- Email: [email protected]
Quantitative studies of mitoses in cerebral hemispheres of fetal rats
306
Developmental Brain Research, 20 (1985) 306-309 Elsevier
BRD60076
Quantitative studies of mitoses in cerebral hemispheres of fetal rats* STEPHEN ZAMENHOF Brain Research Institute and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted February 5th, 1985) Key words: mitosis --cerebral hemisphere - - fetal rat
In continuation of our study of 15-day fetal rat brain development, evidence was obtained of a single layer of mitoses, situated away from the ventricle at a mean depth of 75% of the thickness of the cell layer of cerebral hemispheres ('deep mitoses'). Their number is 17.9% of ventricular mitoses and their spindles are perpendicular to spindles of ventricular mitoses. Their distribution is not correlated with distribution of ventricular mitoses. The fate and destination of these deep mitotic cells is at present unknown; the possibility that they are mitoses of the capillaries has been discussed. Mitoses in cerebral hemispheres and spinal cords of fetal rats and chick e m b r y o s have been the subject of many publications1-16. The mitoses studied were ventricular and the studies were not quantitative. In the course of investigations of brain development of the fetal rat 16 we have o b t a i n e d evidence of a uniform single layer of mitoses (hereafter called ' d e e p mitoses') which are situated deep inside the wall of the cerebral hemisphere. The subjects of this communication are quantitative studies of these mitoses and of ventricular mitoses in the cerebral hemispheres of 141/2- and 15-day-old rat fetuses. The animals and p r e p a r a t i o n of slides were as described previously16. F o r the present work, we used litters A and B (Table I), with litter sizes of 9 and 11, respectively (a total of 20 fetuses). On the conceptual 141/2 (A) or 15th (B) day of pregnancy, the females were anesthetized and o p e n e d , the uteri were removed and then each individual whole fetus (without dissecting out the brain) was r e m o v e d , weighed and fixed in Bouin's fluid. A f t e r fixation, serial paraffin sections, 10/~m, were m a d e and were stained with cresylecht violet. The sections (Fig. 1 in ref. 16) were parasagittal, through the right or left cerebral hemisphere. The slides were observed under oil immer-
sion (970x); all mitoses (Fig. 1A, 1B) were counted along the ventricular zone and deep zone, from lines A - B to C - D (Fig. 1 in ref, 16), in segments 100 ~zm long (reticule in eyepiece), a d d e d together (Table I) and also plotted separately in blocks of 5 segments (Fig. 2). The reticule in the eyepiece (10-/~m divisions at this magnification) also served to measure the distance of deep mitoses from the ventricular surface as well as the thickness of the cell layer of cerebral hemispheres at each of these locations, to calculate this distance in percent of this thickness. The angles of mitotic spindles were d e t e r m i n e d only in mitotic metaphase, anaphase or telophase, and only when the planes of mitoses made possible their observation: the angles of spindles were d e t e r m i n e d by means of the eyepiece reticule and calibrated rotation of the eyepiece. The results are r e p r e s e n t e d in Table I and on Figs. 1 and 2. The number of deep mitoses ( D M on Fig. 1) is 17.9% of the ventricular mitoses (VM) and they are situated away from the ventricular surface, at a depth of 75% of the thickness of the cell layer of cerebral hemispheres. While 90 + 5% of the ventricular mitoses (in metaphase, anaphase or telophase) have their spindles parallel (tangential) to the ventricular
* An abstract of this work has appeared ~7. Correspondence: S. Zamenhof, Brain Research Institute and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
307
D~M
V VM
B Fig. 1. A: microphotograph and B: drawing. Parasagittal section of the wall of cerebral hemisphere of a 15-day-old rat fetus; higher magnification (1500x) corresponding to Fig. 1 in ref. 16. Deep mitoses (DM, arrow) have spindles perpendicular to spindles of ventricular mitoses (VM, arrow); V, ventricle. There are no mitoses between DM and VM.
308 TABLE I
Mitoses in fetal rat cerebral hemispheres Cells in metaphase, anaphase or telophase only. Litters A (141/2-day fetuses) and B (15 day-fetuses).
