close

Вход

Забыли?

вход по аккаунту

?

H3-thymidine autoradiographic studies on cytokinetic responses to x-ray irradiation and to thio-TEPA in the neural tube of mouse embryos.

код для вставкиСкачать
H3-thymidineAutoradiographic Studies on Cytokinetic
Responses to X-ray Irradiation and to Thio-TEPA
in the Neural Tube of Mouse Embryos '
SETSUYA FUJITA? MASAKIYO HORII,2 TAKASHI TANIMURA3
AND HIDE0 NISHIMURA3
Department of Pathology, Kyoto FTitsu Medical College and Department
of Anatomy, Faculty of Medicine, Kyoto University,s Kyoto, j a p a n
First, using the methods of H3-thymidine autoradiography and counting
ABSTRACT
mitotic index, cytokinetics of the matrix cells were studied in the telencephalon of
normal mouse embryos at ten-days-postconception, and various kinetic constants of
the matrix cells were determined: Generation time, five hours 20 minutes; mitotic
duration, 24 minutes; presynthetic resting time, two hours 36 minutes-one hour 36
minutes; DNA synthetic time, one hour 20 minutes; and post synthetic resting time,
one-two hours. Based on this information, effects of two teratogenetic agents, x-rays
and thio-TEPA, on the cellular proliferation were analyzed. By x-ray irradiation (200 r)
only proliferating matrix cells are damaged in the neural tube, but not neuroblasts.
The radiation induces a temporary block of the flow of the matrix cells through the
cell cycle a t the late t z period so that the mitotic and DNA synthetic cells subsequently
decrease in number. Some of the matrix cells that are captured a t tz period fail to
tolerate the block, degenerate and are eliminated from the matrix layer. On the other
hand, thio-TEPA, which was proved non teratogenetic to the C.N.S.i n this experimental
condition, causes a slight prolongation of t z duration, but does not significantly influence the proliferative process in the neural tube.
Concerning teratogenesis in the central of the proliferating cells in the neural tube
nervous system, it has been generally have remained unknown. As Carlson ('54)
known that many teratogenetic agents pointed out, classical histologists who
evoke primary disturbances in cellular pro- noticed merely increase of the mitotic
liferation in the neural tube (Hicks, De- figures in the neural tube have often
Harport, Johnson and Brown, '55; Hicks, drawn the wholly unjustifiable conclusions
'60; Zwilling, '55). However, controversy that the mitotic activity has been increased
still exists as to the nature of the primary or cellular proliferation has been stimuresponses in the cellular proliferation that lated.
Recently Sauer ('35), Sidman, Miale
finally leads to the malformations; many
embryologists who observed arrest of cellu- and Feder ('59), Sauer and Walker ('59),
lar proliferation and degenerative changes Hicks, D'Amato, Coy, O'Brien, Thurston
in the cell system after the treatment with and Joftes ('61), and Fujita ('60, '62, '63)
teratogenetic agents, conclude that the demostrated that, in the early neural tube
suppression of the cellular proliferation of vertebrates, the matrix cells (synonyms:
and depopulation from the cell system is spongioblasts and germinal cells of HIS,
the primary cause of teratogenesis. On primitive ependymal cells) are the only
the contrary, others such as Patten ('53) cells that are capable of proliferation and
and Murakami ('55) stressed hyperplas- that, between divisions, they perform an
tic malformations. Now it is important elevator movement; nuclei of the matrix
to decide whether arrest of cellular prolif- cells synthesize DNA in the S-zone or in
eration or hyperplasia is evoked in the the deeper half of the matrix layer. ascend
neural tube as the response to teratogene- toward the internal limiting membrane as
tic agents. However, it has been very dif- they finish synthesis, divide directly beficult to demonstrate detailed changes in neath the ventricular surface and, as they
the proliferative activity in the neural tube
investigation was supported by a Grant-inof mammalian embryos, since the mode Aid1 This
for Co-operative Research from the Ministry of
of cellular proliferation and the kinetics Education in Japan.
ANAT. REC.,149: 3748.
37
38
S. FUJITA, M. HORII, T. TANIMURA AND H. NISHIMURA
get through mitosis, descend into the Szone to start their generative cycle again.
