/ . Embryol. exp. Morph. Vol. 55, pp. 65-76, 1980
Printed in Great Britain © Company of Biologists Limited 1980
65
On the role of germ cells in planarian regeneration
.11. Cytopliotometric analysis of the nuclear Feulgen-DNA content
in cells of regenerated somatic tissues
By V. GREMIGNI, 1 C. MICELP AND E. PICANO1
From the Institute of Zoology, University of Pisa, Italy
SUMMARY
Previous findings by our group have shown how primordial male germ cells take part in
regenerative blastema formation in planarians by migrating to the wound.
The role of these cells in rebuilding transected tissues has been investigated in a population
of Dugesia lugubris s.l. which is particularly suited for our purpose. In fact, these planarians
provide a clear karyological marker to distinguish diploid male germ cells (2n = 8) from
tryploid embryonic or somatic cells (3« = 12). In this study we employed the cytophotometric analysis of the nuclear Feulgen-DNA content in order to distinguish non-replicating
male germ cells from reserve and somatic cells. The Feulgen-DNA content in cells from the
gonad-free caudal area was measured after complete regeneration. Most non-replicating
cells (94-95%) were found to have a DNA amount typical of cells previously estimated as
triploid. Some (5-6%) nuclei containing a DNA amount typical of cells previously estimated
as diploid male gonia were also found. These findings seem to support the view that primordial male germ cells also participate in rebuilding somatic tissues according to the field influence they encounter during regeneration. The possibility that metaplasia (or cell transdifferentiation) may occur in planarians is finally discussed.
INTRODUCTION
Regeneration has been extensively investigated in planarians (see for review
the books by Brondsted (1969) and by Chandebois (1976)) because these organisms provide a suitable material for a detailed study of this complex and still
questioned phenomenon involving such general problems as totipotency,
dedifferentiation and metaplasia.
One of the first phases of regeneration in planarians involves the formation
of a blastema that is widely regarded as being composed of an accumulation
of totipotent cells at the wound. Although many experiments have been specially
done and several theories have been proposed to account for the origin of the
regenerative blastema, this is still a controversial issue. One of the more credited
theories on the subject, proposed by E. Wolff and his school (Wolff & Dubois,
1947a, b; Dubois, 1949; Wolff, 1962; Lender, 1962; Gabriel, 1970) states that
the blastema is formed only from embryonic, reserve cells usually referred to as
neoblasts. According to this theory, neoblasts are undifferentiated cells widely
1
Authors' address: The Institute of Zoology, via Volta 4, 56100 Pisa, Italy.
2
Present address: The Institute of Zoology, University of Camerino, Italy.
66
V. GREMIGNI, C. MICELI AND E. PICANO
scattered in the parenchyma of adult worms. Moreover, they would be able to
migrate to the injured area and, being unique in their capacity to divide, would
be responsible for the regeneration of transected or injured tissues. The neoblast
theory is supported, with some variation, by many other authors. Some of them
(Betchaku, 1970; Baguna, 1975), however, deny the migratory capacity of
neoblasts.
An opposing theory, supported among others by Lang (1912), Steinmann
(1925), Hyman (1951), Woodruf & Burnett (1965), Rose & Shostak (1968),
Hay (1968), Coward (1969), states the blastema is formed from already differentiated cells capable of 'abjuring' their specialization as a consequence of a
regenerative stimulus. Due to their dedifferentiation, these cells would thus
lose their structural and chemical specialization, and would reacquire embryonic
characteristics including developmental totipotency typical of blastema cells.
A conciliatory theory was suggested by Betchaku (1970) and Pedersen (1972)
and has been recently reproposed by one of us (Gremigni, 1974). This states that
the blastema can be formed by both types of cells, i.e. neoblasts and dedifferentiated cells. This viewpoint has been supported by a number of observations in
which primordial male or female germ cells take part, along with the so-called
neoblasts, in blastema formation (Vandel, 1921; Fedecka-Bruner, 1965, 1967;
Banchetti & Gremigni, 1973; Gremigni & Puccinelli, 1977; Puccinelli & Gremigni, 1977; Grasso, Gardenghi & Di Giovanni, 1977).
The fact that germ cells take part in blastema formation gives rise to an
important question: are dedifferentiated germ cells only capable of rebuilding
new gonads, or can they undergo ^differentiation into other cell types? The
latter phenomenon would be referred to as metaplasia or cell transdifferentiation.
