Hydrobiologib 227: 13-79, 1991. S. Tyler (ed.), Turbellarian Biology. 0 1991 Kluwer Academic Publishers. Printed in Belgium.

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Monoclonal antibodies as markers of specific cell types and regional antigens in the freshwater planarian Dugesia (G.) tigrina

Rafael Romero, Joan Fibla, David Bueno, Lauro Sumoy’, Marc Aureli Soriano & Jaume Bagufia* Departament de GenPtica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain; ‘present address: 56 Old Mill Lane, West Harford, 06107 Connecticut, USA (*author for correspondence)

Key words: monoclonal antibodies, cell types, regional antigens, Dugesia (G.) tigrina, neoblasts, regeneration

Abstract

We have produced monoclonal antibodies (mAb’s) against antigens of the fresh-water planarian Dugesia (G.) tigrina (Girard) using standard protocols. Labeling these mAb’s with PAP (peroxidase- antiperoxidase) and indirect-immunofluorescence methods, we then determined the distribution of their antigens in the planarian. Out of 112 mAb’s that showed some specificity for restricted parts of the planarian, 7 1 were found to be cell- or tissue-specific - among them 36 for parenchymal cells, 7 for muscle cells, 11 for epidermal cells, 8 for gastrodermis, and 7 to basement membrane. Another 41 showed different, but overlapping, regional specificities, namely to pharynx and parenchyma. So far, we have been unable to isolate specific mAb’s against undifferentiated cells (neoblasts). These mAb’s should be im- portant tools in study of tissue and cell morphology, regeneration, and growth and degrowth.

Introduction

The technique using hybridomas to produce mAb’s (monoclonal antibodies) was first de- scribed by Kohler & Milstein (1975). Although not primarily intended to study embryonic devel- opment or regeneration, its application and influ- ence within these fields has been enormous. mAb’s have been used so far to mark different cell types and tissues and their subsets, to detect an- tigens that are spatially and temporally restricted during developmental processes, and to single out stages of commitment during the determination and differentiation of cells.

Fresh-water planarians are mainly known for their power to regenerate and for their sustained ability for growth and degrowth (see Brondsted,

1969, and Baguna et al., 1990, for references). Both phenomena are fairly well understood at the tissue and cell levels. However, basic questions as to the origin and lineages of cells involved in re- generation and growth as well as to the determi- nation of pattern of structures during regenera- tion, growth, and degrowth are still unanswered or debatable. Moreover, the molecular and ge- netic mechanisms underlying these phenomena are virtually unknown (see Gremigni, 1988; and Bagufia et al., 1988, 1990, for references).

There are several reasons why mAb’s can be good experimental tools in such studies: (1) they show a high degree of sensitivity and specificity to cell types and their subsets; (2) they may serve as early markers of cell determination and differen- tiation of specific cell types; and (3) they can be

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used to detect and characterize topographically (regionally) restricted antigens and to reveal the early stages of pattern formation before overt dif- ferentiation is manifest.

In this paper, we describe several antibodies, raised against the planarian Dugesiu (G.) tigrina, that are cell-specific or exhibit patterns of label- ling that may be of interest for the study of re- generation and growth and degrowth in these or- ganisms.

polyethylene glycol (PEG) (Galfre et al., 1977). After selection in hypoxanthine-, aminopterin-, and thymidine-supplemented medium (HAT), hybridomas were screened by incubating cell- culture supematants on parafhn sections (details below) of 7-mm-long D. tigrina. Hybridomas pro- ducing monoclonal antibodies to cell types or areas of interest were cloned by limiting dilution and stored in liquid nitrogen.

Screening of monoclonul antibodies Materials and methods

Species and culture conditions

The planarians used belong to an asexual race (class C; see Ribas et al., 1989) of D. tigrinu col- lected near Barcelona, Spain. They were main- tained in planarian saline (PS; Sale, 1984) in the dark at 10 “C, fed once a week with beef liver. Specimens chosen for the immunization proce- dure were starved for 15 days before use.

