THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275300-307 (1996)

Incorporation of Bromodeoxyuridine in Regenerating Fin Tissue of the Goldfish C a r a s s i u s a u r a t u s

c ~ . ~ . SANTAMARIA, M. MARI-BEFFA, L. SANTOS-RUIZ, AND J. BECERFU Department of Cell Biology and Genetics, Faculty of Science, University of iklalaga, 29071 Malaga, Spain

ABSTRACT We have investigated the pattern of incorporation of 5-bromo-2'-deoxyuridine-5'- monophosphate (BrdU) by proliferating cells during regeneration of the tail fin of Carassius aura- tus. Fifteen days after amputation, intraperitoneal injection of a single dose of 0.25 mglg wet weight of BrdU and subsequent immunocytochemical detection on sections revealed groups of replicating cells in the blastema and epidermis a t different proximodistal levels. Proliferating blastemal cells were confined to a crowded, compact distal area that lost its replicative ca- pacity laterally, causing the differentiation of scleroblasts, which synthesize the lepidotrichia hemisegments. Proximally, but centrally located, the blastemal cells did not incorporate BrdU and they differentiated giving rise to the mature intraray connective tissue. An independent cell-pro- liferation process was noted in the epidermis. The distal cap did not proliferate; the lateral faces of the epidermis showed high rates of cell replication in the central layer at every level of the regener- ate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorpo- rate BrdU when actinotrichia were present. The possible role of basal epidermal cells in the synthesis of actinotrichia, the contribution of these collagen macrofibrils to the morphogenetic process, and the different pathways of cell differentiation during fin regeneration are discussed. @ 1996 Wiley-Liss, Inc.

Teleost fins are composed of segmented and bi- furcated skeletal elements, the rays or lepidotri- chia, which are made of two tile-like dermal bone hemirays or hemisegments. These structures con- tain connective tissue with nerves and blood ves- sels and are surrounded by the skin. In the interray regions, connective tissue and epidermis on both sides form a thinner layer than in the ray regions (Becerra et al., '83). Each ray is api- cally tapered with a double palisade of long rigid rods called actinotrichia, which may have morpho- genetic activity (Santamaria and Becerra, '91).

After partial amputation, the fins regenerate through a process that closely resembles normal development. After healing of the wound, an api- cal blastema is formed by dedifferentiation of pre- existing mesenchymal Icells, which proliferate and finally differentiate into a number of cell types. Some cells differentiate into lepidotrichia-forming cells or scleroblasts which restore the hemiseg- ments on both sides of the rays; others give rise to fibroblasts to regenterate the intraray connec- tive tissue (Santamaria and Becerra, '91; Mari- Beffa et al., '96). This system provides a valuable model for studies of development and regenera- tion (Mari-Beffa, '87; Molven, '91; Akimenko et al.,

0 1996 WILEY-LISS, INC.

'95). The nerve growth-dependent proliferation of cells in regeneration of tetrapod limbs has been examined in detail both in vivo and in vitro (Boilly et al., '86; Brockes and Kintner, '86; Boilly and Albert, '88; Winner, '89; Goldhamer et al., '92; Mescher, '92; Smith et al., '95). However, little is known about the regenerative and developmen- tal proliferation of cells in fishes, although some preliminary studies have been performed (Graudie, '80; Graudie and Singer, '79; Ferretti and Graudie, '95). The recent introduction of zebrafish mutants has opened a vast field of research in vertebrate development (Mullins et al., '94; Gkraudie et al., '95), in particular in the development and regen- eration of fins. Moreover, gene expression has also been successfully studied in this model system (Sordino et al., '95).

Bromodeoxyuridine (5'-bromo-Z'-deoxyuridine; BrdU), a pyrimidine analog of thymidine, can be used to label the DNA of proliferating cells which can then be detected immunocytochemically with

Received October 11, 1995; revision accepted March 7, 1996. Address reprint requests to Prof. J. Becerra, Departamento de

Biologia Celular y GenBtica, Facultad de Ciencias, U.M.A. Campus Universitario de Teatinos, dn. 29071 Malaga, Spain.

INCORPORATION OF BrdU IN REGENERATING FIN TISSUE 301

a monoclonal antibody against BrdU (Gratzner, '82). This technique was originally used in cell and organ cultures, but it has been successfully used in experiments in vivo for both descriptive and ki- netic studies (Alfei et al., '93; Oinuma et al., '92).