Thus the nuclei of the matrix cells are arranged in well defined M-, I- and S-zones in
the matrix layer owing to their phases in
the generation cycle: cells in the mitotic
phase are in M-zone, cells in the presynthetic and postsynthetic resting periods gather
in I-zone and those in DNA synthetic stage
are crowded in S-zone. In the earlier
stages of development, the matrix cells
form a single homogeneous cell population as demonstrated by autoradiography
(Fujita, '63) and by electron microscopy
(Fujita and Fujita, '63). Recently, a
method of kinetic analysis of the matrix
cells by means of the cumulative labeling
technique of H3-thymidine autoradiography
was reported (Fujita,'62).
Now the question raised above may be
rewritten as: what effect do the teratogenetic agents exert on the kinetics of the
matrix cells? This is now accessible by
aid of the H3-thymidine autoradiography.
Besides, the stratified arrangement of the
nuclei of matrix cells owing to their phases
in the generative cycle seems to facilitate
the analysis. First, the present experiments were planned to analyze the primary cytokinetic responses of the matrix
cells in the neural tubes of mouse embryos
to x-ray irradiation. The authors used
mouse embryos at ten-days-postconception.
It is known that x-ray irradiation at this
date frequently causes malformations of
the central nervous system, such as microcephalia, hydrocephalia, brain hernia
(Murakami, Kameyama, Majima and
Sakurai, '61). On the other hand, Tanimura and Nishimura ('62) reported that
thio-TEPA (triethylenethiophosphoramide;
TESPA), a radiomimetic substance (Pradhan, West, Baird and Stewart, '61), when
given to mouse embryos at ten-days-postconception, results mostly in malformation
of the extremities and never of the central
nervous system. Therefore, the authors
intended secondly to elucidate the mode of
the action of the radiomimetic substance
on the proliferation of the matrix cells,
in comparison with x-ray irradiation and
tried to find the differences in the primary
actions of these two teratogenetic agents
on the cytokinetics in the neural tube.
Fig. 1 Schematic representation of an elevator movement of the matrix cell. M, I and
S indicate M-zone (zone of mitosis), I-zone (intermediate zone) and S-zone (zone of DNA
synthesis) respectively; m, mantle layer; t s , DNA synthetic time; tz postsynthetic resting
time; tM,mitotic time; tl, presynthetic resting time, and t c , generation time.
CYTOKINETIC RESPONSES TO TERATOGENS
MATERIALS AND METHODS
Seventeen CF1 strain pregnant mice
were used at ten-days-postconception. They
were divided into three groups consisting
of five, three and nine mice respectively.
All the animals, except five out of nine in
the group 3 , were treated with H3-thymidine as shown in figure 2. The dosage of
H3-thymidine ( 7 K!/gm body weight) was
determined by taking in consideration of
the findings of previous investigators that
5-7 &/gm body weight is necessary for
optimal trans-placental labeling of fetuses
(Uzman, '60; Miale and Sidman, '61) and
that even in a higher dosage (8.1 &/gm
body weight) no effect of deleterious influence of the radiation on the offspring
was observed in long-term experiments
during three months (Uzman, '60).
All the embryos of the sacrificed animals were fixed in Carnoy's solution, embedded in paraffin and cut in serial sections. Each mitotic index was determined
from the average of counts of 1,000 matrix
cells in Feulgen stained cerebral vesicles of
three survived littermates from five irradiated mothers (no. 9 to 13). The sections
Group i
<
Groupz.
Crraups
J , 7pC/gm.H3-thymidine
C , 5 pq/pthio-TEPA i
; J ; 3.SyC/gm.H1-tkymidine;
I , E O O I - . x-ray;
x
sacrifice.
Fig. 2
Schedule of experiment.
39
of the labeled animals were stained with
Meyer's hematoxylin prior to the autoradiography and suitable sections of the cerebral vesicles were selected and autoradiographed by the usual stripping film technique. The film used was FUJI ET-2cE autoradiographic plates of 15 I-I in thickness.
Exposure duration was ten days. The films
were developed in FD 111 developer at
20°C for four minutes. The processed
films were inverted and stained with
Giemsa solution or again with Meyer's
hematoxylin.
