&p.1:Abstract To obtain specific immunological probes forstudying molecular mechanisms involved in cell renew-al, cell differentiation, and pattern formation in intactand regenerating planarians, we have produced a hybri-doma library specific for the asexual race of the fresh-water planarian Dugesia (Girardia) tigrina. Among the276 monoclonal antibodies showing tissue-, cell-, cellsubtype-, subcellular- and position-specific staining, wehave found monoclonal antibodies against all tissues andcell types with the exception of neoblasts, the undiffer-entiated totipotent stem-cells in planarians. We have alsodetected position-specific antigens that label anterior,central, and posterior regions. Patterns of expression un-covered an unexpected heterogeneity among previouslythought single cell types, as well as interesting cross-re-activities that deserve further study. Characterization ofsome of these monoclonal antibodies suggests they maybe extremely useful as molecular markers for studyingcell renewal and cell differentiation in the intact and re-generating organism, tracing the origin, lineage, and dif-ferentiation of blastema cells, and characterizing thestages and mechanisms of early pattern formation.Moreover, two position-specific monoclonals, the firstones isolated in planarians, will be instrumental in de-scribing in molecular terms how the new pattern unfoldsduring regeneration and in devising the pattern forma-tion model that best fits classical data on regeneration inplanarians.&bdy:

Introduction

Freshwater planarians (Platyhelminthes, Turbellaria, Tri-cladida) have been used as a model system to study pat-tern formation because they offer four main advantages:(1) they have a simple three-layered flattened structure

with marked antero-posterior and dorso-ventral polari-ties; (2) they are in a continuous state of cell renewalwith a single population of proliferating multipotentstem-cells, or neoblasts, which give rise, by differentia-tion, to 12–15 non-proliferating short-lived differentiatedcell types that are continuously replaced during the lifetime of the individual; (3) they may grow or degrow de-pending on the daily balance between cells born by cellproliferation and cells lost by cell lysis and death; and(4) they are able to regenerate the entire animal from anexcised piece of any region of the body (for general re-views, see Brøndsted 1969; Baguñà et al. 1990, 1994).

However, despite these advantages, the number ofmolecular markers for specific tissues, cell types or posi-tions is rather limited: neurotransmitters (serotonin, ace-tylcholinesterase, dopamine) and neuropeptides (FMRF-amide, neuropeptide F, small cardiac peptide, substanceP, and Met and Leu-enkephalin among others) for nervecells (summarized in Gustafsson et al. 1986; Reuter et al.1988; Fairweather and Skuce 1995; Reuter and Gustafs-son 1995) melanin and tyrosinase for pigment cells (Pal-ladini et al. 1979a, b); actin for muscle cells (Pascolini etal. 1992); acid and alkaline phosphatase and some neuro-peptides for intestinal cells (Reuter and Palmberg 1987);eosinophilic and cyanophilic granules for secretory cells(Pedersen 1959a, 1961); rhabdite granules for epithelialcells (Hori 1978); and fibronectin, laminin and collagenIV for basal membrane and the extracellular matrix (Li-ndroos and Wikgren 1987). Moreover, these cytologicaland histochemical markers have, in general, not beenused for studies of cell replacement, cell differentiationor pattern formation. To trace the lineage of cells duringcell renewal in the intact organism or the lineage of blas-tema cells during regeneration, molecular markers ofspecific cell types are clearly needed. In addition, to es-tablish how the lost pattern is first reset and how newstructures and organs appear during regeneration, posi-tion-specific markers of such regions or organs are alsoclearly needed. In both processes, this would allow visu-alization of the pattern or the cell type before morpho-genesis and overt differentiation takes place.

D. Bueno (✉) · J. Baguñà · R. RomeroDepartament de Genètica, Facultat de Biologia,Universitat de Barcelona, Av. Diagonal 645, E-08071 Barcelona,Catalonia, SpainTel. +34-3-4021500 (4021498); Fax +34-3-4110969&/fn-block:

Histochem Cell Biol (1997) 107:139–149 © Springer-Verlag 1997

O R I G I N A L PA P E R

&roles:David Bueno · Jaume Baguñà · Rafael Romero

Cell-, tissue-, and position-specific monoclonal antibodiesagainst the planarian Dugesia (Girardia) tigrina

