also been ascribed to gland functions (Burighel and Cloney, 1997), but whether secretory products are enzymatic, are involved in the above-suggested roles, or have some other functions is uncertain. Monoclonal CF12F6 is the first raised MAb that specifically recognizes cells of a whole, single organ in this group of organisms. With the aid of additional sets of monoclonals to be developed (research in progress), it is expected that the new panel of MAbs will be used as a principal tool in studying botryllid ascidian allorecognition, stem cell biology, and other cellular aspects of their interesting life history portrait.

ACKNOWLEDGMENTS Thanks are due to E. Moiseeva and C. Rabinowitz. This work is part of the study performed in the Minerva Center of Marine Invertebrates Immunology and Developmental Biology and was also supported by grants from the USIsrael Bi-National Science Foundation, by the National Institutes of Health (R01-DK54762), and by Israel Science Foundation (No. 161/98).

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