RESULTS
Control group
In the wall of the cerebral vesicles from
the embryos which had been labeled for
one hour with H3-thymidine, labeled cells
were restricted to the S-zone of the matrix
layer (fig. 7). No mitotic cells in the
M-zone had taken the label. The percentage of the labeled cells counted from eight
littermates was 44.1% and the mitotic
index was 8.1% . In the embryos which
were labeled for two hours the number
of the labeled matrix cells increased and
all mitotic figures were found to be radioactive (fig. 8). Labeled cells were also
found in the I-zone. They are cells in t2
stage, which have finished DNA synthesis
and are ascending toward the M-zone. The
percentage of labeled cells in the matrix
layer counted from six littermates reached
60.4%. In the periphery of the matrix
layer, a thin layer of neuroblasts, i.e. the
mantle layer began to appear but here all
the cells were found completely free from
the radioactivity. After three hours of
cumulative labeling, the percentage of
labeled cells increased to 82.8%. And
after five hours of cumulative labeling,
all matrix cells in the telencephalic wall
had taken the label without discrimination
of M-, I- and S-zones, though the distribution of the silver grains was not uniform.
After seven hours of cumulative labeling
this distribution of the grains became quite
homogeneous over every nucleus of the
matrix cells (fig. 9). The measurements
of percentage of labeled cells in this series
are shown in figure 3 by the curve marked
C which increases fairly linearly with time
and rises to 100% in a time of four hours.
40
S . FUJITA, M. HORII, T. TANIMURA AND H. NISHIMURA
% LC
100
-I
0
1
2
3
4
5
6
7
ht-
Fig. 3 Measurements of the percentage of labeled matrix cells plotted against time.
C indicates the control; T thio-TEPA group; and R irradiated animals.
Group 2 (thio-TEPAgroup)
In the embryos which were in contact
with the H3-thymidine for two hours after
thio-TEPA injection, the labeled cells in
the matrix layer were mostly restricted to
the S-zone but some radioactive nuclei
were found in the I-zone (fig. 10). The
percentage of the labeled cells counted
from six littermates was 61.7%. This
value is approximately the same as in the
control (compare fig. 10 with fig. 8). But
most of the mitotic cells in the M-zone
were still free from radioactivity. On the
other hand, embryos which were labeled
for three hours with H3-thymidine after
thio-TEPA injection, the labeled cells markedly increased (fig. 11) and the percentage
counted from three littennates reached
85.0%. All mitotic figures in the M-zone
were heavily radioactive. In embryos
which were labeled cumulatively for seven
hours by successive injections of H3-thymidine, all matrix cells were densely and uniformly labeled (fig. 12). A few pycnotic
nuclei were found in the matrix layer but
they were not so numerous as in the
embryos irradiated by x-rays nor did they
show any tendency to gather in a certain
zone in the matrix layer. The measurements of percentage of labeled cells in
this thio-TEPA group are shown in figure
3 by the curve marked T .
Group 3 (x-irradiatedgroup)
In the embryos which received the injection of H3-thymidine immediately after
the x-ray irradiation and were killed two
hours later (fig. 1 3 ) , the labeled cells in
the wall of the telencephalon were restricted to the S- and I-zones. In the Mzone, mid-mitotic cells were rare and no
label had appeared over the mitotic figures.
Prophase nuclei directly beneath the internal limiting membrane did not increase in
number. Except for these differences, the
basic pattern of the labeling is quite similar to that of the control (cf. fig. 8). Average counts of the labeled cells from three
surviving littermates was 58.2%. After
three hours of cumulative labeling the
percentage of the labeled cells counted
from five littermates increased to 64.4%,
but this is very low in comparison with
that of the control. After five hours of
cumulative labeling, the percentage of the
labeled cells counted from two littermates
was 74.9%, which is far lower than the
control. Corresponding to this abrupt and
marked arrest of the increase in the percentage of labeled cells, in the embryos
which received H3-thymidine three hours
after the irradiation and were killed two
hours later (fig. 14), the labeled cells were
reduced approximately to 2 0 % . This reduction in number of labeled cells is
CYTOKINETIC RESPONSES TO TERATOGENS
chiefly due to marked depopulation of the
DNA synthetic compartment in the Szone (compare fig. 14 with fig. 8 ) . The
matrix layer also showed marked changes
in cellular morphology. Many rounded
pycnotic cells with condensed cytoplasm
were found in S- and I-zones. No mitotic
figures in the M-zone had incorporated the
label. The neuroblasts in the mantle layer
remained morphologically intact everywhere. In the telencephalic wall of the
embryos which were labeled cumulatively
for seven hours after the irradiation (figs.