&misc:Accepted: 16 September 1996

Our approach has been to isolate monoclonal antibod-ies (mAbs) and use them as markers to study the mecha-nisms underlying these biological processes. mAbs arepowerful tools in developmental studies as: (1) they ex-hibit a high degree of sensitivity and specificity allowingthe detection of small differences between otherwiseidentical cells; (2) they can serve as early markers of celldetermination and differentiation which may give cluesabout transitional stages prior to overt differentiation, aswell as to point out lineage relationships between differ-ent cell populations; (3) they allow the detection andcharacterization of topographically restricted antigensand the visualization of the earliest stages of pattern for-mation before morphological features become evident;and (4) they allow the detection and isolation of un-known substances related to these processes startingfrom complex mixtures of unidentified antigen prepara-tions. mAbs have been used successfully in several de-velopmental systems. Among them we could mentionthe works of Fujita (1987) and Go et al. (1992) in Dro-sophila; Dunne et al. (1985), Heimfeld et al. (1985), Lit-tlefield et al. (1985), Javois et al. (1986), and Bode et al.(1988) in Hydra; Mita-Miyazawa et al. (1987) on ascidi-ans; Wang et al. (1992) on cockroach; Itoh et al. (1988)on Xenopus; and Goldhamer et al. (1989) and Klatt et al.(1992) on newt.

In this paper we describe several antibodies whichspecifically recognize cells and tissues in intact and re-generating planarians. These antibodies are of interest astools to provide new insights into planarian anatomy, his-tology, and patterning mechanisms during the processesof growth/degrowth and regeneration.

Materials and methods

Species and culture conditions

The planarians used belong to an asexual race (class; Ribas et al.1989) of the species Dugesia (Girardia) tigrinacollected nearBarcelona. They were maintained in spring water in the dark at4–6° C and fed once a months with beef liver. Specimens chosenfor immunization and immunocytochemical procedures were shift-ed to 22±1° C and starved for 15 days before use.

Antigen preparation and immunization

We used three different kinds of immunogens: (1) homogenatesobtained from sonicating intact organisms (whole organisms ordifferent regions) or regenerative blastemas in 100 mM phosphate-buffered saline, pH 7.2 (PBS); (2) total cells obtained by macera-tion (Baguñà and Romero 1981) washed in PBS, or by dissociatonin calcium-magnesium-free medium (CMF; Baguñà 1973); and(3) enriched fractions of neoblasts obtained by filtration throughnylon meshes of macerated or dissociated cells (Baguñà et al.1989). In the first two cases, 20 organisms, 7-mm-length, or 5×106

cells in 1 ml PBS or CMF were used, respectively; in the third, 107

neoblasts (purity >80%) in 1 ml PBS or CMF were used. The im-munogens (100 µg homogenate or 106 cells) were injected intra-peritoneally into 8-week-old Balb/C female mice. Injections wererepeated twice, each at 2-week intervals.

Production of monoclonal antibodies

Four days after the last injection, the mouse’s spleen was removed,dissociated, and centrifuged with mouse myeloma NS1 cells, andfused in the presence of polyethylene glycol (PEG; Galfre et al.1977; Harlow and Lane 1988). After selection in HAT medium(hypoxanthine-aminopterine-thymidine), hybridomas producingIgG antibodies were screened using an enzyme linked immunosor-bent assay (ELISA; Miles and Hales 1968a, b) and their specifici-ty tested by immunocytochemical procedures. Hybridomas pro-ducing the antibody of the desired specificity were cloned by lim-iting dilution, amplified, and stored in liquid nitrogen.

Immunocytochemical procedures

Hybridomas were screened for the production of mAbs using indi-rect immuno-fluorescence and the peroxidase-linked avidin-biotincomplex (ABC) method on macerated cells and on paraffin sec-tions.

Paraffin sections

Immunostained paraffin sections were obtained as described inRomero et al. (1991). In brief, intact and regenerating organismswere fixed in 4% paraformaldehyde in PBS (pH 7.4) for 3 h. Afterwashing several times in PBS, they were embedded in 51–53° Cparaffin, cut at 10-mm saggital or transverse sections, and mount-ed on gelatin-coated slides. After deparaffination in xylene and re-hydration through an ethanol series, they were rinsed in PBS andpermeabilized in 0.4% pepsin (Sigma) in 0.1 N HCl (pH 5.0) for15 min at 37° C. After a rinse in PBS, they were blocked by incu-bation in 5% powered defatted milk (Molico, Nestlé) in PBS for 1h at room temperature. They were then incubated in a 1:1 hybrido-ma culture supernatant-PBS solution overnight at 4° C and rinsedin PBS for 48 min (four rinses of 12 min each). The presence oflinked antibody was detected with the peroxidase-conjugated ABCmethod (Vector). After five rinses in PBS (12 min each), antibod-ies were revealed with diaminobenzidine tetrahydrochloride(DAB, Sigma) and the sections rinsed again, dehydrated, andmounted in DPX.