Litter
A
B Mean
Number of
Ventricular mitoses
samples
Hemisphere
Total number per parasagittal section
% With spindle parallel to ventricle
right left right left right and left combined
216 222 223 234 224
95 89 91 87 90
9
9 11 11 40
Deep mitoses
+_ 40 + 34 + 23 + 30 + 31
Total number
% of ventricular mitoses
% With spindle perdicular to ventricle
Location*
79 70 71 74 73.4
77 76 73 75 75
+ 4
37 + 8
17.1
+_ 3 _+ 7 + 6 - 5
42 41 39 40
18.9 18.3 16.7 17.9
+_ 4 + 3.8 + 5.4 _ 4.8
+_ 7 _+ 16 + 8 + 16 + 13
* Away from ventricle (future ependyma), in % of the thickness of the cell layer of cerebral hemispheres at that location.
surface 5,8.12, in the deep mitoses 73.4 + 13% of the spindles are perpendicular to the ventricular surface (i.e. perpendicular to ventricular mitoses, Fig. 1A, B). As can be seen from Table I, the standard deviations are low and there are no significant differences between right and left cerebral hemispheres, between littermates, and between different litters (fetal age difference V2 day). The distribution (density) of ventricular mitoses along the ventricle (future ependyma) follows a characteristic pattern, but the locations of peaks and depressions of densities are different in the two cerebral hemispheres (Fig. 2) and different from animal to animal, even in the same litter. This speaks against the possibility that a specific pattern of distribution serves the general pattern of morphological changes (enlargements) of cerebral hemispheres. The locations of peaks and depressions of mitotic densities of deep mitoses are not correlated with those of ventricular mitoses, even in the same hemisphere of the same animal. While the mean distributions of peaks and depressions of ventricular mitoses along the ventricle in the right and left cerebral hemispheres are not correlated, the mean distributions of deep mitoses in right and left hemispheres are practically identical (Fig. 2). All this suggests that the destination of the cells in deep mitoses is different from that of ventricular mitoses. The origin and the fate of these cells after mitosis can be at present only a subject of conjecture.
Since the deep mitoses are present at fetal day 141/2 and 15, they occur before the appearance of a subventricular zone in terms of Boulder Committee nomenclature 4 and its updated versiontl; they are situated away from ventricular and subventricular layers. The percentage and the location of deep mitoses are entirely different from 'subsurface' mitoses described by Smart 14. Deep mitoses could be glioblasts, especially precursors of astrocytesH; however, although glioblasts can coexist with neuroblasts 9,10, there would only be very few of them at this stage) and there is no obvious reason why they should be sit-
35.
if°x\\ L /1
30.
\
25
E E
~e b
20-
(5 15o "~
I0-
, 025
, 075
, ~25
, t75
, 225
, 275
r 325
, 375
--
, 425mm
Fig. 2. Mean distribution of mitoses along the ventricle in section as in Fig. 1 of this article and Fig. 1 in ref. 16. Abscissa, distance along the ventricle in mm; ordinate, total number of ventricular mitoses (V, upper curves) or deep mitoses (D, lower curves) per block of 5 segments 100 ,urn long each (i.e. per 0.5 mm); R and L, right and left cerebral hemispheres, respectively.
309 uated in one deep layer and be perpendicular to the ventricular mitoses. A n o t h e r possibility is that the deep mitoses are those of capillary cells (J. A l t m a n , personal communication). The mesodermal origin of some 'subventricular' cells has been suggested3, 6, but their mitoses as such have not been detected and studied7,12,15.
13- and i5-day-old mice embryos and killed some of them 1 h later, did find a few labeled cells in the developing cortical plate, and they could well be the deep mitoses studied in this paper, whatever their distination. Since there are no mitoses situated between deep
old rabbit embryo all capillaries appear within 8 h.
mitoses and the ventricular surface, there is no gradient of mitoses; thus, lower n u m b e r s of deep mitoses than ventricular mitoses cannot simply result from
Sauer lz reported that in a pig embryo (10 mm) blood vessels are present in the outer part of the neural tube
pears more likely that the cells in deep mitoses have
Strong15 reported that in the spinal cord of a 13-day-
wall, yet the mitoses are confined to the region near
low penetration of nutrients and/or mitogens. It ap-
the lumen (p. 395). O n the other hand, Angevine and Sidman2, who introduced [3H]thymidine into
specific receptors for special mitogens that may arrive through the ventricle or from outside of the cerebral hemispheres.
