/. Embryol. exp. Morph. Vol. 32, 3, pp. 697-705, 1974
Printed in Great Britain
697
The neural cell cycle in the looptail (Lp)
mutant mouse
By DORIS B. WILSON 1 AND E. M. CENTER 2
From the Department of Human Anatomy,
University of California, Davis, and the Departments of
Biological Sciences and Anatomy, Stanford University
SUMMARY
The cell cycle of mesencephalic ventricular cells was studied by means of tritiated
thymidine radioautography during normal and abnormal development in the looptail (Lp)
mutant mouse. The total generation time, DNA-synthetic (S), premitotic (G2), mitotic
(M), and postmitotic (G]) periods were compared in looptail homozygotes (LpjLp) which
exhibit neural dysraphism and in their normal littermates ( + / + ) at 10 and 11 days'
gestation. Both normal and abnormal embryos showed a chronological lengthening of the
generation time between the 10th and 11th day. However, the generation time in the 10-day
abnormal brains was 4-5 h longer than that in normal littermates, and the difference was
the result of an increase mainly in the M and Gx periods. At 11 days of gestation the
generation time in the abnormal brains increased by 50 h over that of the normal brains.
Since the cell cycle was actually prolonged in the defective brains, the increased numbers
of mitotic figures which characterize the looptail homozygote brain during early development
appear to reflect the lengthening of the mitotic period rather than increased proliferation.
INTRODUCTION
The looptail mutant mouse is characterized by various degrees of twisting
in the tail of the heterozygote (Lp/ + ) and by extensive neural dysraphism in
the homozygote (LpjLp) (Strong & Hollander, 1949). The dysraphism consists
of an open neural tube from midbrain or hindbrain to tail. Increased numbers
of mitotic figures have been noted in the open regions of the midbrain and
hindbrain, and this has led to use of the term 'overgrowth' in descriptions
of this phenomenon (Stein & Rudin, 1953; Stein & Mackensen, 1957). However, an excess of neural tissue has not been demonstrated quantitatively, and
some regions of the open neural tube even show a reduction in cell density
(Stein, Lievre & Smoller, 1960; Smith & Stein, 1962).
Recent cell cycle studies on ventricular cells in dysraphic regions of the
splotch (Sp/Sp) neural tube have shown that the total generation time of
these cells is actually prolonged (Wilson, 1973 a, 1974). Since the increased
1
Author's address: Department of Human Anatomy, School of Medicine, University of
California, Davis, California 95616, U.S.A.
2
Author's address: Department of Biological Sciences, Stanford University, Stanford,
California 94305, U.S.A.
698
D. B. WILSON AND E. M. CENTER
numbers of mitotic figures which characterize the splotch neural defect were
found to be the result of a longer time spent in mitosis, the present radioautographic study was undertaken to obtain quantitative information on the
proliferative process in the neural tube of the looptail homozygote. In this
study comparisons were made of cell cycle data obtained from the midbrain
of normal ( + / + ) and abnormal (Lp/Lp) littermates at 10 and 11 days'
gestation.
MATERIALS AND METHODS
Looptail heterozygotes (Lp/ + ) were obtained from inbred lines maintained
by Dr Kathryn F. Stein at Mount Holyoke College. The animals were kept on
an artificial light-dark cycle (14 h light, 10 h dark), and embryos were obtained
from timed matings in which day 0 was considered as the day on which a
vaginal plug was observed. On either day 10 or day 11 of gestation, pregnant
females were given a single intraperitoneal injection of [3H]thymidine (5 /tCi/g,
specific activity 2-0 Ci/mM). Embryos were removed and fixed in Carnoy's
solution at intervals ranging from 1 to 16 h after injection. Abnormal embryos
showing neural dysraphism were selected along with an equal number of
normal straight-tailed littermates. The embryos were embedded in paraffin,
sectioned at 5 pum, and stained with the periodic acid-Schiff reaction. Radioautographs were prepared by dipping the slides in Kodak NTB-3 emulsion
(Kopriwa & Le Blond, 1962). The slides were exposed in light-proof boxes at
5 °C for 1 month, at which time they were developed in D-19 and stained
lightly with haematoxylin. The radioautographs were examined at x 970 magnification. Because of extremely low background, nuclei containing four or
more grains were considered labelled. Mean mitotic indices were determined
on a total of 1000 ventricular cells in the optic tectum in each of three embryos.