Macerated cells

Planarians were cut in small pieces and dissociated into singlecells in maceration fluid (1:1:13, acetic acid:glycerol:PBS; Bueno1994) for 12 h at room temperature. Macerated cells were distrib-uted dropwise onto microscopic slides, settled for 1–2 h, and fixedwith 4% paraformaldehyde for 1 h. The slides were then washedin PBS for 30–60 min and permeabilized in 0.1% pepsin (Sigma)in 0.1 N HCl (pH 5.0) for 15–30 min at 37° C. Further steps weresimilar to those described for paraffin sections. Slides weremounted in 50% glycerol in PBS.

Nomenclature

We named the mAbs according to a code with two or three letters(the first representing the species, the remainder representing thearea, tissue or cell type) and one or two numbers (the first indicat-ing the number of the fusion and the second the ordinal numberspecific to the area, tissue or cell type within the fusion). For ex-ample, TEPI-15.5 codes for a mAb to D. tigrina (T) epidermis(EPI) which was the fifth specific to epidermal cells to be obtainedin the 15th fusion. The second (ordinal) number was not usedwhen only one mAb with that specificity was obtained in a fusion.

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Results

In cellular terms, freshwater planarians can be consid-ered to be composed of two main compartments: (1) afunctional one, made of 12–15 non-proliferating differ-entiated cell types (approx. 75–80% of the total numberof cells) that are continuously replaced during the lifetime of the individual; and (2) a proliferative compart-ment formed by a single, though heterogeneous, class ofsmall, undifferentiated, totipotent stem-cells (the so-called neoblast; approx. 20–25% of the total cells).These cells give rise to all differentiated functional celltypes, while maintaining their own density by cell prolif-eration (Lange 1968; 1983; Baguñà 1981; Baguñà andRomero 1981; Romero and Baguñà 1991). When tem-perature and food are kept at the optimum, planarians re-main in steady-state since a balance between the numberof cells (neoblasts) produced by proliferation and thenumber of cells lost from the functional compartment ex-ists (Romero 1987). When any of these parameters arefar from optimum, growth or degrowth ensues.

To obtain specific immunological markers to charac-terize the specific cell types and subtypes, and for study-ing molecular mechanisms involved in cell determina-tion/differentiation and cell renewal in the intact and re-generating adult planarian, we immunized mice with to-tal cell or neoblast-enriched cell fractions. Hybridomacell lines were produced and hybridoma culture fluidsamples screened by immunofluorescence microscopy orwith the ABC method. From over 50 successful fusions,a library of 276 mAbs with region-, tissue-, cell- or cellsubtype-specific staining was obtained. All mAbs of thelibrary belong to the IgG class as they were selected bythe ELISA method using an anti-IgG antibody.

Epidermis-specific mAbs

The outermost part of a planarian consists of a layer ofepidermal cells laid over a basement membrane (Fig. 1).The ventral epidermis is ciliated, the dorsal sparsely so.Dorsal epidermal cells bear characteristic cigar-shapedcytoplasmic bodies or rhabdites, whereas only a few ven-tral cells bear them. As summarized in Table 1, from 14epidermis-specific hybridoma clones five, TEPI-15.6,TEPI-30.1 (Fig. 2A, C), TEPI-17.1 (Fig. 2B, D), andTEPI-15.4 (Fig. 2E, F) were of particular interest. Thefirst two stain the whole epidermis, the latter two its dor-sal (TEPI-17.1) or ventral (TEPI-15.4) part. Most anti-bodies stain all epidermal cells, though others (e.g.,TEPI-15.4) show a patchy staining (Fig. 2E, F). ThismAb stains scattered single cells and clusters of three tofive epidermal cells; the antigen seems located at the api-cal surface of the cell (Fig. 2E). In contrast to the otherantibodies that only stain epidermal cells, TEPI-30.1 alsostains rhabdite cells (Fig. 2A). These are parenchymacells in the process of differentiating from neoblasts intoepidermal cells (Skaer 1961; Lentz 1967).