In an attempt to solve this question, we took advantage of the presence in our
laboratory of a population of Dugesia lugubris s.l. which is particularly suited
for our purposes. These organisms have diploid male germ cells and triploid
embryonic or somatic cells. In this study, we tried to demonstrate whether
non-replicating cells coming from the testes can take part in rebuilding somatic
tissues. Our working hypothesis was based on the widely accepted concept that
non-replicating cells contain a fairly constant nuclear DNA amount per haploid
set of chromosomes (Bouvin, Vendrely & Yendrely, 1948; Mirsky & Ris,
1949; Bedi & Goldstein, 1976). Thus, by means of cytophotometric measurements, we first attempted to verify whether male gonia have a nuclear FeulgenDNA content significantly lower than that of somatic cells. Then we attempted
to demonstrate whether non-replicating cells with the typical Feulgen-DNA
content of male gonia were still present in gonad-free areas after complete
regeneration.
Germ cells in planarian regeneration: II
67
MATERIAL AND METHODS
The specimens of Dugesia lugubris s.l. used in this study were originally
collected in the lake Iseo and then maintained in our laboratory. These planarians belong to a polyploid biotype which has a karyological marker which
distinguish somatic from germ cells. In fact, somatic cells are triploid, female
germ cells are hexaploid and male germ cells are diploid. In addition, we obtained another population of the same species, common to streams near Pisa.
These planarians belong to a biotype with both somatic and germ lines diploid.
The two populations have chromosomes with an identical morphology and are
completely cross fertile. The specimens used in the experiments were isolated at
birth and raised untouched and uninjured until sexual maturity whereupon
they were then able to be used.
Experimental procedure
As a first step we had to verify if the cytophotometric technique employed
was reliable for our purposes; i.e. to establish if the resolution provided by
the cytophotometric analysis of the Feulgen-DNA content was suitable for
distinguishing between diploid and triploid cells. For this purpose we measured
the nuclear Feulgen-DNA content in the caudal parenchyma (Fig. 1) or in the
caudal stump blastema (ca.s.b.) (Fig. 2) of both populations. We know that in
the caudal area and consequently in the ca.s.b. of Iseo specimens only triploid
cells are present while in other areas, diploid cells are also present (Gremigni
& Puccinelli, 1977). This is due to the fact that testes are widely scattered throughout the planarian parenchyma.
In order to verify whether male gonia in Iseo specimens have a nuclear
Feulgen-DNA content lower than that of the somatic cells and similar to that
of cells in Pisa specimens, we prepared squashes of parenchyma containing
testes from Iseo specimens (Fig. 3).
Finally to test whether cells of male origin in Iseo population take part in
rebuilding somatic tissues, we made a transection at the caudal level just posterior to the testes. Following complete regeneration, squashes of the caudal area
were prepared for the cytophotometric analysis (Fig. 4). For this purpose we
made the transection with a razor blade on transilluminated worms viewed under
a binocular microscope so that only the caudal area devoid of testes could be
removed. Controls for these experimental samples were obtained from squashes
prepared from the intact caudal area of Iseo specimens (Fig. 1).
Cytophotometric technique
To obtain cytophotometric measurements of the nuclear Feulgen-DNA
content, small fragments of planarians were fixed for lOmin in 4 % formalin
and then squashed according to the dry-ice technique. The Feulgen technique
was then performed according to the method of Itikawa & Ogura (1954).
68
V. GREMIGNI, C. MICELI AND E. PICANO
ca.s.b.
Fig. 1
Fig. 2
Fig. 1. Scheme of either Iseo or Pisa specimens. The encircled caudal area was used
for squash preparations of intact parenchyma.
Fig. 2. Scheme of either Iseo or Pisa specimens. The level of the cut is diagrammed on
the left. The caudal stump blastema (ca.s.b.) is pictured on the right.
1)7
. ph
11""*? —ph
cut
Fig. 3
Fig. 4
Fig. 3. Scheme of an Iseo specimen. The encircled area containing testes was used for
squash preparations.
Fig. 4. The scheme on the left represents an intact Iseo specimen with the cut level
indicated. In the scheme on the right, the hatched area represents the regenerated
caudal fragment used for squash preparations.
In Figs. 1-4. o = ovaries; t = testes; ph = pharynx.
Germ cells in planarian regeneration: II
69
Fig. 5. A light microscope picture of Feulgen processed nuclei from the caudal
parenchyma of an intact specimen of Pisa population. Roundish nuclei with dispersed chromatin (a) and polymorphic nuclei with either dispersed (b) or condensed
chromatin (c) are visible.