In the present study we investigated the incor- poration of BrdU in replicating cells of regenerat- ing tail fins of Curussius uurutus. We report here the localization of proliferating cells on the proxi- modistal axis of a 15-day-old regenerating fin ray. Localized dividing, nondividing, and synchro- nously dividing cells in both the epidermis and blastema are suggestive of a balance between dif- ferentiation and proliferation in the regenerating blastema.

MATERIALS AND METHODS Animals

Specimens of the goldfish C. aurutus, approxi- mately 7 cm in length, were used in this study. The animals were maintained in aquaria that con- tained water filtered over activated charcoal, with constant aeration. The photoperiod was 12 h of light and 12 h of darkness daily, and the tempera- ture ranged from 19 to 22°C in a climate-con- trolled room. The animals were fed regularly with commercial food (Sera Vipan, Sera, Heinsberg, Germany). Each experimental group consisted of four animals.

The animals were anesthetized with tricaine (MS 222, Sigma, St. Louis, MO) dissolved in wa- ter (62 mg/l) and the tail fins were cut with sharp, sterile scissors. After 15 days, the regenerating fins were dissected out, fixed in Bouin's fluid for 24 h in every case, and embedded in paraffin. The blocks were cut into 7-ym-thick sections.

Labeling ofproliferating cells with BrdU DNA in replicating cells incorporated BrdU af-

ter injection of BrdU (Sigma) in Hank's solution at a dose of 0.25 mglg wet weight. This DNA was immunocytochemically detected 1, 7, 24, 48, 120, and 240 h after intraperitoneal injection by use of antibodies against BrdU that had been indirectly labeled with horseradish peroxidase (HRP). Sections were observed under a micro- scope (Microphot FXA, Nikon, Japan) and photo- graphed with Nomarski optics.

Immunocytochemical procedures Mouse monoclonal antibodies against BrdU

(Boehringer-Mannheim, Germany) were used as primary antibody at a dilution of 1500. Slides were incubated for 18 h at 22°C. The second anti-

serum, raised in rabbit against mouse IgG (Sigma), was used at dilution of 1 5 0 with incubation for 60 min at 22°C. Incubation with the peroxidase- antiperoxidase complex (PAP) (1:200; Sigma) was performed for 30 min at 22°C with 3,3'- diaminobenzidine (DAB; Sigma) as the electron donor. All antisera and the PAP complex were di- luted in phosphate-buffered saline (PBS), pH 7.3. Sodium azide (0.1%) and bovine serum albumin (BSA; 0.1%) were added to the first and second solutions of antibodies. For preparation of control sections, incubation with the primary antibodies was omitted.

RESULTS After amputation, regeneration of the fin pro-

ceeds via formation of a blastemal population of undifferentiated cells once the wound is healed. During the first 10 days, the tip of each ray de- veloped a structured cytoarchitecture that re- mained until the appendage had been completed. In a 15-day-old regenerated fin, every element was presented in each ray: the epidermis covered the entire structure and was of special quality at the distal end; a blastema occupied the tip of the mes- enchymal tissue; the actinotrichia were forming a double palisade in the subepidermal space, near the basement membrane; scleroblasts were syn- thesizing the budding lepidotrichial hemiseg- ments; and, in the central mesenchymal region, fibroblasts had initiated the restoration of intraray connective tissue. We present here cell prolifera- tion, through BrdU incorporation, in that balanced growing situation.

The intraperitoneal injection of BrdU 14 days after amputation yielded different intensities of labeling depending on drug concentration and time after injection. Labeled tissue was sampled and studied 1, 7, 24, 48, 120, and 240 h after in- jection of BrdU. After a single injection of a se- lected BrdU concentration, maximum labeling was noted in transverse sections after 24 h and the bulk of results presented in this article refers to such outcomes. A quantitative study of such process at different times and structures will be done later.

Renewal and regeneration may be studied fol- lowing comparison of labeling index and epider- mal cell layer counting, Twenty-four hours showed a maximum labeling at intermediate layers where higher frequencies are observed. This is suggestive of steady-state kinetics exemplified by our figures.