1 Altman, J., Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis, Exp. Neurol., 16 (1966) 263-278. 2 Angevine, J. B. and Sidman, R. L., Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse, Nature (Lond.), 192 (1961) 766-768. 3 Berry, M., Development of the cerebral neocortex of the rat. in G. Gottlieb (Ed.), Aspects of Neurogenesis, Academic Press, New York, 1974, pp.7-67. 4 Boulder Committee, Embryonic vertebrate central nervous system: revised terminology, Anat. Rec., 166 (1970) 257-261. 5 Doig, C. M. and Smart, I. H. M. The location and orientation of mitotic figures in various epithelia, J. Anat., 101 (1967) 634-636. 6 Hamburger, V., The mitotic patterns in the spinal cord of the chick embryo and their relation to histogenetic processes, J. comp. Neurol., 88 (1948) 221-284. 7 Kiss, F. A., Vascularization and tissue differentiation, Stud. biol. hung., 14 (1975) 1-168. 8 Landrieu, P. and Goffinet, A., Mitotic spindle filter orientation in relation to cell migration in the neo-cortex of normal and reeler mouse, Neurosci. Lett., 13 (1979) 69-72. 9 Levitt, P., Cooper, M. L. and Rakic, P., Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis, J. Neurosci., 1 (1981) 27-39.
10 RaKic, P., Cell migration and neuronal ectopias. In D. Bergsma (Ed.), Morphogenedis and Malformation of Face and Brain, Liss, New York, 1975, pp. 95-129. 11 Rakic, P., Early developmental events: cell lineages, acquisition of neuronal positions, and areal and laminar development. In P. Rakic and P. S. Goldman-Rakic (Eds.), Development and Modifiability of the Cerebral Cortex. Neuroscience Research Program, Bulletin 20 (1981-82) 439-451. 12 Sauer, F. C., Mitosis in the neural tube, J. comp. Neurol., 62 (1935) 377-405. 13 Sidman, R. L., Cell proliferation and migration in the developing brain. In G. M. McKhann and S. J. Yaffe (Eds.), Drugs and Poisons in Relation to Developing Nervous System, U.S. Public Health Service Publ. No. 1791, Washington, DC, 1968, pp. 5-11. 14 Smart, I. H. M., Proliferative characteristics of the ependymal layer during the early development of the spinal cord in the mouse, J. Anat., 113 (1972) 109-129. 15 Strong, L. H., The first appearance of vessels within the spinal cord of the mammal: their developing patterns as far as partial formation of the dorsal septum, Acta anat., 44 (1961) 80-108. 16 Zamenhof, S. and Marthens, E., Quantitative study of fetal brain development in individual rats, Develop. Brain Res., 3 (1982) 657-661. 17 Zamenhof, S., Quantitative studies of mitoses in cerebral hemispheres of fetal rat, Neurosci. Abstr., 10 (1984) 287.
Developmental Brain Research, 20 (1985) 306-309 Elsevier
BRD60076
Quantitative studies of mitoses in cerebral hemispheres of fetal rats* STEPHEN ZAMENHOF Brain Research Institute and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted February 5th, 1985) Key words: mitosis --cerebral hemisphere - - fetal rat
In continuation of our study of 15-day fetal rat brain development, evidence was obtained of a single layer of mitoses, situated away from the ventricle at a mean depth of 75% of the thickness of the cell layer of cerebral hemispheres ('deep mitoses'). Their number is 17.9% of ventricular mitoses and their spindles are perpendicular to spindles of ventricular mitoses. Their distribution is not correlated with distribution of ventricular mitoses. The fate and destination of these deep mitotic cells is at present unknown; the possibility that they are mitoses of the capillaries has been discussed. Mitoses in cerebral hemispheres and spinal cords of fetal rats and chick e m b r y o s have been the subject of many publications1-16. The mitoses studied were ventricular and the studies were not quantitative. In the course of investigations of brain development of the fetal rat 16 we have o b t a i n e d evidence of a uniform single layer of mitoses (hereafter called ' d e e p mitoses') which are situated deep inside the wall of the cerebral hemisphere. The subjects of this communication are quantitative studies of these mitoses and of ventricular mitoses in the cerebral hemispheres of 141/2- and 15-day-old rat fetuses. The animals and p r e p a r a t i o n of slides were as described previously16. F o r the present work, we used litters A and B (Table I), with litter sizes of 9 and 11, respectively (a total of 20 fetuses). On the conceptual 141/2 (A) or 15th (B) day of pregnancy, the females were anesthetized and o p e n e d , the uteri were removed and then each individual whole fetus (without dissecting out the brain) was r e m o v e d , weighed and fixed in Bouin's fluid. A f t e r fixation, serial paraffin sections, 10/~m, were m a d e and were stained with cresylecht violet. The sections (Fig. 1 in ref. 16) were parasagittal, through the right or left cerebral hemisphere. The slides were observed under oil immer-