Production of monoclonul antibodies

We followed a protocol modified from that de- scribed by Harlow & Lane (1988). For immuni- zations, we used three different kinds of immu- nogens: (1) homogenates obtained sonicating intact organisms in 10 mM PBS (phosphate- buffered saline; pH 7.2); (2) total cells obtained by maceration (Bagufia & Romero, 198 l), washed in PBS and sonicated; and (3) enriched fractions of live neoblasts obtained after the procedure de- scribed by Bagtia et al. (1989). In the former two cases, 20 organisms, 10 mm in length, or 5 million sonicated cells in 1 ml PBS were used, respec- tively; in the last, 10 million live neoblasts in 1 ml PBS were used. The preparations were injected each intraperitoneally into a 6- to-g-week-old BALB/C female mouse. The injection was re- peated twice, each at two-week intervals. Four days after the last injection, the mouse’s spleen was removed, dissociated, and centrifuged with mouse myeloma NSl cells in the presence of

Hybridomas were screened using the indirect- immunofluorescence and the PAP (peroxidase- antiperoxidase) methods on paratfm sections. The sections, 10 pm thick sagittal sections mounted on gelatin-coated slides, were prepared from or- ganisms fixed in 4% paraformaldehyde in PBS, dehydrated through an ethanol series, cleared in xylene, and embedded in soft paraffin. To assay for the presence of cell- and tissue-specific mAb’s, the sections were rehydrated, rinsed in 100 mM TBS (Tris-buffered saline), and permeabilized in 0.4% pepsin (Sigma, London) in 0.1 N HCl (pH 5.0) for 15 minutes at 37 “C, blocked of non- specific absortion by incubation in 10 y0 powdered milk (Molico, Nestle) in TBS for 30 minutes at room temperature, and then incubated in hybri- doma culture supernatant overnight at 4 o C. After three rinses in TBS (5 minutes each), they were incubated 1 h in FITC-conjugated goat anti- mouse Ig (Nordic) diluted 1:50 in TBS with 0.25% Triton X-100. Following a brief rinse in TBS (3 x 5 min) sections were mounted in 50% glycerol in 100 mM TBS and examined for fluorescence-labelled cells using a Leitz photomi- croscope (Dialux 20) equipped with epifluores- cence. The PAP method was used as described in Reuter et al. (1984).

Nomenclature

We named the mAb’s according to a code with two or three letters (the first for the species, the latter for the area, tissue, or cell type) and two

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numbers (number of fusion and a within-fusion- type specifying number). For example, ‘TE-12.1’ codes for an mAb to D. tigrina (T) epidermis (E) obtained in the 12th fusion, the first case.

Results

hence, it stained all ectodermal (E) and mesoder- mal (parenchymal, P) derivatives. Other mAb’s of interest showing overlapping expression in sev- et-al tissues were TGE-15.1, which stained gas- trodermis and epidermis, and TEMb- 13.1, which reacted with epidermis and basement membrane.

From 15 successful fusions, 112 mAb’s showing area-, tissue- or cell-type-specific staining were obtained and studied (Table 1). The largest group belonged to antigens expressed by different classes of parenchymal secretory cells. These cells are laden with high numbers of secretory granules that may constitute a high percentage of total protein which may be highly immunogenic. The patterns of staining by these mAb’s were diverse (see Fig. 5). The next group comprised mAb’s recognizing tissue antigens (epidermal, gastroder- mal, and parenchymal) and non-cellular struc- tures such as the basement membrane. Although our primary goal was to produce mAb’s against undifferentiated cells (neoblasts), none has been found so far, even when fairly enriched neoblast fractions were used as immunogen (see Discus- sion).

mAb’s to several tissues (pawnAb’s)

The mAb TEP-7.1 recognized an antigen present in all planarian tissues except the gastrodermis;

Table 1. Monoclonal antibodies against Dugesia (G.) tigrina.

Sites against which specific mAb’s have been found

Number of mAb’s

Cell- or tissue-specific mAb’s Epidermis Parenchymal cells Muscle cells Gastrodermis Basement membrane All cell types except gastrodermal cells (Pan-mab)

Region-specific (multi-tissue) mAb’s

mAb’s to gastrodermis

The antibody TG-13.1 stained gastrodermis throughout the body (Fig. 1) and was entirely negative for ectodermal and mesodermal deriva- tives. This pattern was complementary to that of TEP-7.1. Within the gastrodermis, both intestinal and club (goblet) cells were stained.

mAb’s to epidermis

The antibody TE-13.2 recognized an antigen found all over the epidermis (dorsal and ventral). Other mAb’s stained a more restricted popula- tion; TE-17.1, for example, only reacted to dor- sal epidermis and to inner and outer epithelia of the pharynx; Of particular interest is the mAb TE-15.4 which showed a patchy staining along the ventral epidermis (Fig. 2). This mAb stained

11 36

1 8 1

2

41

Total 112

Fig. I. Staining pattern of antibody TG-13.1. Sagittal paraf- fin section of D. tigrina stained by indirect immunofluores- cence. Positive reaction is restricted to gut diverticula (G). Reactive areas in the epidermis (E) are due to autofluores-

cence of rhabdoids. x 120

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Fig. 2. Staining pattern of antibody TE-15.4 in ventral epi- dermal cells. Tangential paraffm section of ventral epidermis of D. tigrina stained by the PAP method. Dark grey areas are single (thin arrows) or clustered (thick arrows) reactive cells.