Endoderm (gut)-specific mAbs

The gut in planarians is blind, consisting of a highlyramified epithelium (Fig. 1) formed by two cell types:the gastrodermal or digestive cell and the goblet (granu-lar, club or Minot) secretory cell type. Thirteen mAbsstained specifically both components of the gut, and an-

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Fig. 1 Diagram summarizing the basic structure and morphologyof an asexual specimen of Dugesia (Girardia) tigrina. A Dorsalview showing the brain and the nerve cords (black) and the threemain branches of the gut (gray). B Sagittal section. Anterior is tothe left and dorsal at the top. (b Brain ganglia, e eye, g gut diver-ticulum, n longitudinal ventral nerve cord, ph pharynx)&/fig.c:

Table 1 Summary of the number, relative percentage, and speci-ficities of the monoclonal antibodies obtained in this work&/tbl.c:&tbl.b:

Specificity Number Percentage

General (all 8 2.9tissues)Parenchyma 13 4.7+epidermisCo-expressed 28 10.1in twocell typesEpidermis 14 5.0Gastrodermis 13 4.7Musculature 37 13.4Nervous 5 1.8systemSecretory cells 8 2.9(acidophilic)Secretory cells 81 29.3(cyanophilic)Goblet cells 11 4.0Pharynx 2 0.7epitheliumFlame cells 4 1.4Pigmentary cells 1 0.4Fixed paren- 3 1.1chyma cellsSecretory cells 25 9.0(unclassified)Nuclei 7 2.5Basement 14 5.0membraneRegional 2 0.7Total 276 100

&/tbl.b:

other 11 were found to be specific for goblet cells. NomAb specific to gastrodermal cells has been produced.The mAb TG-42 (Fig. 3A) stains both cell types withinthe gut throughout the body. The goblet-specific TGLO-13 (Fig. 3B, C) stains goblet cells throughout the gastro-dermis, but does not stain digestive cells. In both cases,these mAbs label cytoplasmic material around phagocyt-ic or excretory vacuoles, which are unlabelled. Somecross-reaction exists between several gut-specific mAbsand other body regions: goblet/dorsal epidermis (TGOB-23.3), whole gut/epidermis (TG-15.2, TG-53.1, TG-53.2,TG-53.3), whole gut (nerve cells (TNER-47), as well aswith several parenchyma secretory cells.

Muscle-specific mAbs

Muscle cells in planarians are all mesodermally ar-ranged, lying entirely beneath the epidermal cell layer.Muscle cells are 150–200 µm long and 5–10 µm wide(Baguñà and Romero 1981), and each muscle fiber ismade of several tandemly aligned cells. Muscle fibersare disposed in circular, longitudinal, and diagonal ar-rays. Within the mesoderm or parenchyma run dorso-ventral fibers and transverse fibers. In addition to longi-tudinal and circular fibers, specific organs such as thepharynx have arrays of radial fibers that connect the in-ner and outer epithelia. We have produced 37 muscle-specific mAbs. The bulk of them, such as TMUS-13, la-bel all muscle cells (Fig. 4A, B). They reveal unexpect-

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Fig. 2A–F Epidermal-specificmonoclonal antibodies (mAbs)in D. tigrina. Epithelial cellslabelled with the mAbs TEPI-30.1 (A, C all epidermal andrhabdite cells), TEPI-17.1(B, D dorsal epidermal cells),and TEPI-15.4 (E, F ventralepidermal cells). Sagittal paraf-fin sections; anterior is to theleft, dorsal at the top (except inF). A TEPI-30.1 labelling epi-dermal and rhabdite cells(arrows). B TEPI-17.1 label-ling dorsal epidermal cells.C Magnification of A showinglabelled rhabdite cells (r).D Magnification of TEPI-17.1labelling the dorsal epidermalcells. E TEPI-15.1 labellingsingle and clustered (arrow-heads) ventral epidermal cells.F tangential section at the ven-tral epithelium labelled withthe mAb TEPI-15.1 showingtheir patchy staining(arrowheads). (g Gut diverticu-lum, p parenchyma, r rhabditecells) BarsA, B 1 mm, C, E, F0.1 mm, D 0.5 mm&/fig.c:

Fig. 4A–E Muscle-specificmAbs in D. tigrina musclecells labelled with the mAbsTMUS-13 (A, B all musclecells) and TMUS-46 (C–E lon-gitudinal muscle cells). Sagittalparaffin sections. Anterior is tothe left and dorsal at the top.A TMUS-13 labelling the phar-ynx and the pre-pharyngeal re-gion. B Magnification of thepre-pharyngeal region in Ashowing the longitudinal, circu-lar, and dorso-ventral (long ar-row) muscle fibers. Note thefine fibes wrapping the gut di-verticula (arrowhead). Dorsalto the upper left, ventral to thelower right. C Longitudinalmuscle fibers labelled with themAb TMUS-46. D Longitudi-nal muscle fibers in the head la-belled with the mAb TMUS-46. E Magnification of D (eEye, g gut diverticulum, phpharynx) BarsA, C 1 mm,B 0.25 mm, D 0.5 mm&/fig.c:

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Fig. 3A–C Endoderm (gut)-specific mAbs in D. tigrina.Gut cells labelled with themAbs TG-42 (A gastrodermaland goblet cells) and TGLO-13(B, C goblet cells). Sagittalparaffin sections. Anterior is tothe left and dorsal at the top.A Gut diverticula stained withTG-42. B Goblet cells (arrow-heads) labelled with the mAbTGLO-13. Dashed lineindi-cates the outer boundary of thegut. C Magnification of Bshowing labelled goblet cells.(g Gut diverticulum)BarsA 0.5 mm, B 0.25 mm,C 0.1 mm&/fig.c:

edly fine fibers which seem to wrap the gut diverticula(Fig. 4B) as well as to travel within the parenchyma inall directions. TMUS-13 has a clear cytoplasmic loca-tion, the nuclei being unstained. The epitope recognizedby TMUS-13 is specific to organisms of the phylumPlatyhelminthes (data not shown). Two muscle-specificmAbs, TMUS 46.1 and TMUS-46.2, have more restrict-ed specificities, only labelling the longitudinal fibersboth of the body wall and of the pharynx (Fig. 4D–F).TMUS-13 and TMUS-46.2 also label muscle precursorcells (myoblasts) during regeneration.

Nerve-specific mAbs

Planarians belong to the most primitive metazoan phy-lum, exhibiting true bilateral symmetry and a bilaterallysymmetrical central nervous system (CNS). This consistsof an anterior concentration of nerve cells (“the brain”)and one or several pairs of longitudinal nerve cords. InD. tigrina the single pair of longitudinal nerve cords areconnected by commisures in a ladder-like configuration.The peripheral nervous system (PNS) consists of a mesh-work of nerve fibers and bi- or multipolar nerve cellsforming nerve plexuses interconnected with the CNS.We have produced five specific mAbs against the ner-vous system (see Table 1). These mAbs stain all nervecells and fibers of the CNS (see TNER-46, Fig. 5A, B),as well as the sub-muscular plexuses of the body and thepharynx. A fifth nerve-specific monoclonal, TNER-47,also stains the gastrodermis (Fig. 5C).

Parenchyma-specific mAbs

The so-called planarian parenchyma or mesenchyme is asolid mass of tissue that fills the space between the mono-

stratified ciliated epidermis, the gut, and the internal or-gans. It consists of several, highly polymorphic, non-mitot-ic, differentiated cell types and a particular class of undif-ferentiated, mitotic, and multipotential stem cells (neo-blasts). The main class of differentiated cells are secretorycells which, on the basis of their staining patterns, havebeen traditionally classified as acidophilic or cyanophilic.We have produced eight antibodies against acidophiliccells and 81 against cyanophilic cells. Whereas the first donot show regional specificities, the latter are more complexthan expected, 59 of them showing several different region-al specificities. Figure 6A shows the staining pattern ofTCIAN-9 which labels a group of dorsal and ventral secre-tory cells located in the parenchyma of the pre-pharyngealregion and in the pharynx. In contrast, TCIAN-23 (Fig.6B) labels a group of secretory cells at both anterior andposterior ends of the body. Whereas at the head these cellssurround the bain and have a central-ventral position, thoseat the tail have mainly dorsal and ventral distribution. Oth-er mAbs (not shown here) have other interesting regionalpatterns. For example, TCIAN-34, TCIAN-7.5, TCIAN-13.15, and TCIAN 17.10, among many others, only stainthe head in a pattern similar to that of TCIAN-23. In con-trast, TCIAN-13.9 and TCIAN-13.19 only stain secretorycells of the tail. Moreover, TCIAN-13.7 stains only cellslocated at the dorsal parenchyma, TCIAN-7.4 and TCIAN-17.1 those at the ventral side, and TCIAN-15.2 some secre-tory cells clustering around the gut diverticula.

mAbs against extracellular components

Fourteen mAbs have been found to identify extracellularmaterials, namely the basement membrane. For example,the antigen identified with TMB-13.1 (Fig. 6C) stains themembrane that separates the epidermis from the underly-ing muscular layers.