sion (970x); all mitoses (Fig. 1A, 1B) were counted along the ventricular zone and deep zone, from lines A - B to C - D (Fig. 1 in ref, 16), in segments 100 ~zm long (reticule in eyepiece), a d d e d together (Table I) and also plotted separately in blocks of 5 segments (Fig. 2). The reticule in the eyepiece (10-/~m divisions at this magnification) also served to measure the distance of deep mitoses from the ventricular surface as well as the thickness of the cell layer of cerebral hemispheres at each of these locations, to calculate this distance in percent of this thickness. The angles of mitotic spindles were d e t e r m i n e d only in mitotic metaphase, anaphase or telophase, and only when the planes of mitoses made possible their observation: the angles of spindles were d e t e r m i n e d by means of the eyepiece reticule and calibrated rotation of the eyepiece. The results are r e p r e s e n t e d in Table I and on Figs. 1 and 2. The number of deep mitoses ( D M on Fig. 1) is 17.9% of the ventricular mitoses (VM) and they are situated away from the ventricular surface, at a depth of 75% of the thickness of the cell layer of cerebral hemispheres. While 90 + 5% of the ventricular mitoses (in metaphase, anaphase or telophase) have their spindles parallel (tangential) to the ventricular
* An abstract of this work has appeared ~7. Correspondence: S. Zamenhof, Brain Research Institute and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
307
D~M
V VM
B Fig. 1. A: microphotograph and B: drawing. Parasagittal section of the wall of cerebral hemisphere of a 15-day-old rat fetus; higher magnification (1500x) corresponding to Fig. 1 in ref. 16. Deep mitoses (DM, arrow) have spindles perpendicular to spindles of ventricular mitoses (VM, arrow); V, ventricle. There are no mitoses between DM and VM.
308 TABLE I
Mitoses in fetal rat cerebral hemispheres Cells in metaphase, anaphase or telophase only. Litters A (141/2-day fetuses) and B (15 day-fetuses).
Litter
A
B Mean
Number of
Ventricular mitoses
samples
Hemisphere
Total number per parasagittal section
% With spindle parallel to ventricle
right left right left right and left combined
216 222 223 234 224
95 89 91 87 90
9
9 11 11 40
Deep mitoses
+_ 40 + 34 + 23 + 30 + 31
Total number
% of ventricular mitoses
% With spindle perdicular to ventricle
Location*
79 70 71 74 73.4
77 76 73 75 75
+ 4
37 + 8
17.1
+_ 3 _+ 7 + 6 - 5
42 41 39 40
18.9 18.3 16.7 17.9
+_ 4 + 3.8 + 5.4 _ 4.8
+_ 7 _+ 16 + 8 + 16 + 13
* Away from ventricle (future ependyma), in % of the thickness of the cell layer of cerebral hemispheres at that location.
surface 5,8.12, in the deep mitoses 73.4 + 13% of the spindles are perpendicular to the ventricular surface (i.e. perpendicular to ventricular mitoses, Fig. 1A, B). As can be seen from Table I, the standard deviations are low and there are no significant differences between right and left cerebral hemispheres, between littermates, and between different litters (fetal age difference V2 day). The distribution (density) of ventricular mitoses along the ventricle (future ependyma) follows a characteristic pattern, but the locations of peaks and depressions of densities are different in the two cerebral hemispheres (Fig. 2) and different from animal to animal, even in the same litter. This speaks against the possibility that a specific pattern of distribution serves the general pattern of morphological changes (enlargements) of cerebral hemispheres. The locations of peaks and depressions of mitotic densities of deep mitoses are not correlated with those of ventricular mitoses, even in the same hemisphere of the same animal. While the mean distributions of peaks and depressions of ventricular mitoses along the ventricle in the right and left cerebral hemispheres are not correlated, the mean distributions of deep mitoses in right and left hemispheres are practically identical (Fig. 2). All this suggests that the destination of the cells in deep mitoses is different from that of ventricular mitoses. The origin and the fate of these cells after mitosis can be at present only a subject of conjecture.
Since the deep mitoses are present at fetal day 141/2 and 15, they occur before the appearance of a subventricular zone in terms of Boulder Committee nomenclature 4 and its updated versiontl; they are situated away from ventricular and subventricular layers. The percentage and the location of deep mitoses are entirely different from 'subsurface' mitoses described by Smart 14. Deep mitoses could be glioblasts, especially precursors of astrocytesH; however, although glioblasts can coexist with neuroblasts 9,10, there would only be very few of them at this stage) and there is no obvious reason why they should be sit-
35.
if°x\\ L /1
30.