15, 16), the upper half of the matrix layer
consisted of the matrix cells, which are
morphologically relatively normal, and
98% of the nuclei in this portion had
incorporated the label. However, the silver
grains were not as uniform or densely distributed as in the control (compare fig. 15
with fig. 9). This uneven distribution of
the radioactivity among the matrix cells
in this layer indicates that some of them
had not entered into the DNA synthetic
stage during the last seven hours. On the
other hand, in the deeper half of the
matrix layer, many cells showed marked
regressive changes in their nuclei and
cytoplasm, but most of them had taken the
label heavily as shown in figure 16. The
measurements of the percentage of labeled
cells in this irradiated group are shown
in figure 3 by the curve marked R and
changes in mitotic rates in various times
after x-ray irradiation are illustrated in
figure 4.
41
linearly with time (cf. curve C in fig. 3 ) .
This finding indicates that the matrix cells
form a homogeneous cell population in
mice, also, as in chick embryos (Fujita,
'63).
Therefore, the method of kinetic analysis previously described with the chick
embryo (Fujita, '62) can be applied to
this matrix cell system of the mouse. Thus
the generation time and DNA synthetic
time of the matrix cell of the mouse embryo at ten-days-postconception can be determined from figure 3 as follows. The
increase of labeled cells in the control
group (indicated by the curve C in fig. 3 )
may be redrawn schematically as figure 5.
The curve starts from a at time zero,
increases linearly and reaches 100% at
time b. Here a, the fraction of matrix
cells which take up the label at the time
of H3-thymidine injection are the fraction
of cells that are in the phase of DNA synthesis at that time. Thus in an asynchronous population of cells such as matrix
cells, the percentage a represents the relative length of ts the period of DNA synthesis as a fraction of the generation time
tc, i.e. a = 100 X ts/tc. The time b cor-
% LC
100 -
%MI
41
1
/
hr
0
0
1
2
3
4
5
6
7
24
Fig. 4 Mitotic indices of the matrix cells in
the cerebral walls at different times after x-ray
irradiation. MI in the control is 8 . 1 % .
DISCUSSION
In the normal embryos, the percentage
of labeled matrix cells increased nearly
Fig. 5 Schematic figure showing increase of
labeled matrix cells in control animals during
cumulative labeling. Ordinate, percentage of
labeled matrix cells; abscissa, time in hours.
The labeled cells increase linearly and soon
reach 100%.
s.
42
FUJITA,
M. HORII, T . T A N I M U R A AND H. N I S H I M U R A
responds to ( t -~ t s ) , as Fujita reported
previously ('62).
As seen in figure 5,
u:IOO = C : C
While,
Thus,
(100
+ b.
a = 100 x t s / t c , and
b = tc - t s .
x t S / t G ) :loo = c:c + ( t C - t S ) .
tG= c + b, and ts = c .
Direct measurements of ( c b ) and c
from the figure 3 gave t G = 5 hours 20
minutes, and ts = 1 hour 20 minutes.
Mitotic time, t M , postsynthetic resting
time, te, and presynthetic resting time,
ti, of the matrix cell were calculated as
described previously (Fujita, '62). Namely,
mitotic index = 100 X t M / t cthus
,
mitotic
time, tM = mitotic index X t c / l O O = 8.1
X 5*/3/100 = 0.4 hours = 24 minutes.
Duration of tz can be determined from the
time required for labeled DNA to appear
in the mitotic figures in the M-zone (cf.
fig. 1 ) . Since the labeled mitosis appeared
from 1 to 2 hours after the injection of
the label, tz was estimated to be 1 to 2
hours, and ti was calculated from ti = t c ( tz tS tw ) = 1 hour 36 minutes - 2
hours 36 minutes. Thus each fraction of
the cell cycle for the matrix cells in the
cerebral vesicle of the mouse embryo at
ten-days-postconception was determined as
is summarized in figure 6.