For further details, see text. x 800

Fig. 3. Expression of the antigen recognized by mAb TM- 13.1 in muscle cells. Sag&al paraffin section of D. tigrimz after PAP staining. Circular and longitudinal muscles of the body wall (thick arrows), dorsoventral fibers (dv), and thin fibers (thin arrows) wrapping the gut diverticula (G) are stained.

x400

scattered single cells and clusters of three to five cells, and the antigen seemed to be located at the apical surface of the cell. A similar pattern was seen along the inner pharyngeal epithelium with mAb TEF-17.2.

mAb’s to musculature

The antigen recognized by the mAb TM-13.1 is found in most muscle cells of D. tigrina (Fig. 3). Longitudinal, circular, and diagonal muscle fibers of the body wall as well as dorsoventral and transverse (right-left) fibers running through the parenchyma were clearly stained. Thin muscle fibers enveloping the gut diverticula are of partic- ular interest since to our knowledge this is the first time that a loose arrangement of muscle fibers around the gut of a Dugesia species has been demonstrated, though some other triclads and other turbellarians do have such musculature (Rieger et al., 1990).

Fig. 4. Staining pattern of antibody TM-13.1 in the pharynx. Sag&al pa&in section stained by the PAP method. Anterior (cephalic) end is to the right. im, inner musculature; mo, mouth opening; om, outer musculature; po, pharynx opening. x 300

TM-13.1 located within the elongated contractile portion of the cell (Bueno & Romero, unpub- lished observations). Western blotting showed the molecular weight of this antigen to be around 116 kD.

Although musculature of the pharynx was also stained by TM-13.1, not all pharyngeal muscle fibers reacted (Fig. 4). mAb’s to basement membrane

Preliminary immunocytochemical localization The mAb’s TMb-13.1, -13.2, -13.3, and -13.4 re- in dissociated cells showed the antigen to mAb cognized components of the basement membrane

throughout the body. In all cases, staining was limited to a thin line within the dense region of the basement membrane apposed to the proximal re- gion of the epidermal cells.

mAb’s to parenchyma cells

The staining patterns of mAb’s to parenchymal cells (including at least four to six cell types ac- cording to morphological criteria of Bagufia & Romero, 198 1) are bewildering in their complex- ity.

TP- 13.14 stained dorsally and ventrally situ- ated peripheral cells (Fig. 5a); TP-13.12 stained only dorsal cells (Fig. 5b); TP-15.2 stained cells peripheral to the gastrodermis (Fig. 5~); and TP- 7.1 revealed parenchyma cells scattered only within the pharynx and in the pre-pharyngeal and pharyngeal areas (Fig. 5d). Other mAb’s (not shown here) reacted in interesting regional (area) patterns. For example, mAb’s TP-7.5, TP-13.15 and TP-17.10 stained cells located at the very anterior end of the organism. By contrast, mAb’s TP-13.9 and TP-13.19 stained cells located in the

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caudal area, whereas mAb’s TP- 13.8 stained both ends of the body.

Cell-spec$c mAb’s

Although most of the mAb’s already mentioned can be considered cell-specific in that they stained muscle, epidermal, gastrodermal, or specific pa- renchymal cells, there is still a high degree of un- certainty on the specific cell types actually stained. Nevertheless, the mAb’s TM-13.1 for muscle cells (Figs 3, 4), TE-15.4 for specific epidermal cells (Fig. 2), and most parenchyma-specific mAb’s (see Fig. 4) can be considered cell-specific. In ad- dition, the monoclonal TG- 15.3, which selectively stained the club or goblet cells of the gastroder- mis, can be added to the growing list of cell- specific mAb’s detected in planarians.

Discussion

The monoclonal antibodies we have produced to D. tigrina recognize tissue-, cell- and region-

d

“RMX

NERVE CORD NERYE CORD

Fig. 5. Diagrammatic representation of staining patterns of antibodies recognizing some regionally restricted antigens of paren- chymal cells as seen in sagittal sections ofD. tigrina. Darkened areas represent reactive sites. a) mAb TP-13.14; b) mAb TP-13.12;

c) mAb TP-15.2; d) mAb TP-7.1.