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Fig. 5A–C Nervous system-specific mAbs in D. tigrina.Nerve cells and processes la-belled with the mAbs TNER-46(A, B) and TNER-47 (C, alsolabelling the gut). Sagittal par-affin sections. Anterior is to theleft and dorsal at the top.A TNER-46 labelling the brainganglia (b) and the longitudinalventral nerve cords. Note theimmunostaining in the pharyn-geal plexuses (arrowhead).B Magnification of A. Note la-belling in the dorsal plexus (ar-rowhead) and in a dorso-ven-tral nerve fiber (arrow).C TNER-47 labelling the lon-gitudinal ventral nerve cord,dorsal plexus (arrowhead), andgut. (b Brain ganglia, e eye; phpharynx) BarsA 1 mm,B, C 0.5 mm&/fig.c:

Nuclear- and DNA-specific mAbs

Seven mAbs labelled the nuclei of all cells throughoutthe body (Fig. 6D). One of them, TNU-46, was studiedfurther. Planarian homogenates digested with proteasesor DNases were fixed onto nitrocellulose membranes and

stained with TNU-46. DNase-treated homogenates gaveno reaction, whereas protease-treated homogenates gavesimilar, or even stronger, labelling levels as untreatedcontrols. This means that the antigen is DNA. Indeed,DNAs of different sources (planarian Dugesia (S) medit-erranea, Drosophila melanogaster, salmon sperm, hu-

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Fig. 6 Secretory cells labelledwith the mAbs TCIAN-9 (A)and TCIAN-23 (B), basementmembrane labelled with themAb TMB-13.1 (C), and nucleilabelled with the mAb TNU-46(D). Sagittal paraffin sectionsof D. tigrina. Anterior is to theleft and dorsal at the top, exceptin D. A TCIAN-9 immuno-staining in cyanophilic secreto-ry cells of the pre-pharyngealand pharyngeal regions.B TCIAN-23 immunostainingin cyanophilic secretory cells atthe cephalic and caudal re-gions. Note immunoreactivecells wrapping the brain gan-glia (arrow). C Ventral base-ment membrane labelled withthe mAb TMB-13.1. D DNAimmunostaining in the nuclei(arrowheads) with the mAbTNU-46. Anterior is to the topand dorsal to the left. (e Eye, pparenchyma, ph pharynx) BarsA, B 1 mm, C, D 0.25 mm&/fig.c:

Fig. 7A, B Regional-specificmAbs in D. tigrina. Sagittalparaffin sections labelled withthe mAbs TCEN-49 (A centralarea) and TNEX-59 (B anteriorand posterior areas). Anterior isto the left and dorsal at the top.Arrows indicate the boundariesof these molecular regions inboth A and B. (e Eye, ph phar-ynx) Bars1 mm&/fig.c:

man, and lambda phage) are all labelled with TNU-46.The antigenic nature of the nuclear-specific mAbs otherthan that of TNU-46 has not been studied.

Region-specific mAbs

We have isolated two mAbs which label specific areas ofthe body. One mAb, TCEN-49, detects an antigen thatlabels all cells from the cental area of the body with theexception of epidermal cells (Fig. 7A). Anterior (head)and posterior (tail) cells are not stained. Labelled cellsare not related by lineage but by position. The othermAb, TNEX-59, has an apparently complementary la-belling to TCEN-49 as it stains the nuclei of all cells inthe cephalic and caudal regions with a distal-proximalgradient in intensity (Fig. 7B), as well as all epidermalcells. Again, cells within these regions are not related bylineage.

Discussion

Extent and features of the collection

The objective of this study was to establish a hybridomalibrary specific for cells, tissues, organs, and regions ofplanarians, in particular of the asexual race of D. tigrina.As described, we have obtained 276 mAbs, fulfilling thisobjective (Table 1). So far, almost all differentiated celltypes and tissues are recognized by at least three mAbs.The largest set is formed by those labelling the so-calledparenchyma secretory cells, namely the cyanophiliccells. These cells, loaded with huge numbers of secretorygranules filled with mucopolysaccharides and glycopro-teins, are highly immunogenic. The next set is formed bythose reacting with the muscle system which is alsohighly immunogenic, followed by mAbs against epider-mis and gastrodermis. Other tissues and cells, such as thenervous system (five mAbs), pigment cells (one mAb),flame cells (four mAbs), and fixed parenchyma cells(three mAbs) have been, in our experiments, much lessimmunogenic. The only cell types for which no mAbsare available are the sex cells and the undifferentiatedcells or neoblasts.