+
+ +
matrix cell is not primarily affected by
irradiation, and ( 2 ) cellular feed from
tI into ts compartment is not markedly suppressed by x-rays for at least two hours
after irradiation, since the labeled cells
increased at the normal rate in the meantime.
The mitotic frequency curve (fig. 4 )
showed one phasic change, it abruptly
dropped immediately after irradiation and
recovered to the normal level in three
hours or so time after the irradiation.
Similar changes in the mitotic frequency
curve after irradiation have been repeatedly reported by previous investigators in
various cell systems (Tansely, Spear and
Glucksmann, '37; Carlson, '54). The fact
that no time lag is observed between the
irradiation and the drop of the mitotic
frequency curve suggests that the irradiation arrested the cellular flow in the cell
cycle at the period just prior to mitosis,
that is, at the late tz period. While Lajtha
('60) reported a block of cell flow at late
tl period in various cell systems. If, however, the generation cycle of the matrix
cell were blocked at late ti period, the drop
of the mitotic frequency curve in this case
would first appear 3 to 4 hours after the
irradiation, since the matrix cells that were
in t2 and ts periods at the time of irradiation should proceed normally in their cell
cycle and continue to enter into mitotic
phase with normal rate so that the mitotic
frequency curve would be kept at normal
level for tz
ts = 1:36-2:36
2:30 =
3-4 hours. Such a time lag has never
been observed in our experiments nor in
other cell systems as mentioned above.
The hypothesis of a late &block is also
inconsistent with the above conclusions
(1)-(2) drawn from the autoradiographic
data.
On the other hand, the percentage of
the labeled matrix cells increased almost
normally for the first two hours after
irradiation, but this tendency changed
rather abruptly thereafter (cf. curve R in
fig. 3); the rate of the increase is greatIy
reduced for the next three hours (from
2 to 5 hours after the irradiation). Short
term labeling during two hours in this
period (fig. 14) revealed that cellular feed
into the DNA synthetic compartment in
the meantime was much reduced; the per-
+
Fig. 6 Cell cycle of the matrix cells in the
cerebral wall of mouse embryo at ten-days-postconception. The numbers in the figure indicate
hours :minutes.
In the irradiated telencephalon which
were exposed to H3-thymidine for two
hours immediately after irradiation, the
percentage of labeled matrix cells was
nearly identical with that of the control
(compare the curve R with the curve C,
in fig. 3 ) . This finding indicates that:
( 1 ) the process of DNA synthesis of the
+
CYTOKINETIC R E S P O N S E S T O TERATOGENS
43
centage of labeled cells after two hours of externally from the matrix layer. The basic
the labeling in this case was very low phenomenon common in both these cases
(20% ) in comparison with that in the nor- is their loss of contact with the internal
mal animal (60.4% ). This abrupt reduc- limiting membrane, though the necrotic
tion of cellular feed into the DNA syn- cells are not differentiating neuroblasts as
thetic compartment that appeared two discussed later. Therefore, by analogy,
hours after irradiation can be interpreted though superficial, it is inferred that the
reasonably on the assumption of the late matrix cells performing the elevator move&-block,as we will discuss below. Accord- ment pump out the neuroblasts just like
ing to the tz-block hypothesis, the cells the necrotic cells from the matrix layer
that were in t~ and M
t periods at the time that have lost their contact with the inof the irradiation are saved from damage ternal limiting membrane.