Fig. 6. A light microscope picture of Feulgen processed nuclei from caudal regenerated parenchyma of an Iseo specimen. Three regularly shaped nuclei with
dispersed chromatin (d) and two polymorphic nuclei (e) are visible.
Slides were left for 60 min in 5 N-HC1 at room temperature (22 °C) and rinsed
briefly in distilled water. They were then stained for 60 min with the Schiff's
reagent. Following three 10 min washes in freshly prepared bisulphite solution,
the squashes were dehydrated in a graded series of ethyl alcohol and mounted
with the D.P.X. mounting medium (B.D.H.). Measurements of the nuclear
Feulgen-DNA content were carried out with a Barr and Stroud (Glasgow)
integrating micro-densitometer, type GN2.
Each slide contained experimental samples of the two populations employed.
Five slides were prepared for each type of sample and approximately 100 nuclei
were analysed on each slide. Blastema samples were prepared 60 h after the
transection.
RESULTS
In the caudal parenchyma of either Iseo or Pisa specimens, we could distinguish the following major types of interphase nuclei on the basis of their
morphological and staining characteristics: regularly shaped, roundish nuclei
with randomly dispersed chromatin (a), polymorphic nuclei with either dispersed
(b) or condensed chromatin (c) (Fig. 5). A few mitotic nuclei were also observed.
In the same sample, nuclei with condensed chromatin were usually smaller than
70
V. GREMIGNI, C. MICELI AND E. PICANO
180 i
180 ...
160
160 •
!40 •
140 •
120 •
120
'_S •
o
<! 100
o
g 100
o
1 80
•g 80
<u
Z
60
60
40
40
20
20
0
20
40
60 80
Feulgen-DNA
Fig. 7
100 a.u.
0
20
40 60 80 100 a.u.
Feulgen-DNA
Fig. 8
Fig. 7. Distribution of optic absorbance in arbitrary units (a.u.) of nuclei in the
caudal parenchyma of intact Iseo specimens.
Fig. 8. Distribution of optic absorbance in a.u. of nuclei in the caudal parenchyma of
intact Pisa specimens.
those with dispersed chromatin. Differences in size were also noted between
nuclei of Iseo specimens and those of Pisa specimens, the former being larger.
The basic value for the Feulgen-DNA content was obtained by analysing only
those nuclei which appeared homogeneous in shape and in chromatin distribution (a). This was done in both intact and regenerated parenchyma.
In the ca.s.b. of either Iseo or Pisa specimens two main types of non-dividing
nuclei were also distinguished according to previously stated ultrastructural
characteristics (Gremigni & Puccinelli, 1977). Regularly shaped nuclei with
randomly dispersed chromatin (from undifferentiated cells) (d); polymorphic
nuclei with condensed chromatin clumps adjacent to the nuclear envelope
(from dedifferentiating cells) (e). Some very dense and often vacuolated nuclei
with an irregular contour (from degenerating cells), and some mitotic nuclei were
also observed. The Feulgen-DNA content was measured only in the d-type
nuclei (Fig. 6).
The results of the measurements of the Feulgen-DNA content in parenchymal
nuclei from intact Iseo or Pisa specimens are shown in Figs. 7 and 8. In the
histogram of Fig. 7 a very high peak can be seen which corresponds to the lower
Germ cells in planarian regeneration: II
180
11
180 -i
160 .
160 -L
140 •
140 S
120 •
120 -
100 •
100 •
80 •
80 -
60
60 -
40 •
40 -
20 •
0
20
40 60 80
Feulgen-DNA
Fisu 9
100 a.u.
0
r1
20
'A
-40 60 80
Feulgen-DNA
100
Fig. 10
Fig. 9. Distribution of optic absorbance in a.u. of nuclei in the caudal stump blastema (ca.s.b.) of Iseo specimens.
Fig. 10. Distribution of optic absorbance in a.u. of nuclei in the caudal stump
blastema (ca.s.b.) of Pisa specimens.
value of nuclear D N A amount (46-13 = 0-26 a.u.) measured in Iseo samples.
This peak identifies the cell class containing the 3« = 3c DNA amount in this
population. The second and very small peak identifies those cells in which the
DNA amount is double that of the previous one. Most of these cells are likely
to be considered in the G2 period of interphase. It must be noted that even in
non-injured adult worms, undifferentiated cells undergoing division are scattered
throughout the parenchyma (Lange, 1967; Best, Hand & Rosenvold, 1968;
Pedersen, 1972; Baguna, 1975; Hay & Coward, 1975).
