/ . Embryol. exp. Morph. Vol. 29, 3, pp. 745-751, 1973
745
Printed in Great Britain
Chronological changes in the cell cycle of chick
neuro epithelial cells
By D. B. WILSON 1
From the Departments of Surgery and Biology,
University of California, San Diego
SUMMARY
Radioautographic data obtained from a total of 103 chick embryos injected with
[3H]thymidine suggest that the generation time of neuroepithelial cells in dorsolateral
regions of the optic tectum increases from approximately 8 h at 3 days of incubation to
15 h at 6 days of incubation. The most marked change occurs between the 4th and 5th day,
when the generation time increases from 9 to 13 h, respectively. Between the 3rd and 6th day
of incubation the length of the DNA-synthetic (S) period and of the premitotic (G2) period
remains fairly constant; however, the duration of mitosis (M) and of the postmitotic (Gj)
period appears to be prolonged.
INTRODUCTION
Proliferation in the embryonic central nervous system has been studied by
various methods, including radioautography (Sidman, Miale & Feder, 1959;
Fujita, 1962, 1963; Kallen & Valmin, 1963; Martin & Langman, 1965; Langman, Guerrant & Freeman, 1966; Kauffman, 1968) and the use of mitotic
inhibitors (Watterson, Veneziano & Bartha, 1956; Jelinek, 1959; Jelinek &
Klika, 1961; Kallen, 1961, 1962; Langman et al. 1966). Absolute counts of
mitotic figures, as well as calculations of mitotic indices and mitotic densities
have also provided information on neural proliferation (Hamburger, 1948;
Kallen, 1961; Cowan, Martin & Wenger, 1968). Chronological changes have
been observed in the duration of the cell cycle in chick and mouse spinal cord
(Fujita, 1962; Kauffman, 1968) and in the length of mitosis in chick spinal cord
and optic tectum (Jelinek, 1959; Jelinek & Klika, 1961; Fujita, 1962; Kallen,
1962). A gradual increase in the absolute number of mitotic figures has been
noted in the chick optic tectum until the sixth day of incubation, after which
a sharp decline occurs (Cowan et al. 1968), whereas colchicine studies show
a peak in 'relative mitotic rate' at approximately 3-5-4 days of incubation
(Kallen, 1961).
The present radioautographic study was undertaken in order to analyse
cellular proliferation with respect to chronological changes in the duration of
1
Author's address: Department of Human Anatomy, School of Medicine, University of
California, Davis, California 95616, U.S.A.
746
D. B. WILSON
Table 1. Number of embryos labeled with [3H]thymidine for varying periods of
time between the 3rd and 6th day of incubation
Incubation age (days)
Duration (h)
of labeling
1
1-5
2
4
6
8
9
10
11
12
13
14
15
16
18
Total
c
3
4
5
6
2
3
2
3
2
2
2
2
2
1
0
1
0
1
0
23
2
3
2
3
2
2
2
2
2
1
0
1
0
1
0
23
2
2
2
1
1
1
2
2
2
2
3
3
2
2
1
2
3
2
1
1
1
2
2
2
2
2
3
3
2
1
29
28
each phase in the cell cycle of neuroepithelial cells in the chick embryo. Since
different regions of the central nervous system may show varying degrees of
maturity during embryogenesis, this study was confined to neuroepithelial cells
in the dorsolateral region of the optic tectum from the third through the sixth
day of incubation. The generation time and the length of each phase in the
cell cycle were determined by the cumulative labeling method described by
Fujita (1962).
MATERIALS AND METHODS
White Leghorn eggs were incubated at 38 °C for approximately 40 h, at
which time rectangular windows were made in the shell of each egg. The
embryos were stained lightly with neutral red and staged according to the criteria
of Hamburger & Hamilton (1951). Embryos of the same developmental age
were selected, the shell apertures were covered with transparent tape, and the
eggs were returned to the incubator. A single injection of [3H]thymidine
(25/tCi/embryo, specific activity 2-0mCi/mM) was made through the shell
window at 3, 4, 5 or 6 days of incubation. Embryos were immediately returned
to the incubator and later fixed in Carnoy's fluid at varying intervals ranging
from 1 to 18 h after injection. Numbers and ages of embryos obtained at each
stage are listed in Table 1.
The embryos were processed according to standard histological procedures,
serially sectioned at 5 /*m, and prestained with the periodic acid-Schiff (PAS)
Cell cycle of chick neuroepithelial cells
141
reaction. The slides were dipped in NTB-3 gel, exposed at 5 °C for 2 weeks,
developed in Kodak D-19, and lightly stained with Harris's hematoxylin.