No replicating cells were observed in the first transverse sections, which included the apical epi- dermis (Fig. 1). When a narrow layer of mesen-

302 JA. SANTAMAR~AET AL.

Fig. 1. Transverse section of a 15-day-old regenerated cau- dal fin through the apical epidermis. No incorporation of BrdU is detected a t this level. Label on the right side corresponds to the adjacent ray initial blastema. x100.

Fig. 2. Transverse section of a regenerating ray that includes mesenchymal cells (arrows) fixed 24 h after in- jection of BrdU. Subepidermal basement membrane (small arrows) can be clearly identif ed. The left side (asterisk) cor- responds to a presumptive interray field. Labeled mitotic figures can be observed in the epidermis (arrowhead). x340.

Fig. 3. Transverse section showing an overview of two re- generating rays. The blastemal cells have widely incorporated BrdU (stars) and the actinotrichia palisade (arrows) have been consolidated. At this level, with undifferentiated rays, the in- terray space contains an unlabeled mesenchymal tissue (as- terisk). The basal epidermal layer and peridermis have not incorporated BrdU. The epidermis seems to be slightly la- beled in the presumptive interray field. x160.

Partial transverse section of a regenerating ray showing the epidermis (without labeling of the peridermis

Fig. 4.

INCORPORATION OF BrdU IN REGENERATING FIN TISSUE 303

chymal tissue appeared at more proximal levels, few blastemal cells were labeled, and some labeled and heterogeneously distributed cells in the cen- tral layers of the epidermis were observed (Fig. 2). When the mesenchymal area became wider in the prospective ray tissue, blastemal cells were extensively labeled between both palisades of ac- tinotrichia (Fig. 3). The epidermis at this level showed a constant pattern of medial replicating cells; the peridermis and basal layer remained unlabeled (Fig. 3). The prospective interray areas usually appeared free of label, both in mesenchy- ma1 and epidermal tissue (Fig. 3).

The majority of cells that were located beneath the basement membrane at this level incorporated BrdU (Fig. 4). In that location, the first differen- tiated scleroblasts will appear. Here, an unlabeled central blastemal region could also be observed. No changes in the epidermis were observed at this level.

The number of labeled cells in the ray mesen- chymal tissue decreased drastically when the scle- roblasts (unlabeled) started to synthesize the lepidotrichia (Fig. 5). Labeled cells were now con- centrated within the interray region at this level, both in the epidermis and in the connective tissue (Fig. 5). At sites where new lepidotrichial material was deposited, scleroblasts were not labeled and the intraray region contained few and dispersed repli- cating cells (Fig. 6). At this level some cells of the basal epidermal layer incorporated BrdU.

and basal layer), basement membrane (arrows), and an acti- notrichia palisade (small arrows) with labeled blastemal cells. Between the basement membrane and the actinotrichial pali- sade, some labeled cells can be seen yet. The differentiation of the first scleroblasts will occur in this location. Underneath (asterisk), a zone of scant labeling can be seen occupying the central region of the blastema. x450.

Fig. 5. Transverse section showing a regenerating ray. La- beling of blastemal cells has been significantly reduced. Nev- ertheless, cells in the interray region (arrows), in both the basal epidermis and mesenchyme, are strongly labeled. Note the outline of the lepidotrichium hemisegments (small arrows) below the unlabeled basal epidermis (stars) and scleroblasts (asterisks). x200.

Transverse section of a regenerating ray at the consolidated lepidotrichial level (arrows). The majority of basal epidermal cells is now labeled. Some labeled cells in the in- traray tissue are associated with the edges of the lepidotri- chia (arrowheads). No actinotrichia profiles can be seen. x200.

At this level in the transverse section, very little label is visible in the connective tissue (both intra- and inter- ray). There is a small number of positive outer scleroblasts (arrows). The epidermis remains strongly labeled. x200.

Fig. 8. Transverse section of a control ray. Cells in a very thin epidermis are the only structure that incorpo- rates BrdU. x170.

Fig. 6.

Fig. 7.

At sites of consolidated lepidotrichial segments, no replication of scleroblasts was observed and very little label was detected at the edges of the hemisegments and in the connective tissues of the intraray (Fig. 7). Epidermal cells labeled in these areas were homogeneously distributed, both in the ray and interray regions. Labeled scleroblasts and fibroblasts infiltrating through the subepidermal space could be seen. Cell proliferation in the basal epidermal stratum was also clearly detected in an epidermis that, in this level, presented a re- duced number of cell layers (Fig. 7).