After seven hours of cumulative labeland proceed in the cell cycle at a normal
rate so that the cellular feed into the DNA ing, the majority of the pycnotic nuclei
synthetic compartment is kept normal for which accumulated in the periphery of the
M
t 4- tl hour after irradiation, i.e. one hour matrix layer had heavily incorporated the
24 minutes-two hours 24 minutes. This label (fig. 16). This finding clearly indicorresponds approximately to the interval cates that they degenerated after they had
of two hours between the irradiation and normally synthesized DNA. This concluthe abrupt reduction in the cellular feed sion strongly favors the assumption that
into the DNA synthetic compartment that further progress of the matrix cells
was observed in our experiments. And through the cell cycle is blocked and the
after this time, the cellular feed into the degeneration is caused at the postsynthetic
DNA synthetic compartment is abruptly period, tz. If the block and the degenerareduced since the cells that were at tr tion took place just before DNA synthesis,
period at the time of the irradiation are i.e. at the late tl period, as Lajtha (’60)
arrested at the late t 2 period and cannot believed, the majority of the necrotic cells
follow the preceding cells. From 5 to 7 should not be labeled with H3-thymidine
hours after the irradiation the labeled cells since the cells that were at tl, tM and tz
again increased at nearly normal rate periods at the time of irradiation must
(fig. 3 ) . This may be due to the recovery suffer the block at the next late tl period
and they could not incorporate the label
of cells from &block.
Pycnotic cells appeared first in the (cf. fig. 1 and fig. 6).
All our observations in the neural tube
I-zone as early as two hours after the
irradiation, and greatly increased in num- of mice as well as those previously reber by four hours and gradually accumu- ported with mouse cells in vitro (Whitlated in the most peripheral zone of the more, Stanners, Till and Gulyas, ’61) supmatrix layer. Since the functional damage port the view that radiation causes a late
of the cells observed so far is restricted to &block and the &degeneration which rethe late t2 stage, the degenerative cells that sults in temporary inhibition of the proliffirst appeared in the I-zone seem to corre- eration and partial elimination of the prospond to the cells that were arrested at the liferating matrix cells. Processes at tM,tl
late tz period and failed to recover from and ts periods are relatively insensitive to
the block. These necrotic cells are rounded radiation, though several investigators asand lose their contact with the internal sume that radiation affects ts (Sherman
limiting membrane. They seem to be and Quastler, ’59) or tl period (Lajtha, ’60).
Hicks, DeHarport, Johnson and Brown
pushed externally from the matrix layer
as the matrix cells perform their up and (’55) believed that the necrotic cells cordown movement. This process of outward respond to the “primitive differentiating
migration of these necrotic cells, though neuroblasts.” However, these necrotic cells
it is a passive one, reminds us of the mode cannot be differentiating neuroblasts, since
of neuroblast production in the neural tube they synthesized DNA after x-ray irradia(Fujita, ’63). In the course of develop- tion. If the differentiating neuroblasts but
ment of the central nervous system, newly not the matrix cells were vulnerable to
formed neuroblasts rapidly migrate out x-rays, they would degenerate without syn-
44
S. FUJITA, M . HORII, T. TANIMURA AND H. NISHIMURA
thesizing labeled DNA because the neuroblast does not carry out further DNA synthesis (Fujita, '63), and the necrotic cells
would be completely free from the radioactivity. Or, even if we assumed that
x-rays block the process of differentiation
of the matrix cells to neuroblasts and
damage the cells at this stage, the majority
of the necrotic cells should be free from
radioactivity; if x-rays affected the process
of differentiation, the matrix cells that
were at t,, tMand t zperiods at the time of
the irradiation and destined to become
neuroblasts should be captured and degenerate without synthesizing labeled DNA at
the time of differentiation, i.e. at the next
tL period (Fujita, '63). Thus the capture
and degeneration of the unlabeled differentiating cells should continue for t l
t M
t z hours, i.e. four hours, so that examination of the necrotic cells after seven
hours of cumulative labeling should reveal
that four-sevenths of the necrotic cells
are unlabeled and the remaining threesevenths are labeled. But, our findings
indicate that most of the necrotic cells
had heavily incorporated the label (figs.
15, 16) so that the possibility of selective
damage to the differentiating neuroblasts
is excluded.
In contrast with the marked cytokinetic
responses against x-rays, matrix cells in
the thio-TEPA group take up H3-thymidine
at almost normal rate except for a slight
prolongation of the time t z ; tz became
longer than two hours but remained still
shorter than three hours. Thus it is concluded that the thio-TEPA exerts negligible
influence on the cytokinetics of the neural
tube in this experimental condition. This
conclusion may be correlated with the
fact reported by Tanimura and Nishimura
('62) that no malformations in the central nervous system were found with this
dosage of thio-TEPA.
