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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275300-307 (1996)
Incorporation of Bromodeoxyuridine in
Regenerating Fin Tissue of the Goldfish
Carassius auratus
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 auratus. 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 capacity 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-proliferation 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 regenerate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorporate 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 bifurcated skeletal elements, the rays or lepidotrichia, which are made of two tile-like dermal bone
hemirays or hemisegments. These structures contain connective tissue with nerves and blood vessels 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 apically tapered with a double palisade of long rigid
rods called actinotrichia, which may have morphogenetic 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 apical blastema is formed by dedifferentiation of preexisting 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 hemisegments on both sides of the rays; others give rise
to fibroblasts t o regenterate the intraray connective tissue (Santamaria and Becerra, '91; MariBeffa et al., '96). This system provides a valuable
model for studies of development and regeneration (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 developmental 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 regeneration 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
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 kinetic studies (Alfei et al., '93; Oinuma et al., '92).
In the present study we investigated the incorporation of BrdU in replicating cells of regenerating tail fins of Curussius uurutus. We report here
the localization of proliferating cells on the proximodistal axis of a 15-day-old regenerating fin ray.
Localized dividing, nondividing, and synchronously dividing cells in both the epidermis and
blastema are suggestive of a balance between differentiation and proliferation in the regenerating
blastema.
MATERIALS AND METHODS
Animals
Specimens of the goldfish C. aurutus, approximately 7 cm in length, were used in this study.
The animals were maintained in aquaria that contained water filtered over activated charcoal, with
constant aeration. The photoperiod was 12 h of
light and 12 h of darkness daily, and the temperature ranged from 19 t o 22°C in a climate-controlled 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 water (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 ofproliferatingcells with BrdU
DNA in replicating cells incorporated BrdU after 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 microscope (Microphot FXA, Nikon, Japan) and photographed 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-
301
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 peroxidaseantiperoxidase 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 diluted 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 proceeds via formation of a blastemal population of
undifferentiated cells once the wound is healed.
During the first 10 days, the tip of each ray developed a structured cytoarchitecture that remained until the appendage had been completed.
In a 15-day-oldregenerated 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 mesenchymal tissue; the actinotrichia were forming
a double palisade in the subepidermal space, near
the basement membrane; scleroblasts were synthesizing the budding lepidotrichial hemisegments; and, in the central mesenchymal region,
fibroblasts had initiated the restoration of intraray
connective tissue. We present here cell proliferation, 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 injection of BrdU. After a single injection of a selected BrdU concentration, maximum labeling was
noted in transverse sections after 24 h and the bulk
of results presented in this article refers t o such
outcomes. A quantitative study of such process at
different times and structures will be done later.
Renewal and regeneration may be studied following comparison of labeling index and epidermal 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 epidermis (Fig. 1).When a narrow layer of mesen-
302
JA. SANTAMAR~AET
AL.
Fig. 1. Transverse section of a 15-day-old regenerated caudal 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 injection of BrdU. Subepidermal basement membrane (small
arrows) can be clearly identif ed. The left side (asterisk) corresponds t o a presumptive interray field. Labeled mitotic
figures can be observed in the epidermis (arrowhead). x340.
Fig. 3. Transverse section showing an overview of two regenerating rays. The blastemal cells have widely incorporated
BrdU (stars)and the actinotrichia palisade (arrows) have been
consolidated. At this level, with undifferentiated rays, the interray space contains an unlabeled mesenchymal tissue (asterisk). The basal epidermal layer and peridermis have not
incorporated BrdU. The epidermis seems to be slightly labeled in the presumptive interray field. x160.
Fig. 4. Partial transverse section of a regenerating ray
showing the epidermis (without labeling of the peridermis
INCORPORATION OF BrdU IN REGENERATING FIN TISSUE
chymal tissue appeared at more proximal levels,
few blastemal cells were labeled, and some labeled
and heterogeneously distributed cells in the central 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 actinotrichia (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 mesenchyma1 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 differentiated 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 mesenchymal tissue decreased drastically when the scleroblasts (unlabeled) started t o synthesize the
lepidotrichia (Fig. 5). Labeled cells were now concentrated 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 replicating cells (Fig. 6). At this level some cells of the
basal epidermal layer incorporated BrdU.