More proximally, as the complete differentiation of rays was achieved, replicating cells were re- stricted to the thinner epidermis (Fig. 8).

Summing up, during regeneration, the subapi- cal blastemal cells proliferated, incorporating BrdU in their nuclei. Rays differentiate proximal to their blastema and BrdU-labeled cells gradu- ally diminished in number as they adjoined to the subepidermal basement membrane and differen- tiated into scleroblasts. Cells in the intraray con- nective tissue differentiated at a higher rate than epidermal cells, because the incorporation of BrdU remained in the epidermal cell nuclei at the level where mesenchymal cells were already unlabeled. All of these features are summarized in Figure 9.

DISCUSSION Cell proliferation and growth control are cru-

cial during fin development and regeneration but little is known about this subject (Geraudie, '80; Ferretti and Geraudie, '95; Akimenko et al., '95). Fins of fishes, resembling other organs, have a morphology (size, shape, bifurcation, segmenta- tion, etc.) that is species-specific and, therefore, under genetic control. Results of experimental ma- nipulations such as partial, hemiray, or individual ray amputations and chemical administration (re- tinoic acid, etc.) which cause alterations in the regenerate fin, such as loss of bifurcation (White et al., ,941, fin rays fusions (Santamaria et al., '93; Geraudie et al., ,941, loss of synchronization in the segmentation of both hemisegments and loss of segmentation and bifurcation (Mari-Beffa, '87), suggest that emerging cell-cell and cell-matrix mesenchyme interactions (Oster et al., '85) act as mechanisms that control morphology, as it has also been proposed in other systems (Nijhout, '90).

We have described here the pattern of incorpo- ration of BrdU in the tissues that have been im- plicated in tail fin regeneration, in an attempt to understand proliferation of cells in this system. Our information provides wild-type parameters

304 J . k SANTAMAFtfA ET AL,.

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Fig. 2

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Fig. 4

Fig. 5

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Fig. 7

Fig. 8

Fig. 9. Drawing of a 15-day-old regenerated ray showing a current model of cell proliferation and differentiation dur- ing fin regeneration. The proliferation of blastemal cells is concentrated in a conspicuoufi population of cells at the tip of each regenerating ray. Arrow heads represent different paths of cell migration and/or differentiation. From the proximal area of the proliferating blastema and in its central region, the loss of proliferating cells and the morphological changes observed support differentiati on to fibroblasts via a prefibro-

blast state with eventual proliferation (arrows). These fibro- blasts would be responsible for the restoration of intraray connective tissue. From the lateral side of the blastema and through the actinotrichial region, another migratioddiffer- entiation occurs (arrowheads). These cells eventually prolif- erate to form both the cells adjoining subepidermal basement membrane and those starting the reconstruction of the he- misegment (scleroblasts). Proximally, some scleroblasts or their precursor cells would migrate to colonize the subepi-

INCORPORATION OF BrdU IN REGENERATING FIN TISSUE 305

that can be used as controls in selective ablation or chemical experiments, as well as a basis for comparation with results obtained from other spe- cies (Geraudie and Ferreti, ’95). The present re- sults show high, selective, and clear label of cells, however, we have detected a great variability among both animals and species. Quantitative studies are required to definitively establish the exact proliferative pattern of the different cell types, locations, and times of regeneration.

Blastemal proliferation Blastemal cells are characterized by their ab-

sence of differentiation. They are densely packed, with extensive cell-cell contacts of the gap-junc- tion type and high rates of proliferation. Break- age of cell contacts, synthesis of extracellular molecules (collagen I, collagen 11, proteoglycans, etc.), decreased cell proliferation, and initiation of cell differentiation (Zimmermann and Thies, ’84) characterize their disappearance.