Our present experiments revealed that,
with H3-thymidine cumulative labeling
technique, we can analyze in detail the
cytokinetics of matrix cells of the developing central nervous system of mouse
embryos in normal and teratogenetic conditions. It becomes clear that x-ray irradiation in teratogenetic dosage suppresses the
proliferative activities of the matrix cells
+
+
and partially depopulates them, but no
hyperplastic reaction was evoked as far
as the observations shortly after the treatment are concerned. In order to elucidate
the actions of the teratogenetic agents,
however, it will be still necessary to pursue
further cytokinetic reactions in the successive stages following the primary damage, since the primary damage acts indirectly through intermediary processes to
lead to final malformations (Hicks, '55).
Analysis of cytokinetic changes of matrix
cells in subsequent days to the teratogenetic treatments is now in progress in
our laboratory.
LITERATURE CITED
Carlson, J. G. 1954 Radiation Biology. Ed. by
A Hollaender. McGraw Hill Company, New
York. Vol. 1, part 2, pp. 763-824.
Fujita, S. 1960 Mitotic pattern and histogenesis of the central nervous system. Nature,
185: 702-703.
1962 Kinetics of cellular proliferation.
Exptl. Cell Research, 28: 52-60.
1963 The matrix cell and cytogenesis
in the developing central nervous system. J.
Comp. Neur., 120: 37-42.
Fujita, H., and S. Fujita 1963 Electron microscopic studies on the neuroblast differentiation
in the central nervous system of domestic fowl.
Zeitschr. f . Zellforsch., 60: 463-478.
Hicks, S . P. 1960 Acute necrosis and malformation of developing mammalian brain
caused by x-ray. Proc. SOC.Exp. Eiol. Med.,
75: 485-489.
Hicks, S. P., C. J. DAmato, M. A. Coy, E. D.
OBrien, J. M. Thurston and D. L. Joftes 1961
Migrating cells in the developing central nervous system studied by their radiosensitivity
and tritiated thymidine uptake. Fundamental
Aspects of Radiosensitivity: Brookhaven Symposia in Biology No. 14. Upton, N.Y., pp.
246-261.
Hicks, S. P., C. E. DeHarport, L. A. Johnson and
B. L. Brown 1955 The neurochemical significance of experimentally induced malformations in mammals. Biochemistry of the Developing Nervous System. Ed. by H. Waelsch.
Academic Press, New York, pp. 491-498.
Lajtha, L. G. 1960 The Nucleic Acids. Ed. by,
E. Chargaff and J. N. Davidson. Academic
Press, New York. Vol. 3, pp. 527-546.
Murakami, U. 1955 Experimental embryologic
and pathologic study on malformation of the
central nervous system. Acta Pathol. Japon.,
5: 495-513.
Murakami, U., Y. Kameyama, A. Majima and T.
Sakurai 1961 Patterns of radiation malformations of the mouse fetus and subjected stage
CYTOKINETIC RESPONSES TO TERATOGENS
of development. Annual Report of the Research
Institute of the Environmental Medicine, 9:
71-81.
Patten, B. M. 1953 Embryological stages in
establishing of myeloschisis with spinal bifida.
Am. J. Anat., 93: 365-395.
Pradhan, S. N., W. L. West, G. M. Baird and
J. D. Stewart 1961 Effect of thio-TEPA on
the synthesis of protein and nucleic acid in
tumor bearing mice. Cancer Res., 21: 984-988.
Sauer, F. C. 1935 Mitosis i n the neural tube.
J. Comp. Neur., 62: 3 7 7 4 0 5 .
Sauer, M. E., and B. E. Walker 1959 Radioautographic study of interkinetic nuclear migration in the neural tube. Proc. SOC.Exp. Biol.
N. Y.,101: 557-560.
Sherman, F. G . , and H. Quastler 1960 DNA
synthesis in irradiated intestinal epithelium.
Exptl. Cell Research, 19: 343-360.
Sidman, R. L., I. L. Miale and N. Feder 1959
Cell proliferation and migration i n the primitive ependymal zone : A n autoradiographic
45
study of histogenesis in the nervous system.
Exptl. Neurol., I : 322-333.