Observations were confined to dorsolateral portions of the tectum midway
between its cranial and caudal ends. A total of 60 abnormal and 60 normal
embryos served as a basis for this investigation, and the length of each period
was determined by means of observations on the appearance of labelled mitoses
at different times after injecting radioactive thymidine (Fujita, Horii, Tanimura
&Nishimura, 1964; Kauffman, 1968, 1969; Hoshino, Matsuzawa & Murakami,
1973; Wilson, 1974).
RESULTS
Litters obtained from looptail heterozygous matings (Lp/ + x Lp\ +) showed
the following characteristics. Approximately 25 % of each litter consisted of
straight-tailed normal homozygotes ( + / + ) , 50% were loop-tailed heterozygotes (Lp/+) and 2 5 % were loop-tailed homozygotes (Lp/Lp) with rachischisis extending from midbrain to varying levels of the tail (Figs. 1, 2).
Although the gross and histological features of the central nervous system
were normal in the + / + and Lpj + embryos, only the straight-tailed individuals
( + / + ) were used as normal controls for the abnormal homozygotes (Lp/Lp).
Cell cycle in looptail mice
699
3
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Fig. 1. Looptail heterozygote (Lpj + ) at 11 days' gestation.
Fig. 2. Looptail homozygote (LpjLp) with open neural tube at 11 days' gestation.
Fig. 3. 10-day normal midbrain 7 h after injection with [3H]thymidine. Arrows
indicate unlabelled mitoses. V, ventricle.
Fig. 4. 10-day abnormal midbrain 7 h after injection with [3H]thymidine. Note large
number of labelled mitoses at ventricular border. V, ventricle.
10-day normal and abnormal embryos
In the normal 10-day embryos approximately 16 % of the ventricular mitotic
nuclei were labelled 1 h after injection of the isotope. The percent labelled
mitotic nuclei dramatically increased during the next 4 h , at which time
700
D. B. WILSON AND E. M. CENTER
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Fig. 5. The cell cycle in the normal ( + / + ) midbrain (continuous line) and in the
abnormal (LpjLp) midbrain (broken line) at 10 days' gestation. Abscissa: hours
after injection with [3H]thymidine. Ordinate: mean percent labelled mitoses.
approximately 94 % were labelled. In the abnormal embryos, 10 % of the
mitotic figures were labelled 1 h after injection, and 95 % were labelled after
4h.
Seven hours after injection distinct differences were noted when the percent
labelled mitoses dropped to 20 % in the normals in contrast to 60 % in the
abnormals (Figs. 3, 4). Few labelled mitoses were seen in the normals 8 h after
injection whereas approximately 40 % were still labelled in the abnormals.
A second wave of labelled mitoses began in the normal brains 9 h after
injection, and by 14 h 85 % were labelled. In the abnormal brains the second
wave of mitotic labelling occurred at 13-5 h, and only 15 % were labelled at
16 h. Fig. 5 shows the percent labelled mitoses plotted graphically against
time after injection for normal and abnormal embryos at 10 days' gestation.
The gestation time read directly from the graph was 9-0 h for the normal
midbrain, and the interval between the 50 % points on the ascending and
descending limbs of the curve indicated a DNA-synthetic (S) period of 5-0 h.
The mitotic index (MI) was 12-1 % (S.E. ±0-23). The duration of mitosis (M)
was thus MI/100 x generation time or approximately 1-1 h. Since the 50%
point on the ascending curve is equal to the premitotic (G2) period plus \ the
length of mitosis (M), G2 was calculated as 0-9 h (1-5 h minus \ x 1-1 h). The
postmitotic (Gt) period was determined by subtracting the sum of S, M and
G2 from the total generation time and was equal to 2-0 h.