A histogram similar to the previous one has been obtained by analysing nuclei
in the caudal parenchyma of Pisa specimens (Fig. 8). However, the higher peak
in this histogram appears to reach 30-41 ±0-19 a.u. and identifies the cellular
class with the DNA amount 2/i = 2c typical of this population. The second
peak corresponds to cells with a DNA amount double that of the previous
one. The value of the DNA amount expressed in arbitrary units (a.u.) observed
in parenchymal nuclei from Pisa specimens is nearly equal to two thirds of the
amount observed in nuclei from Iseo specimens.
72
V. GREMIGNI, C. MICELI AND E. PICANO
180 -
180 -,
160 -
160 •
s\
140 -
140 •
120
120 •
'2,
100
i IOO •
'o
80
f 80 z
60
60 -
40
40 •
20
20 -
0
20
40 60 ' 80
Feulgen-DNA
Fig. 11
100 a.u.
0
20
40 60 80 100 a.u.
Feulgen-DNA
Fig. 12
Fig. 11. Distribution of optic absorbance in a.u. of nuclei in the parenchyma containing testes of Iseo specimens.
Fig. 12. Distribution of optic absorbance in a.u. of nuclei in the regenerated caudal
area of Iseo specimens.
In the nuclei of blastema cells of either Iseo or Pisa specimens (Figs. 9-10)
two distinct peaks were also obtained. The mean value of the DNA amount
of the higher peak in nuclei from Iseo specimens (Fig. 9) was 47-06 ± 0-38 a.u.
It identifies the cell class with the DNA amount 3n = 3c. In the same histogram
a second and smaller peak identifies nuclei with a DNA amount double that of
the previous one.
In the nuclei of Pisa specimens (Fig. 10) the mean value of the DNA amount
of the higher peak was 31-15 ±0-25 a.u. It identifies the cellular class with the
DNA amount (2n = 2c) typical of this population. The second and smaller
peak corresponds to the DNA amount that is double that of the basic one.
In the blastema samples of both Iseo or Pisa specimens the percentage of
nuclei possessing a DNA amount double that of the basic values is higher
than that of the corresponding nuclei in parenchymal samples. This can be due
to the fact that the number of cells undergoing division in the blastema is greater
than that in the parenchyma of non-injured worms as widely demonstrated (see
Br0ndsted, 1969; Pedersen, 1972 for review). The same interpretation can be
Germ cells inplanarian regeneration: II
13
made for the absence of a clear gap between the two peaks. This can be due to
the presence in the blastema of a number of cells in the S period of the interphase.
Three peaks can be seen in the histogram of Fig. 11 which refers to a small
area of uninjured Iseo specimens containing testes. The major peak corresponds
to the 3« = 3c value of the amount of DNA typical of this population. This is
likely due to non-replicating somatic and/or reserve cells. Another very small
peak corresponds to the 6c DNA amount and is probably due to cells in the Ga
period. The third peak, located at the left of the histogram, corresponds to the
2n — 2c value of the DNA amount as measured in nuclei from Pisa specimens.
This peak identifies, in our opinion, the male non-replicating gonia. This is,
in fact, the unique cell type with a diploid chromosome set in Iseo specimens.
This peak includes about 16% of the total number of nuclei analysed in the
samples. This percentage was obtained including in the 2n = 2c class the nuclei
with a range of Feulgen-DNA content (from 18-49 to 41-73 a.u.) within the 2c
value plus two times its standard deviation (S.D. = 5-81) obtained from Pisa
parenchymal nuclei.
The results of the measurements made in the regenerated caudal area of
lseo specimens are shown in Fig. 12. In the histogram a pattern distribution
similar to that of Fig. 11 can be noted, in that three peaks corresponding to the
2c, 3c and 6c values of DNA amount are represented. The 2c peak, however,
comprises only a 5-6 % of the nuclei analysed. Taking into account that testes
are absent in the regenerated caudal area of Iseo specimens, those nuclei with a
2n = 2c DNA amount are most likely interpreted as belonging to cells of male
origin which have migrated to this area during regeneration.