303
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 reduced number of cell layers (Fig. 7).
More proximally, as the complete differentiation
of rays was achieved, replicating cells were restricted to the thinner epidermis (Fig. 8).
Summing up, during regeneration, the subapical blastemal cells proliferated, incorporating
BrdU in their nuclei. Rays differentiate proximal
to their blastema and BrdU-labeled cells gradually diminished in number as they adjoined to the
subepidermal basement membrane and differentiated into scleroblasts. Cells in the intraray connective 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 crucial during fin development and regeneration but
little is known about this subject (Geraudie, '80;
and basal layer), basement membrane (arrows), and an acti- Ferretti and Geraudie, '95; Akimenko et al., '95).
notrichia palisade (small arrows) with labeled blastemal cells. Fins of fishes, resembling other organs, have a
Between the basement membrane and the actinotrichial pali- morphology (size, shape, bifurcation, segmentasade, some labeled cells can be seen yet. The differentiation
tion, etc.) that is species-specific and, therefore,
of the first scleroblasts will occur in this location. Underneath
under
genetic control. Results of experimental ma(asterisk), a zone of scant labeling can be seen occupying the
nipulations such as partial, hemiray, or individual
central region of the blastema. x450.
Fig. 5. Transverse section showing a regenerating ray. La- ray amputations and chemical administration (rebeling of blastemal cells has been significantly reduced. Nev- tinoic acid, etc.) which cause alterations in the
ertheless, cells in the interray region (arrows), in both the
regenerate fin, such as loss of bifurcation (White
basal epidermis and mesenchyme, are strongly labeled. Note
the outline of the lepidotrichium hemisegments (small arrows) et al., ,941, fin rays fusions (Santamaria et al., '93;
below the unlabeled basal epidermis (stars) and scleroblasts Geraudie et al., ,941, loss of synchronization in the
(asterisks). x200.
segmentation of both hemisegments and loss of
Fig. 6. Transverse section of a regenerating ray at the segmentation and bifurcation (Mari-Beffa, '87),
consolidated lepidotrichial level (arrows). The majority of basal
suggest that emerging cell-cell and cell-matrix
epidermal cells is now labeled. Some labeled cells in the inmesenchyme
interactions (Oster et al., '85) act as
traray tissue are associated with the edges of the lepidotrichia (arrowheads). No actinotrichia profiles can be seen. x200. mechanisms that control morphology, as it has
Fig. 7. At this level in the transverse section, very little also been proposed in other systems (Nijhout, '90).
label is visible in the connective tissue (both intra- and interWe have described here the pattern of incorporay). There is a small number of positive outer scleroblasts
ration
of BrdU in the tissues that have been im(arrows). The epidermis remains strongly labeled. x200.
plicated
in tail fin regeneration, in an attempt to
Fig. 8. Transverse section of a control ray. Cells in a
very thin epidermis are the only structure that incorpo- understand proliferation of cells in this system.
rates BrdU. x170.
Our information provides wild-type parameters
304
J . k SANTAMAFtfA ET AL,.
f- Fig. 1
Fig. 2
<-
Fig. 3
Fig. 4
Fig. 5
4-
Fig. 6
Fig. 7
Fig. 8
Fig. 9. Drawing of a 15-day-old regenerated ray showing
a current model of cell proliferation and differentiation during fin regeneration. The proliferation of blastemal cells is
concentrated in a conspicuoufipopulation 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 fibroblasts would be responsible for the restoration of intraray
connective tissue. From the lateral side of the blastema and
through the actinotrichial region, another migratioddifferentiation occurs (arrowheads). These cells eventually proliferate t o form both the cells adjoining subepidermal basement
membrane and those starting the reconstruction of the hemisegment (scleroblasts). Proximally, some scleroblasts or
their precursor cells would migrate to colonize the subepi-
INCORPORATION OF BrdU IN REGENERATING FIN TISSUE
305
cal techniques. Some embryological data support
the hypothesis that basal epidermal cells synthesize 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 laterally flanked by actinotrichia. Some cells colonize the space between actinotrichia and the
basement membrane at this level, retaining in an
Blastemal proliferation
active proliferative state. No experimental data
Blastemal cells are characterized by their ab- definitively support the involvement of actinotrisence of differentiation. They are densely packed, chia in the differentiation of scleroblasts but a
la~t~~~
with extensive cell-cell contacts of the gap-junc- close relationship between “ p r e ~ ~ l e r ~ band
tion type and high rates of proliferation. Break- actinotrichia has been reported in fin regeneraage of cell contacts, synthesis of extracellular tion and development (Santamaria and Becerra,
molecules (collagen I, collagen 11, proteoglycans, ’91; Wood and Thorogood, ’84).