It has been well established that, 4 or 5 days after amputation, a blastema forms at the tip of each ray, with unclear limits. According to our data, the blastema extends for a few micrometers beneath the apical epidermis. Previous morpho- logical and histochemical analyses defined larger domains (Santaman’a and Becerra, ’91; Toole et al., ’84). A very distal area of limited incorporation of BrdU was found. Such cells could be collaborat- ing with the apical epidermis in the reconstruction of basement membrane, in the synthesis of acti- notrichia, and even in the formation of the microen- vironment for formation of the whole blastema. Localized turnover of tritiated proline incorporated by actinotrichia suggests the existence of a popu- lation of blastemal cells implicated in the selec- tive degradation of actinotrichia (Mari-Beffa et a]., ’89) which has not been detected by histochemi-

dermal space and could be responsible for the external accretive growth of the hemisegments (curved arrows). These cells eventually proliferate and become flattened (sclerocytes) when the hemisegment is consolidated. The epidermis ap- pears to be influenced by another proliferative process. The apical epidermis does not proliferate and its lateral faces pro- liferate only in the medial layers. This proliferation process could provide cells for distal enlargement of epidermis and its superficial renewal. Below the dotted line, the control situ- ation is represented. Here, cell proliferation only occurs in some epidermal cells (including basal ones) for the renewal of the epidermis. Shadowing represents proliferation of cells. Figures at the right side indicate the corresponding levels of the shown histological sections.

cal techniques. Some embryological data support the hypothesis that basal epidermal cells synthe- size the actinotrichia during fin development (Geraudie, ’77, ’80). The use of antibodies against actinotrichia should help us to identify the cells that are actually synthesizing the actinotrichia and their possible role in fin morphogenesis.

The blastema is well developed when it is lat- erally flanked by actinotrichia. Some cells colo- nize the space between actinotrichia and the basement membrane at this level, retaining in an active proliferative state. No experimental data definitively support the involvement of actinotri- chia in the differentiation of scleroblasts but a close relationship between “ p r e ~ ~ l e r ~ b l a ~ t ~ ~ ~ and actinotrichia has been reported in fin regenera- tion and development (Santamaria and Becerra, ’91; Wood and Thorogood, ’84).

Cell proliferation in the interray area for sev- eral micrometers proximally is probably necessary for expansion of interray spaces, to supply cells that contribute to enlargement of the lepidotri- chium hemisegments dorsally and ventrally, and perhaps to provide “prefibroblasts” to replace the subepidermic connective tissue at the site of the ray. In the central region of the blastema, where the hemisegments are already visible, a de- crease in BrdU incorporation occurred, probably because another route of differentiation was in progress, namely construction of the intraray connective tissue.

Epidermal Proliferation During fin regeneration, the epidermis has its

own proliferation dynamics. Some correlations can be made between labeled cells in the epidermis and connective tissue. The apical cap of the fin and limb buds has been reported to have specific morphological and physiological characteristics. It forms an apical ridge which in fins is called the “pseudoapical ridge” (GBraudie and Franqois, ’73) and it always exhibits an unusual behavior as far as proliferation is concerned. In several re- ported cases, low mitotic activity has been noted in the epidermal cells that cover the apical ridge (Geraudie, ’80). Although in fin regeneration a pseudoapical ridge cannot be detected, a thicker nonproliferative epithelium appears. However, cells of the subapical epithelium of the regenerat- ing ray proliferate, even though the basal and peridermal layers were unable to incorporate BrdU. The absence of proliferation at the pseudo- apical ridge has been related by (Xraudie (’80) to the synthesis of actinotrichia in fin development

306 J.A. SANTAMARfA ET AL.

because the formation of these macrofibrils occurs inside the fin fold, where no mesenchymal cells are present (Bouvet, '74). Although in fin regen- eration actinotrichia are formed at sites where mesenchymal cells are present, two of our results support those of GBraudie ('80): 1) the basal epi- dermis remains as a nonreplicating layer in the epidermis covering the ray region and these cells proliferate at levels where actinotrichia are ab- sent (see Figs. 6, and '7); and 2) the cells in the basal layer of the epidermis that covers the interray areas (without actinotrichia) incorporate BrdU from very distal levels (see Fig. 5).

Cells at the basal layer in the rayhnterray boundary do incorporate BrdU, suggesting these cells are probably generative of both regions. This is suggestive of a local proliferation in both re- generation and renewal of this tissue. Samplings from 24 h incorporate more BrdU than 1 or 7 h samplings. That scarce incorporation does not sup- pose labeling of the basal layer and precludes a slower rate of proliferation for these cells. Probably apical incorporation ma,y occur but in any case re- newal by proliferation att suprabasal layers is more important than regeneration at the basal layer.

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