701
Cell cycle in looptail mice
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Fig. 6. The cell cycle in the normal ( + / + ) midbrain (continuous line) and in the
abnormal (Lp/Lp) midbrain (broken line) at 11 days' gestation. Abscissa: hours
after injection with [3H]thymidine. Ordinate: mean percent labelled mitoses.
Table 1. Duration of generation time, DNA-synthetic (S), premitotic (G2),
mitotic (M), and postmitotic (Gx) periods for normal ( + / + ) and abnormal
(Lp/Lp) embryos
Duration (h)
time
S
G2
M
Gx
.10 days
Normal
Abnormal
90
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50
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0-9
0-8
11
2-3
20
4-9
11 days
Normal
Abnormal
10-5
15-5
60
80
10
0-6
10
1-9
2-5
50
Cell cycle data for the 10-day abnormal midbrain were as follows. The
generation time was 13-5 h, S was 5-5 h, MI 17-2 % (s.E. ±0-29), M was 2-3 h,
G 2 0-8 h, and Gx 4-9 h.
11-day normal and abnormal embryos
Labelled mitotic figures were not observed 1 h after injection in either
normal or abnormal brains. However, at 2 h after injection approximately
80 % were labelled in the normals and 74 % in the abnormals. A peak of
approximately 90 % labelled figures was attained by both groups at 4 h and
45
EMB
32
702
D. B. WILSON AND E. M. CENTER
remained at this level until 6 h, after which the percent dropped dramatically
to 44 % at 8 h in the normal brain, although the abnormals still showed a high
percent (76 %) of labelling.
The percent labelled mitoses dropped to a low point of 5 % in the normal
embryo at 10 h, while the percent in the abnormal brain did not reach the
low point until 14 h after injection. Whereas the percent abruptly increased
after 10 h in the normals, it showed a gradual climb after 15 h in the abnormals.
These data are plotted graphically in Fig. 6.
The 11-day cell cycle data were determined as above for the 10-day embryos.
In the normal 11-day brains the generation time was 10-5 h, S was 6-0 h,
MI 9-9% (S.E.±0-16), M 1-0 h, G 2 1-0 h and Gx 2-5 h. For the abnormal
11-day brains the generation time was 15-5 h, S was 8-0 h, MI 12-3 % (S.E. ± 0-30),
M 1-9 h, G2 0-6 h and Gx 5-0 h.
Table 1 summarizes the data for the normal and abnormal brains at 10 and
11 days of gestation.
DISCUSSION
Tritiated thymidine radioautography has been used to obtain quantitative
data on the cell cycle during normal development of the mouse spinal cord
(Kauffman, 1968) and telencephalon (Hoshino et al. 1973), although only
approximate values can be determined for the length of each period in the
cycle. For example, the duration of mitosis (M) depends on calculations of
mitotic index x generation time, and the postmitotic (G r ) and premitotic (G2)
periods are calculated indirectly by subtraction. However, despite such limitations with the in vivo pulse labelling technique, valuable data have been
obtained particularly with respect to comparisons of the cell cycle during
normal and abnormal development (Fujita et al. 1964; Kauffman, 1969;
Konyukhov & Sazhina, 1971; Wilson, 1973a, 1974).
In the present study the results on the cell cycle of ventricular cells in the
tectum of the normal littermates ( + / + ) of loop tail homozygotes (Lp/Lp) are
similar to those obtained for normal littermates ( + / + , Sp/+) used as controls
for splotch homozygotes (Sp/Sp) in previous studies (Wilson, 1973 a, 1974).
For example, the generation time at 10 days' gestation in normal littermates
of looptail and splotch embryos was 9-0 h and 8-5 h, respectively. The normal
embryos also showed a chronological increase in the generation time between
the 10th and 11th day of gestation, and this increase resulted primarily from
a lengthening of the S and Gx periods. Similar chronological increases in the
S and Gx periods have been observed in mouse thoracic spinal cord (Kauffman,
1968) and telencephalon (Hoshino et al. 1973). Although cell cycle studies on
the chick mesencephalon likewise have shown a chronological increase in the
generation time between the 3rd and 6th day of incubation this was due to
a lengthening of Gx and M (Jelinek, 1959; Jelinek & Klika, 1961; Kallen,
1961, 1962; Wilson, 1973ft).