Cell proliferation in the interray area for sevetc.), decreased cell proliferation, and initiation
of cell differentiation (Zimmermann and Thies, eral micrometers proximally is probably necessary
’84) characterize their disappearance.
for expansion of interray spaces, to supply cells
It has been well established that, 4 or 5 days that contribute t o enlargement of the lepidotriafter amputation, a blastema forms at the tip of chium hemisegments dorsally and ventrally, and
each ray, with unclear limits. According t o our perhaps t o provide “prefibroblasts” to replace the
data, the blastema extends for a few micrometers subepidermic connective tissue at the site of the
beneath the apical epidermis. Previous morpho- ray. In the central region of the blastema, where
logical and histochemical analyses defined larger the hemisegments are already visible, a dedomains (Santaman’a and Becerra, ’91; Toole et crease in BrdU incorporation occurred, probably
al., ’84).A very distal area of limited incorporation because another route of differentiation was in
of BrdU was found. Such cells could be collaborat- progress, namely construction of the intraray
ing with the apical epidermis in the reconstruction connective tissue.
of basement membrane, in the synthesis of actiEpidermal Proliferation
notrichia, and even in the formation of the microenvironment for formation of the whole blastema.
During fin regeneration, the epidermis has its
Localized turnover of tritiated proline incorporated own proliferation dynamics. Some correlations can
by actinotrichia suggests the existence of a popu- be made between labeled cells in the epidermis
lation of blastemal cells implicated in the selec- and connective tissue. The apical cap of the fin
tive degradation of actinotrichia (Mari-Beffa et a]., and limb buds has been reported to have specific
’89) which has not been detected by histochemi- 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
dermal space and could be responsible for the external far as proliferation is concerned. In several reaccretive growth of the hemisegments (curved arrows). These ported cases, low mitotic activity has been noted
cells eventually proliferate and become flattened (sclerocytes) in the epidermal cells that cover the apical ridge
when the hemisegment is consolidated. The epidermis ap(Geraudie, ’80). Although in fin regeneration a
pears to be influenced by another proliferative process. The
apical epidermis does not proliferate and its lateral faces pro- pseudoapical ridge cannot be detected, a thicker
liferate only in the medial layers. This proliferation process nonproliferative epithelium appears. However,
could provide cells for distal enlargement of epidermis and cells of the subapical epithelium of the regeneratits superficial renewal. Below the dotted line, the control situ- ing ray proliferate, even though the basal and
ation is represented. Here, cell proliferation only occurs in
peridermal layers were unable to incorporate
some epidermal cells (including basal ones) for the renewal
of the epidermis. Shadowing represents proliferation of cells. BrdU. The absence of proliferation at the pseudoFigures at the right side indicate the corresponding levels of apical ridge has been related by (Xraudie (’80) t o
the shown histological sections.
the synthesis of actinotrichia in fin development
that can be used as controls in selective ablation
or chemical experiments, as well as a basis for
comparation with results obtained from other species (Geraudie and Ferreti, ’95). The present results 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.
J.A. SANTAMARfA ET AL.
306
because the formation of these macrofibrils occurs
inside the fin fold, where no mesenchymal cells
are present (Bouvet, '74). Although in fin regeneration actinotrichia are formed at sites where
mesenchymal cells are present, two of our results
support those of GBraudie ('80):1) the basal epidermis remains as a nonreplicating layer in the
epidermis covering the ray region and these cells
proliferate at levels where actinotrichia are absent (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 regeneration and renewal of this tissue. Samplings
from 24 h incorporate more BrdU than 1 or 7 h
samplings. That scarce incorporation does not suppose 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 renewal by proliferation att suprabasal layers is more
important than regeneration at the basal layer.
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