The neural cell cycle in the looptail (Lp) mutant mouse

Transcription

1 /. Embryol. exp. Morph. Vol. 32, 3, pp , Printed in Great Britain 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 (G 2 ), 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 G x 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.

2 698 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 [ 3 H]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 25% 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).

3 Cell cycle in looptail mice 699 3,4 Fig. 1. Looptail heterozygote (Lpj + ) at 11 days' gestation. Fig. 2. Looptail homozygote (LpjLp) with open neural tube at 11 days' gestation. Fig day normal midbrain 7 h after injection with [ 3 H]thymidine. Arrows indicate unlabelled mitoses. V, ventricle. Fig day abnormal midbrain 7 h after injection with [ 3 H]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 4h, at which time

4 700 D. B. WILSON AND E. M. CENTER /* / / / i / ' / ( / I / i ' /? / J I! it Ti \v T\ \ \ \ \ 1 * i 1 * / \ ^ / 1 t / \ \ J \ s f ^ \ \ / I ^ / \ N 1r \ N / / \ / > i i i i i i IS Time (h) 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 [ 3 H]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 (G 2 ) period plus \ the length of mitosis (M), G 2 was calculated as 0-9 h (1-5 h minus \ x 1-1 h). The postmitotic (G t ) period was determined by subtracting the sum of S, M and G 2 from the total generation time and was equal to 2-0 h.

5 Cell cycle in looptail mice 701 IVIW d 4() f - < \\ \ y/ i i i i i i i i i / " * - - ' , Time (h) 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 [ 3 H]thymidine. Ordinate: mean percent labelled mitoses. Table 1. Duration of generation time, DNA-synthetic (S), premitotic (G 2 ), mitotic (M), and postmitotic (G x ) periods for normal ( + / + ) and abnormal (Lp/Lp) embryos Duration (h) time S G 2 M Gx.10 days Normal Abnormal days Normal Abnormal 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 h, and G x 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

6 702 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 h and G x 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, G h and G x 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 (G 2 ) 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 G x periods. Similar chronological increases in the S and G x 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 G x and M (Jelinek, 1959; Jelinek & Klika, 1961; Kallen, 1961, 1962; Wilson, 1973ft).

7 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 G x. 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 G x 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 G x (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 G x 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 G x 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

8 704 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 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. REFERENCES FUJITA, S., HORII, M., TANIMURA, T. & NISHIMURA, H. (1964). H 3 -thymidine autoradiographic studies on cytokinetic responses to X-ray irradiation and to thio-tepa in the neural tube of mouse embryos. Anat. Rec. 149, HOSHINO, K., MATSUZAWA, T. & MURAKAMI, U. (1973). Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon. Expl Cell Res. 11, JELINEK, R. (1959). Proliferace v centralnim nervovem kufecfch zarodku. I. Doba trvani mitosy v germinalni zone michy od 2. do 6. dne zarodecneho vyvoje. Cslkd Morf. 1, JELINEK, R. & KLIKA, E. (1961). Proliferacni aktivita pri tzv. 'pferustani' neuralnf trubice. Cslkd Morf. 9, 406^14. KALLEN, B. (1961). Studies on cell proliferation in the brain of chick embryos with special reference to the mesencephalon. Z. Anat. EntwGesch. 122, KALLEN, B. (1962). Mitotic patterning in the central nervous system of chick embryos: studied by a colchicine method. Z. Anat. EntwGesch. 123, KAUFFMAN, S. L. (1968). Lengthening of the generation cycle during embryonic differentiation of the mouse neural tube. Expl Cell Res. 49, KAUFFMAN, S. L. (1969). Cell proliferation in embryonic mouse neural tube following urethane exposure. Devi Biol. 20, KONYUKHOV, B. V. & SAZHINA, M. V. (1971). Genetic control over the duration of G x phase. Experientia 27, KOPRIWA, B. & LEBLOND, C. P. (1962). Improvements in the coating technique of radioautography. /. Histochem. Cytochem. 10, SMITH, L. J. & STEIN, K. F. (1962). Axial elongation in the mouse and its retardation in homozygous looptail mice. /. Embryol. exp. Morph. 10, STEIN, K. F. & MACKENSEN, J. A. (1957). Abnormal development of the thoracic skeleton in mice homozygous for the gene for looped-tail. Am. J. Anat. 100, STEIN, K. F. & RUDIN, I. A. (1953). Development of mice homozygous for the gene for looptail. /. Hered. 44, STEIN, K. F., LIEVRE, F. & SMOLLER, C. G. (1960). Abnormal brain differentiation in the homozygous loop-tail embryo. Anat. Rec. 136, STRONG, L. C. & HOLLANDER, W. F. (1949). Hereditary loop-tail in the house mouse. /. Hered. 40,

9 Cell cycle in looptail mice 705 VAN ABEELEN, J. H. F. (1966). Behavioural profiles of neurological mutant mice. Genetica 37, VAN ABEELEN, J. H. F. (1968). Behavioural ontogeny of looptail mice. Anim. Behav. 16, 1-4. VAN ABEELEN, J. H. F. & RAVEN, S. M. J. (1968). Enlarged ventricles in the cerebrum of loop-tail mice. Experientia 24, WILSON, D. B. (1973a). The cell cycle in the mesencephalon of two neurological mutants of the mouse. Anat. Rec. 175, 471. WILSON, D. B. (19736). Chronological changes in the cell cycle of chick neuroepithelial cells. /. Embryol. exp. Morph. 29, WILSON, D. B. (1974). Proliferation in the neural tube of the splotch (Sp) mutant mouse. /. comp. Neurol. 154, (Received 19 February 1974, revised 13 June 1974)