Tanimura, T., and H. Nishimura 1962 Teratogenic effect of thio-TEPA, a potent antineoplastic compound, upon the offspring of pregnant
mice. Acta Anat. Nippon., 37: 66-67 (Japanese).
Tansely, K., F. G. Spear and A. Gliicksmann
1937 An effect of gamma rays on cell division
in the developing rat retina. Brit. J. Ophthalmol,. 21: 273-298.
Uzman, L. L. 1960 The histogenesis of the
mouse cerebellum as studied by its tritiated
thymidine uptake. J. Comp. Neur., 114: 137160.
Whitmore, G. F., C. P. Stanners, J. E. Till and
S. Gulyas 1961 Nucleic acid synthesis and
the division cycle in x-irradiated L-strain
mouse cells. Biochim. Biophys. Acta, 47: 66-67.
Zwilling, E. 1955 Analysis of Development.
Ed. by B. H. Willier, P. Weiss and V. Hamburger. Saunders Book Co., Philadelphia.
PLATE 1
EXPLANATION O F FIGURES
Figs. 7-9 Autoradiographs of cerebral walls of mouse embryos at
ten-days-postconception.
7
One hour after injection of H3-thymidine. Labeled nuclei are restricted to the S-zone and the mitotic figures i n the M-zone (indicated
by arrows) are completely free from the radioactivity. x 600.
8
Two hours after injection of H”-thymidine. Labeled cells increase
markedly i n number and all mitotic figures in the M-zone (arrows)
have taken the label. But the cells i n the mantle layer (below) are
free from the radioactivity. X 600.
9
After seven hours of cumulative labeling. All nuclei of the matrix
cells have uniformly incorporated the label. X 600.
Figs. 10-12 Autoradiographs of cerebral walls of mouse embryos at
ten-days-postconception which received injection of thio-TEPA two hours
before exposure to H”-thymidine.
10
Two hours after exposure to H3-thymidine. Few labeled cells appear
in the M-zone but the percentage of the labeled matrix cells is not
significantly reduced as in the control (cf. fig. 8 ) . X 600.
11 Three hours after injection of H3-thymidine. Labeled cells increase
and all mitotic figures i n the M-zone (arrows) are heavily labeled.
X 600.
12
46
After seven hours of cumulative labeling. All matrix cells are labeled.
The pattern is quite similar to that of the control shown in figure 9.
X 600.
CYTOKINETIC RESPONSES TO TERATOGENS
Setsuya Fujita, Masakiyo Horii, Takashi Tanimura and Hideo Nishimura
PLATE 1
47
CYTOKINETIC RESPONSES TO TERATOGENS
Setsuya Fujita, Masakiyo Horii, Takashi Tanimura and Hideo Nishimura
PLATE 2
Figs. 13-16 Autoradiographs of cerebral walls of mouse embryos irradiated by x-rays at ten-dayspos tconception.
Two hours after exposure to H3-thymidine which was injected immediztely after x-ray irradiation. Most of the labeled cells remain in the S-zone. Labeled cells have not reached the M-zone.
Mid-mitotic cells are rare. Percentage of the labeled matrix cells is not significantly reduced as
in the control. x 600.
14 Two hours after exposure to H3-thymidine which was injected three hours after the x-ray irradiation. Labeled cells greatly decrease in number, especially those in the S-zone. Many pycnotic
cells appear i n the I- and S-zones. x 600.
15 Autoradiograph after seven hours of cumulative labeling. The matrix cells near the ventricular
surface retain relatively normal morphology and 98% of the cells are labeled. In the deeper
half of the matrix layer many pycnotic cells accumulate. Most of them have incorporated the
label. The portion framed in the figure is shown at greater magnification in figure 16. x 600.
16 Framed portion in figure 15. Most pycnotic nuclei are heavily labeled. A few unlabeled ones
show more advanced stages of necrosis, such as karyorrhexis. x 1,500.
13
48
Документ
Категория
Без категории
Просмотров
2
Размер файла
977 Кб
Теги
thymidine, embryo, mouse, thiol, irradiation, cytokinetic, response, neural, studies, autoradiographic, ray, tube, tepa
1/--страниц
Пожаловаться на содержимое документа