Cell cycle in looptail mice
703
In the abnormal looptail embryos (Lp/Lp) the generation time of tectal
ventricular cells was 4-5 h and 5 h longer than that of their normal littermates
at 10 and 11 days of gestation, respectively. The prolongation in the 10-dayold abnormal embryos was the result of increases primarily in M and Gx.
Lengthening of these two periods of the cell cycle also was responsible for the
increased generation time observed in the 10-day-old abnormal splotch embryos
(Wilson, 1974). At 11 days of gestation the increased generation time of the
abnormal looptail brains resulted from lengthening of the S, M, and Gx
periods; the 11-day-old abnormal splotch brains showed similar changes in
the cell cycle (Wilson, 1974). The generation time also increased in retinal
cells in the mouse mutants ocular retardation (or)and fidget (fi), although
this was attributable to a lengthening primarily of Gx (Konyukhov & Sazhina,
1971).
Urethane treatment likewise produced a prolongation of the generation
time in the 10-day mouse spinal cord, but the increase occurred in the S,
G 2 and Gx periods, while M showed no change (Kauffman, 1969). Exogenous
teratogenic agents, however, are often cytotoxic (Fujita et al. 1964), whereas
cell damage or increased cell death could not be detected in the early mutant
embryos of the present study. Mutant genes or teratogenic substances thus
may influence various portions of the cell cycle to produce an overall lengthening of the generation time, although Gx appears to be most commonly
affected.
The elevated mitotic index in the looptail homozygotes (Lp/Lp) corroborates
earlier observations on an absolute increase in the number of mitotic figures
(Stein et al. 1960; Smith & Stein, 1962). However, the cell cycle data of the
present study indicate that the cells spend longer periods of time in mitosis
and that the proliferative process is actually prolonged. This would explain
the discrepancy between the 'overgrowth' described in earlier studies and the
failure to observe an increase in total cell number in the hindbrain (Stein et al.
1960). The presence of increased numbers of mitotic figures thus is not necessarily an indication of increased proliferation, and the length of the entire
generation cycle, including mitosis, must be taken into account in order to
obtain a true indication of proliferative activity. Whether or not the prolongation
results in a marked decrease in cell number is currently under investigation
by means of DNA determinations.
Although little is known about the origin of the neural defect in the looptail
homozygotes, a basic failure in proper axial elongation of the neural tube and
notochord has been postulated (Smith & Stein, 1962). Whether or not the
retardation in the neural cell cycle is a cause or an effect of the abnormality
remains to be determined; however, quantitative studies on cellular kinetics
of the neural tube and notochord during the 8th and 9th days of gestation
may eventually provide an answer to this question. Since the body weight of
the abnormal embryos is less than that of normal littermates, especially at
45-2
704
D. B. WILSON AND E. M. CENTER
later stages of gestation, it is possible that the cell cycle in other organ systems
may also be affected.
Of interest also is the question of whether the single dose of the looptail
gene in the heterozygote has any effect on the neural cell cycle, especially since
behavioural deficits and structural abnormalities of the lateral ventricles have
been described in postnatal and adult looptail heterozygotes (Van Abeelen,
1966, 1968; Van Abeelen & Raven, 1968). However, since a prolongation of
only 4 h in the generation time is associated with severe closure defects in the
homozygote, it is possible that lesser effects on the cell cycle in the heterozygote
may not be detectable with current in vivo radioautographic techniques.
This work was supported by NIH research grant HD 08216-01 from the National Institute
of Child Health and Human Development, United States Public Health Service and by
American Cancer Society Institutional Grant No. 1N32-N. The authors wish to express
appreciation to Dr Kathryn F. Stein, Mount Holyoke College, for generously providing
the mutant looptail stock used in the present study.
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3
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(Received 19 February 1974, revised 13 June 1974)