Observations were made on dorsolateral portions of the tectum midway
between its cranial and caudal boundaries. Nuclei with four or more grains
were considered labeled. To determine mean mitotic index, a total of 1000
neuroepithelial tectal cells was counted in each of three embryos. Because of the
imprecision of the cumulative labeling technique, only approximate values could
be obtained for the length of each period in the cell cycle.
OBSERVATIONS
In the 3-day embryo, labeled nuclei occurred only in the periphery of the wall
of the optic tectum 1 h after injection of tritiated thymidine. Mitotic figures
along the ventricular border remained unlabeled. The first labeled mitotic
figures appeared 1-5 h after injection. This was roughly equivalent to the length
of the premitotic (G2) period. Four hours after injection of tritiated thymidine
all nuclei contained label, although some were less heavily labeled than others.
The time at which all nuclei became labeled was equal to the sum of the premitotic (G2) period, mitosis (M), and postmitotic (Gi) period. Eight hours after
injection all nuclei were evenly labeled; thus the total generation time for
neuroepithelial cells in the dorsolateral region of the tectum was considered
to be 8 h.
The length of the DNA-synthetic (S) period was determined by subtracting
the time at which all nuclei first became labeled (the sum of G2, M, and Gj)
from the time at which all nuclei showed homogeneous labeling (total generation
time) and was therefore equal to approximately 4 h. The mitotic index (MI) of
the neuroepithelial cells at 3 days of incubation was 3-89 ± S.E. 0-072. The length
of the mitotic period was calculated as MI/100 x generation time and was equal
to approximately 0-3 h or 18 min. The length of the postmitotic (Gx) period
was calculated to be 2-2 h by subtracting the sum of M and G2 (0-3 + 1-5) from
the time at which all neuroepithelial nuclei first became labeled (4 h).
In the 4-day embryonic tectum the label was confined to the periphery of the
wall until 1-5 h after injection of tritiated thymidine, at which time labeled
mitotic nuclei appeared in the ventricular zone. G2 was thus equal to
approximately 1-5 h. Four hours after injection all nuclei became labeled, and
by 9 h all nuclei were homogeneously labeled. The total generation time was
therefore 9 h. However, a few cells peripheral to the ventricular zone remained
unlabeled even as late as 18 h after injection, and these cells were identified as
neuroblasts which had begun to differentiate and migrate peripherally. These
neuroblasts were excluded from any calculations of the cell cycle, since they
were no longer capable of mitotic activity.
The length of the S-period in the 4-day tectum was 5 h. The mitotic index
was 4-78 + 0-193, and the length of mitosis was calculated as 0-4 h (24 min).
Gx was 2-1 h.
748
D. B. WILSON
Fig. 1. Five-day optic tectum 13 h after injection with [3H]thymidine. Note peripheral
unlabeled neuroblasts (N). x 200.
Fig. 2. Six-day optic tectum 16 h after injection with [3H]thymidine. Labeled nuclei
confined to ventricular zone except for labeled endothelial nuclei of vascular
elements. Note wide band of unlabeled neuroblasts (N), some of which are migrating
peripherally, x 275.
At 5 days of incubation a well-defined peripheral layer of unlabeled neuroblasts was apparent (Fig. 1). A few labeled cells among the neuroblasts were
identified as capillary endothelial cells. The total generation time of the remaining
neuroepithelial cells was approximately 13 h. The S-period was 4h and G 2
was 1-5 h. The MI had increased to 6-21 ±0-079 and the duration of mitosis
was approximately 0-8 h (48 min). Gx was calculated as 6-7 h.
In the 6-day optic tectum a thick layer of unlabeled neuroblasts was found
peripheral to the proliferative cells in the ventricular zone (Fig. 2). Numerous
neuroblasts also were in the process of migrating to the marginal zone of the
tectum. Except for vascular elements, labeled nuclei were confined to the ventricular zone. Among these proliferative ventricular cells, the total generation
time had increased to approximately 15 h. The S-period was 5 h and G2 was
1-5 h. The mitotic index was 9-48 + 0-088 and the length of mitosis was 1-4 h.
Gx was approximately 7-1 h.
The data on the length of each period of the cell cycle for each developmental
stage are summarized in Table 2.
Cell cycle of chick neuroepithelial cells
749
Table 2. Approximate duration {h)for each period of cell cycle of neuroepithelial
cells in chick optic tectum between the 3rd and 6th day of incubation.