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specific antigens in this planarian. To our knowl- edge, this report and Shirakawa’s et al.‘s (this vol- ume) on mAb’s to Phugocatu vividu are the first, albeit at a basic descriptive level, of mAb’s against planarian cells.

These mAb’s and others we are now isolating can be applied in two important ways. First, they may be used as markers of specific tissues and cell types to better characterize, both structurally and functionally, the main cell types in fresh-water planarians. The array of mAB’s to parenchymal cells, for example, may reflect an unexpected functional heterogeneity among parenchymal cells or transient differential functional stages in a sin- gle strain; both possibilities deserve further inves- tigation. Second, those marking regionally re- stricted parts of the intact organism can be used to monitor early stages of pattern formation dur- ing regeneration as well as to monitor the subtle tuning that seems to occur during the processes of growth and degrowth. For example, the mAb TEP-7.1 could be a useful marker to follow the process of differentiation from neoblast to any of the gastrodermal cell types. Also, perhaps the dif- ferential staining of pharyngeal musculature by TM- 13.1 can be related to differences in either the embryonic origin (Hyman, 1951) or the mecha- nisms of renewal of body-wall and pharyngeal muscle cells.

The collection of mAb’s obtained so far in D. tigrina reacts with antigens present in the major tissues and cell types of this organism. However, whereas some tissues and cells have induced a relatively large number of mAb’s (e.g., the highly heterogeneous parenchymal cell types), others, often highly important in functional consider- ations, are under-represented or absent in the repertoire of mAb’s induced. Strikingly absent are mAb’s to components of the nervous system, to the variety of sense organs, to the excretory system, and to specific cell types such as flame cells, rhabdite cells, and, most important to our purposes in studying regeneration, the undiffer- entiated cells or neoblasts.

To obtain mAb’s reacting with the whole ner- vous system or with some specific subsets of nerve cells would be of prime importance to understand

the early stages of pattern formation during head regeneration. Indeed, head determination has been shown to be a very early process (within 6 to 24 h of wounding) involving commitment of new nerve cells that will form the cerebral ganglia (Sal& 1984). Hence, the finding of specific mAb’s to nerve cells would be a key to defining the early stages of head formation before overt differenti- ation and morphogenesis takes place, as has been done with the process of hypostome determina- tion in hydra (Javois, 1990).

The failure to find mAb’s reacting specifically to neoblast antigens was rather unexpected as this type of cell represents 20-30x of the total cell population, depending on body size, temperature, and nutritional conditions (Bagufia & Romero, 1981). Several arguments can be advanced to ex- plain this failure. First, though the neoblast is the most numerous cell type in intact and regenerat- ing planarians, it represents a mere 4-6% of total body volume (BaguAa, 1973); therefore, its spe- cific antigens, if any, could be diluted in a total homogenate. Second, neoblasts are simple cells bearing only a small amount of cytoplasm; hence, even when enriched fractions of neoblasts are used as homogenates (having 15-20% contami- nating cells, some of them parenchymal cells) neoblast-specific antigens, if any, may still be under-represented. Third, the main screening method used here, staining of paraffin sections after paraformaldehyde fixation, is inadequate to single out scattered small cells within a mass of parenchymal tissue made of different cell types with complex and interdigitating morphologies. Finally, the present failure may be a consequence of a common misconception; this is to consider neoblasts as a homogeneous population of cells instead of a heterogeneous class comprising a few totipotent stem-cells and different subpopulations of cells progressively committed along different pathways of differentiation (Baguna et al., 1990). If the latter is true, neoblast-specific antigens will be even harder to find.

To overcome this impasse, we are making a renewed effort to obtain pure populations of neo- blasts by filtering them through nylon meshes with further purification using Ficoll or sucrose gradi-

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ents to rid them of membranes, vesicles, and de- bris of contaminating differentiated cells. In ad- dition, the detection of 7 neoblast-specific polypeptides out of 850 by two-dimensional elec- trophoresis (Collet, 1990) may make it possible, using current methods of extraction and purifica- tion of polypeptides from two-dimensional gels, to obtain antibodies against them, Finally, a promising approach that can be entertained is screening of cDNA libraries made from enriched populations of neoblasts and differentiated cells; cDNAs specific to the neoblast could be obtained in this way.

Acknowledgements

This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS 87/ 1533 and 88/0968) to J.B.

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