Whereas we could not expect to find mAbs againstsex cells from asexual organisms, the absence of mAbsreacting specifically to neoblast antigens was rather un-expected as this type of cell represents 20–30% of the to-tal cell population, depending on body size, temperature,and nutritional conditions (Baguñà and Romero 1981).Several arguments could be advanced to explain our in-ability to identify mAbs against neoblasts. First thoughthe neoblast is the most numerous cell type in intact andregenerating planarians, it represents a mere 4–6% of to-tal body volume (Baguñà 1973); therefore, its specificantigens, unless they are highly antigenic, would be di-luted in a total homogenate. Second, neoblasts are simplecells bearing only a small rim of cytoplasm, hence, even

when enriched fractions of neoblasts are used as homog-enates (actually, having 15–20% contaminating cells,some of them parenchymal cells), neoblast-specific anti-gens, if any, may still be underrepresented. Finally, ourinability to identify mAbs against neoblasts may be aconsequence of a common misconception: this is to con-sider neoblasts as a homogeneous population of cells in-stead of a heterogeneous class comprising a few totipo-tent stem-cells and different subpopulations of cells pro-gressively committed along different pathways of differ-entiation (Baguñà et al. 1990). If the latter is true, neo-blast-specific antigens will be even harder to find.

More than half of our hybridoma library recognizesdifferent subsets or cell subtypes, disclosing an unex-pected heterogeneity within apparently homogeneouscell types according to histochemical criteria. Conven-tional histological techniques (Pedersen 1959a, b, 1961)and quantitative maceration methods (Baguñà andRomero 1981) list, at the most, 20 different cell types inplanarians. Ultrastructural studies, however, have report-ed more than seven different classes of secretory gran-ules within the so-called secretory cells of the pharynx(Pascolini et al. 1971; Baguñà 1973), at least two typesof cyanophilic and acidophilic parenchyma secretorycells, several types of neurons and the presence of glialcells (Golubev 1988), and a large variety of epithelialcells. The finding of 59 mAbs which label different butoverlapping sets of cyanophilic cells was unexpected.The head, the pre-pharyngeal region, and the pharynx arethe regions showing the most diversity. This may reflectan unexpected functional heterogeneity among parenchy-ma secretory cells or transient differential functionalstages in a single or a few cell types or subtypes. Secre-tory cells in planarians have cell bodies within the paren-chyma and long and branched processes usually project-ing to the epidermal surface. They make up 8–20% ofthe total cells of the body (Baguñà and Romero 1981),are organized in different sorts of gland types or lie scat-tered throughout the body, and their products serve sev-eral functions, among others, adhesion, secreting slimefor locomotion, or as a protective coating. A substantialnumber of them, however, release their contents withinthe parenchyma (Baguñà 1973), their function being un-known. The complexity of secretory cells in planariansdiclosed by our hybridoma library deserves further in-vestigation.

Of the total mAbs, 10% showed cross-reactivity withtwo different tissues or cell types, and an additional 7%labelled all cell types or a substantial number of them.Several antibodies belonging to all the different cross-re-activity groups were brought to monoclonality. Hence,cross-reactivity could not be due to the presence of twoor more antibodies in the hybridoma supernatants ana-lyzed. Instead, conservation of a common epitope (eitherconformational or sequential) in an otherwise different(or similar) molecule or expression of the same moleculein two different cell lineages may explain such cross-re-activity. Gastrodermis, namely goblet cells, is the tissuewith the highest cross-reactivity as it co-labels with epi-

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dermis, fixed parenchyma cells, secretory cells, nervecells, and epithelial cells of the pharynx. Whereas cross-reactivity between gastrodermis and epidermis may reston their common “epithelial” features and in similar pro-cesses of absorption (by microvilli) and secretion, thatbetween goblet cells and nerve cells may depend on thepresence of common neuropeptides and neurotransmit-ters (Reuter and Gustafsson 1989). Immunoreactivityagainst bovine pancreatic peptide (BPP), FMRFamide,and vasotocin-like peptide have been demonstrated ingoblet cells of several turbellarian orders (Reuter andPalmberg 1987). More difficult to understand are fea-tures in common between gastrodermis and the so-calledfixed cells and secretory cells of the parenchyma. In con-trast, five mAbs labelled the gastrodermis as well as theepithelium of the pharynx, whereas only two mAbs la-belled specifically the epithelium. Actually, this epitheli-um has both absorptive and secretory functions and itsinner proximal part is structurally very similar to gastro-dermal cells (Baguñà 1973; Bowen and Ryder 1973).