Duration (h)
(days)
Generation
time
S
G2
M
Gx
3
4
5
6
8
9
13
15
4
5
4
5
1-5
1-5
1-5
1-5
0-3
0-4
0-8
1-4
2-2
21
6-7
7-1
age
DISCUSSION
Although the cumulative labeling technique can provide only approximate
values for the length of each period in the cell cycle, the radioautographic results
showed that the cell generation time for neuroepithelial cells in the optic tectum
increased from 8 h at 3 days of incubation to 15 h at 6 days. This agrees with
comparable results which indicate an increase in the total generation time in
mouse thoracic neural tube between 10 and 11 days of gestation (Kauffman,
1968). Fujita (1962) noted an increase in generation time from approximately
5 h in the 1-day chick neural tube to 16 h in the 6-day parietal mesencephalon.
The generation time of 15 h in the 6-day tectum of the present study compares
favorably with Fujita's results (1962), despite possible subjective variations in
determining the time at which homogeneous labeling occurs.
The length of the DNA-synthetic (S) period remained relatively constant
(4-5 h) between the 3rd and 6th day of incubation. This rather short time was
found to be characteristic of newly hatched chick somatic cells (5-6 h) in contrast
to the slightly longer time of 6-8 h in mouse embryonic somatic cells (Cameron,
1964). In the present study the duration of the premitotic (G2) period also
remained constant at 1-5 h, whereas the length of the postmitotic (Gx) period
increased from 2-2 h at 3 days of incubation to 7-1 h at 6 days.
The mitotic time gradually increased from approximately 0-3 h at 3 days of
incubation to 1-4 h at 6 days. The length of mitosis in Fujita's study (1962) was
1 h in the 6-day chick mesencephalon, and the mitotic index was 6-8 %, in contrast to the mitotic index of 9-4% obtained in the 6-day tectum of the present
study. A gradual lengthening of mitotic time was described by Jelinek (1959)
using colchicine to arrest mitosis in the chick spinal cord between 2 and 6 days
of incubation. Also, in the 4-5-day mesencephalon the length of mitosis was
43 min, while at 6 days of incubation mitosis had increased to 133 min (Jelinek
6 Klika, 1961). However, in the present study the duration of mitosis was
approximately 24 min at 4 days and 84 min at 6 days.
The different absolute values obtained in various studies of the cell cycle may
be explained by variations in determining incubation ages of chick embryos,
750
D. B. WILSON
by the use of different techniques, and by the use of data obtained from different
portions of the tectum. As indicated by Cowan et al. (1968), the tectum exhibits
a rostrocaudal and ventrodorsal gradient so that rostral and ventral portions
of the tectum are approximately 2 days more advanced than caudal and dorsal
regions. Since the cell cycle has been shown to change with increasing embryonic
age, one would expect to find such differences in the cycle in regions of differing
maturity.
A noticeable lengthening occurred in the generation time between 4 and 5 days
of incubation. This corresponded with the period during which numerous neuroblasts were differentiating in dorsolateral regions of the tectum (Cowan et al.
1968; LaVail & Cowan, 1971; Wilson, 1971). It is not possible at the present
time to determine whether the change from a homogeneous population of
proliferating cells to a heterogeneous one with proliferating and differentiating
cells results in a change in the actual length of the cell cycle. As suggested by
Kauffman (1968), it is possible that the initial population contains cells with
different generation cycles and that the early differentiation of short-cycle cells
into neuroblasts may leave behind proliferating cells with longer cycles.
Although Cowan et al. (1968) describe a peak in the absolute number of
mitotic figures in the optic tectum at 5-5-6 days of incubation, the increase was
found to be slight when they took into consideration the increase in area of the
tectal wall. Moreover, as indicated by Jelinek & Klika (1961), an absolute increase in the number of mitotic figures may actually reflect an increase in the
duration of mitosis. Thus a consideration of the generation time as well as the
length of each period in the cycle would seem to be a better indicator of mitotic
activity. The results of the present radioautographic study suggest that the most
active proliferation of tectal neuroepithelial cells occurs during the 3rd and 4th
days of incubation when the length of the generation time is short, and that the
increase in the mitotic index observed on the 5th and 6th days of incubation
most likely reflects the prolongation of mitosis and of the total generation time.
This work was supported by research grant HD 04820 from the National Institute of
Child Health and Human Development, United States Public Health Service. The author
wishes to express appreciation to Barbara Gee for technical assistance.
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{Received 31 October 1972)