DISCUSSION
To test the reliability of the technique used to distinguish non-dividing somatic cells from male gonia in Iseo population, we compared the values of the
nuclear amount of DNA in triploid cells from Iseo specimens with that of
diploid cells from Pisa specimens. The latter population could, in fact, provide a
reference value of the nuclear DNA amount for cytophotometric identification
of non-dividing male gonia in Iseo specimens. This experimental procedure
proved suitable and necessary, as in squashes of Iseo specimens, nuclei from
spermatogonia can hardly be distinguished from nuclei of reserve cells and some
somatic cells. This is due to the fact that nuclei become widespread in squashes
and no demarcation remains between testes and the surrounding parenchyma.
The results obtained by cytophotometric analysis of the nuclear FeulgenDNA content show that the mean values of the DNA amount corresponding to
the triploid chromosome set in parenchymal (46-13 + 0-26 a.u.) and in blastema
cells (47-06 + 0-38 a.u.) in Iseo specimens were significantly higher than those
obtained from the same cell types in Pisa specimens (30-41 ± 0-19 a.u. for paren-
74
V. GREMIGNI, C. MICELI AND E. PICANO
chymal cells and 31-15 ± 0-25 a.u. for blastema cells). This finding was expected
on the basis of the different chromosome numbers of the two populations.
Using as reference the In = 2c value of the DNA amount, measured in Pisa
specimens, we could then easily recognize male gonia in Iseo specimens. As a
final step in this investigation, we aimed at checking whether or not nondividing cells with a In = 2c DNA amount were still present in the regenerated
gonad-free caudal area in Iseo specimens. The data obtained showed that 5-6 % of
cells in this regenerated area had a DNA amount equal to that of male germ cells.
The results may be interpreted as an indication that the cut performed at the
caudal level causes the male gonia to move from the testes towards the blastema as
previously demonstrated by the karyological technique (Gremigni & Puccinelli,
1977 ;Gremigni,Miceli&Puccinelli, 1980). During regeneration these cells of male
origin, worked together with other blastema cells of different origin, chromosome
number and nuclear DNA amount, to rebuild the previously removed tissues.
As to the role of male germ cells in planarian regeneration, two questions
need primarily be presented. The first is: can male gonia be considered as differentiated cells, able to dedifferentiate in forming the blastema? Generally,
planarian spermatogonia are poorly differentiated cells and are hardly distinguishable from undifferentiated cells by their morphological, histochemical
and ultrastructural characteristics (Fedecka-Bruner, 1967; Franquinet &
Lender, 1973; our unpublished observations). On the other hand, spermatogonia in Iseo specimens exhibit clear signs of differentiation allowing us to distinguish them from either the embryonic or reserve cells. Primordial male germ
cells, in fact, exhibit a diploid chromosome set due to the fact that triploid
undifferentiated cells eliminate a haploid set in male territory (Benazzi, 1957).
The second question concerns the real significance of cells with a. 2n = 2c
DNA amount in the regenerated caudal area of Iseo specimens. One could
hypothesize that none of these interphase cells are differentiated or differentiating cells. In other words, all cells of male origin could be interpreted as resting
cells which 'not having found' the male teritory in the regenerated tissue, remain undifferentiated and inactive. According to this hypothesis they would be
unipotent cells. This fact would be in contrast with the widely accepted concept
that all blastema cells are pluripotent and capable of differentiating into any
cell type according to the field influence that they encounter during regeneration
(Child, 1920; Wolff, Lender & Ziller-Sengel, 1964). It is also worth noting that
it has been proven that a whole planarian can be rebuilt even from very small
fragments (Montgomery & Coward, 1974) where it is unlikely that 'stem cells'
for every tissue could exist.
In conclusion, a possible interpretation of the regenerative event in the case
studied is the following: reserve cells and/or dedifferentiated somatic cells,
both having 3« = 3c DNA amount, play a major role in planarian regeneration.
Our findings indicate that cells with 2n = 2c DNA amount, coming from the
male territory also participate in rebuilding gonad-free tissues.
Germ cells in planarian regeneration: II
75
Whether or not these cells redifferentiate into cell types different from the
original ones has not been directly demonstrated as yet. However, the planarian
biotype (Iseo) we are working with at present, provide a suitable system where
this question could be tested by analysing the DNA content of specialized cells.
We are grateful to Professor F. D'Amato (Director of the Institute of Genetics) for having
allowed us to use the micro-densitometer and for his helpful suggestions. We wish also to
thank Dr Cionini and Dr Cremonini for their assistance in the use of the instrument and
Mr. Irving Kaufman for his help in translating the paper.
This work was supported by the CNR of Italy.
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{Received 6 June 1979, revised 20 My 1979)