Applications of the hybridoma collection to celldifferentiation and pattern formation in planarians

Planarians have two features that make them ideal mod-els in developmental biology. First, they can grow anddegrow depending on size, temperatue, and food avail-ability (Baguñà and Romero 1981). Second, they can re-generate the entire animal from an excised piece of anyregion of the body (for general reviews, see Brøndsted1969; Baguñà et al. 1990, 1994). Quantitative cellularanalysis has shown that growth or degrowth depends onthe daily balance between cells born by cell proliferationand cells lost by cell lysis and death (Romero andBaguñà 1991). This means that adult planarians are in acontinuous state of cell renewal. Several lines of evi-dence indicate that the neoblasts of the intact organismgive rise to blastema cells during regeneration (Baguñàet al. 1989). Hence, both in growth-degrowth and regen-eration, cell differentiation processes are always active inplanarians. Moreover, during regeneration, lost structuresare determined in precise temporal and spatial sequenceswithin the blastema and post-blastema well before theybecome morphologically visible (Saló 1984; Baguñà etal. 1994).

The hybridoma collection reported here can be usedto address some of the problems posed by growth, de-growth, and regeneration. First, cell type-specific mAbs(e.g., those labelling nerve cells, muscle cells, epidermalcells) may be used to monitor, both in intact and regener-ating organisms, when and where precursors of specificcell types first appear and, in th case of regenerants, tomonitor their sequence of differentiation within the newtissues (distal-proximal or proximal-distal sequence).TMUS-13, labelling all muscle cells, has been used tomonitor the presence of myoblast-like cells in intact or-ganisms and to detect any differences of myoblast differ-entiation along anterior, central, and posterior areas of

the body. The results show the presence of such cells insimilar proportions (1 myoblast/90–100 mature musclecells) in heads, tails as well as in the central body area(Bueno 1994). In addition, TMUS-13 has been used tomonitor the appearance and differentiation of myoblastsin the regenerating pharynx (Espinosa 1993; D. Bueno etal., submitted). It is presently being used, jointly with thelongitudinal muscle-specific TMUS-46.2 to study howthe new pattern of the body wall muscles appears withinthe blastema and, most importantly, howe the new andold muscle patterns interconnect (F. Cebrià, D. Bueno, J.Baguñà, and R. Romero, work in progress). A further ex-ample of how mAbs of this collection may be used tostudy cell renewal and differentiation is by using TEPI-30.1 as a differentiation marker of dorsal epidermal cellsas it also labels the rhabdite cells (parenchyma cells inthe process of differentiating from neoblasts into epider-mal cells; Skaer 1961; Lentz 1967; Hori 1978).

The second and, to us, main application of mABs isas markers of specific areas or regions along the anterior-posterior and dorsal-ventral axes during regeneration.This allows us to assess when specific regions are deter-mined well before specific morphological features ap-pear. In particular, information on the specific order ofdetermination of regions, monitored by the labelling ofspecific mAbs, will be instrumental for knowing whetherthe order of appearance of proximal-distal or distal-prox-imal and to devise the best model of pattern formation inplanarians. In this context, two of the mAbs obtained inthe planarian D. tigrina, are particularly relevant. OnemAb, TCEN-49, detects an antigen, TCEN-49 Ag, whichis expressed in the central area of the body (Fig. 7A).The other mAb, TNEX-59, stains all nuclei in the headand tail regions in a pattern complementary to TCEN-49(Fig. 7B). Changes in TCEN-49 Ag expression havebeen monitored during both anterior and posterior regen-eration at different axial levels, varying in agreementwith a molecule conferring postitional identity (Bueno etal. 1996). In addition, the kinetics of fading and reex-pression of TCEN-49Ag agree with a recent version ofthe reaction-diffusion model (Meindhardt 1993), thatpictures pattern formation as a hierarchical chain of localactivators and inhibitors spreading from distal to proxi-mal regions. As TNEX-59 has a complementary patternof expression to TCEN-49 and TCEN-49-labelled cellsare not related by lineage but by position, TNEX-59 anti-gen may confer positional identity to both head and tailareas. It will be very interesting to examine the changesin its staining pattern in a set of regeneration experi-ments similar to those made with TCEN-49 (Bueno et al.1996), as well as to perform double labelling experi-ments with both antibodies (D. Bueno, work in pro-gress). A development of this work will be the cloning ofthe genes coding for TCEN-49 and TNEX-59 antigens,as well as other interesting mAbs reported here, usingexpression libraries (R. Romero et al., work in progress).

&p.2:Acknowledgements We would like to thank Lluis Espinosa, Edu-ard Batlle, and Marc Aureli Soriano for their invaluable help and

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technical expertise in monoclonal antibody screening and immu-nocytochemical work, and Phillip Newmark for helpful discus-sion. This work was supported by grants from the Comisión Ases-ora de Investigación Científica y Técnica (Ministerio de Edu-cación y Ciencia, España; PB89-0249 and PB 92-0532) to J.B..

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