\
25
E E
~e b
20-
(5 15o "~
I0-
, 025
, 075
, ~25
, t75
, 225
, 275
r 325
, 375
--
, 425mm
Fig. 2. Mean distribution of mitoses along the ventricle in section as in Fig. 1 of this article and Fig. 1 in ref. 16. Abscissa, distance along the ventricle in mm; ordinate, total number of ventricular mitoses (V, upper curves) or deep mitoses (D, lower curves) per block of 5 segments 100 ,urn long each (i.e. per 0.5 mm); R and L, right and left cerebral hemispheres, respectively.
309 uated in one deep layer and be perpendicular to the ventricular mitoses. A n o t h e r possibility is that the deep mitoses are those of capillary cells (J. A l t m a n , personal communication). The mesodermal origin of some 'subventricular' cells has been suggested3, 6, but their mitoses as such have not been detected and studied7,12,15.
13- and i5-day-old mice embryos and killed some of them 1 h later, did find a few labeled cells in the developing cortical plate, and they could well be the deep mitoses studied in this paper, whatever their distination. Since there are no mitoses situated between deep
old rabbit embryo all capillaries appear within 8 h.
mitoses and the ventricular surface, there is no gradient of mitoses; thus, lower n u m b e r s of deep mitoses than ventricular mitoses cannot simply result from
Sauer lz reported that in a pig embryo (10 mm) blood vessels are present in the outer part of the neural tube
pears more likely that the cells in deep mitoses have
Strong15 reported that in the spinal cord of a 13-day-
wall, yet the mitoses are confined to the region near
low penetration of nutrients and/or mitogens. It ap-
the lumen (p. 395). O n the other hand, Angevine and Sidman2, who introduced [3H]thymidine into
specific receptors for special mitogens that may arrive through the ventricle or from outside of the cerebral hemispheres.
1 Altman, J., Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis, Exp. Neurol., 16 (1966) 263-278. 2 Angevine, J. B. and Sidman, R. L., Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse, Nature (Lond.), 192 (1961) 766-768. 3 Berry, M., Development of the cerebral neocortex of the rat. in G. Gottlieb (Ed.), Aspects of Neurogenesis, Academic Press, New York, 1974, pp.7-67. 4 Boulder Committee, Embryonic vertebrate central nervous system: revised terminology, Anat. Rec., 166 (1970) 257-261. 5 Doig, C. M. and Smart, I. H. M. The location and orientation of mitotic figures in various epithelia, J. Anat., 101 (1967) 634-636. 6 Hamburger, V., The mitotic patterns in the spinal cord of the chick embryo and their relation to histogenetic processes, J. comp. Neurol., 88 (1948) 221-284. 7 Kiss, F. A., Vascularization and tissue differentiation, Stud. biol. hung., 14 (1975) 1-168. 8 Landrieu, P. and Goffinet, A., Mitotic spindle filter orientation in relation to cell migration in the neo-cortex of normal and reeler mouse, Neurosci. Lett., 13 (1979) 69-72. 9 Levitt, P., Cooper, M. L. and Rakic, P., Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis, J. Neurosci., 1 (1981) 27-39.
10 RaKic, P., Cell migration and neuronal ectopias. In D. Bergsma (Ed.), Morphogenedis and Malformation of Face and Brain, Liss, New York, 1975, pp. 95-129. 11 Rakic, P., Early developmental events: cell lineages, acquisition of neuronal positions, and areal and laminar development. In P. Rakic and P. S. Goldman-Rakic (Eds.), Development and Modifiability of the Cerebral Cortex. Neuroscience Research Program, Bulletin 20 (1981-82) 439-451. 12 Sauer, F. C., Mitosis in the neural tube, J. comp. Neurol., 62 (1935) 377-405. 13 Sidman, R. L., Cell proliferation and migration in the developing brain. In G. M. McKhann and S. J. Yaffe (Eds.), Drugs and Poisons in Relation to Developing Nervous System, U.S. Public Health Service Publ. No. 1791, Washington, DC, 1968, pp. 5-11. 14 Smart, I. H. M., Proliferative characteristics of the ependymal layer during the early development of the spinal cord in the mouse, J. Anat., 113 (1972) 109-129. 15 Strong, L. H., The first appearance of vessels within the spinal cord of the mammal: their developing patterns as far as partial formation of the dorsal septum, Acta anat., 44 (1961) 80-108. 16 Zamenhof, S. and Marthens, E., Quantitative study of fetal brain development in individual rats, Develop. Brain Res., 3 (1982) 657-661. 17 Zamenhof, S., Quantitative studies of mitoses in cerebral hemispheres of fetal rat, Neurosci. Abstr., 10 (1984) 287.