Secondary expansion of the transient subplate zone in the developing cerebrum of human and nonhuman primates Alvaro Duquea,b, Zeljka Krsnikc, Ivica Kostovicc, and Pasko Rakica,b,1 a Department of Neuroscience, Yale University, New Haven, CT 06510; bKavli Institute for Neuroscience, School of Medicine, Yale University, New Haven, CT 06510; and cCroatian Institute for Brain Research, School of Medicine, University of Zagreb, Zagreb 10000, Croatia
Contributed by Pasko Rakic, June 22, 2016 (sent for review May 26, 2016; reviewed by Heiko Luhmann and Zoltan Molnar)
The subplate (SP) was the last cellular compartment added to the Boulder Committee’s list of transient embryonic zones [Bystron I, Blakemore C, Rakic P (2008) Nature Rev Neurosci 9(2):110–122]. It is highly developed in human and nonhuman primates, but its origin, mode, and dynamics of development, resolution, and eventual extinction are not well understood because human postmortem tissue offers only static descriptive data, and mice cannot serve as an adequate experimental model for the distinct regional differences in primates. Here, we take advantage of the large and slowly developing SP in macaque monkey to examine the origin, settling pattern, and subsequent dispersion of the SP neurons in primates. Monkey embryos exposed to the radioactive DNA replication marker tritiated thymidine ([3H]dT, or TdR) at early embryonic ages were killed at different intervals postinjection to follow postmitotic cells’ positional changes. As expected in primates, most SP neurons generated in the ventricular zone initially migrate radially, together with prospective layer 6 neurons. Surprisingly, mostly during midgestation, SP cells become secondarily displaced and widespread into the expanding SP zone, which becomes particularly wide subjacent to the association cortical areas and underneath the summit of its folia. We found that invasion of monoamine, basal forebrain, thalamocortical, and corticocortical axons is mainly responsible for this region-dependent passive dispersion of the SP cells. Histologic and immunohistochemical comparison with the human SP at corresponding fetal ages indicates that the same developmental events occur in both primate species.
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ecause of its exceptionally large size, it may not be coincidence that the subplate (SP) zone was originally discovered (1) and its function proposed on the basis of analysis of the developing cortex in human and nonhuman primates (NHPs) (2–4). For example, examination of the embryonic cerebral wall in the macaque monkey showed that thalamic axons form transient synapses with SP neurons before entering the superjacent cerebral cortex, inspiring the term waiting compartment (5). In addition, subsequent research in humans and NHPs indicated that the SP is particularly large subjacent to the association areas, such as the prefrontal cortex, and that a substantial number of SP cells survive and remain scattered within the white matter of the adult telencephalon as interstitial neurons (3, 4). Despite intensive research in various mammalian species in the last 40 y (reviewed in refs. 5–11), the origin as well as evolutionary and developmental mechanisms that underlie the extraordinary expansion and regional diversification of the SP in human are not well understood (10, 12). A generally accepted hypothesis, based on studies of the human fetal brain, is that the SP evolves from the deeper stratum of the primordial preplate (PP) after its separation (split) from the marginal zone (MZ) situated above by arrival of the cortical plate (CP) neurons from the proliferative ventricular zone (VZ) (13). However, the “splitting hypothesis,” for example, cannot explain the large (5–10-fold) difference in the width of the SP subjacent to 9892–9897 | PNAS | August 30, 2016 | vol. 113 | no. 35
various cytoarchitectonic areas of the human fetal cerebrum so salient during midgestation (Fig. 1), and it also stands in contrast to experimental data obtained in macaque monkey, using tritiated thymidine ([3H]dT, or TdR) to label the time and place of neuronal origin (3, 5). Part of the problem has been that most subsequent research on the genesis, function, and fate of SP cells was carried out in nonprimate species, such as rodents, where the SP is small and relatively uniform in width. Therefore, the origin, regional evolutionary expansion, and elaboration of SP in humans remain elusive. Because of the potential significance of the SP in the pathogenesis of many developmental brain disorders (10, 14–16), it is important to experimentally resolve these conceptual matters and discrepancies in the scientific literature. Here we examined the origin, migration pathway, initial settling position, and secondary dispersion of SP cells in the expanding SP zone of NHP (macaque) embryos. TdR was used as a marker of the last mitotic division of the neural stem cells and neuronal progenitors and positions of postmitotic cells plotted at different postinjection intervals in regions subjacent to cortical areas with different SP zone widths. Our major goal was to determine whether the VZ subjacent to the cortical areas with wider SP produces more SP neurons or whether, as hypothesized before (5), regions with wider SP (d in Fig. 1) are the result of neuronal dispersion within a larger neuropil. Our second goal was to verify whether the SP originates by the split of the cells in the MZ, a hypothesis that was based on static human histological material (13). Our third goal was to determine experimentally whether cells bound for deep cortical layers, born at the same time as SP cells, are phenotypically different, and thus would not be affected by downward dispersion and would remain as a distinct undisturbed band at the CP–SP border. Finally, we used sections of Significance The subplate zone, a transient cellular compartment of the embryonic cerebrum, has expanded in size and complexity during primate evolution, culminating in humans. Here, the application of multiple methods, including labeling time and place of neuronal origin and subsequent changes in their positions in macaque monkey embryos, and the use of histo- and immunochemistry in human fetal cerebral tissue of comparable prenatal ages reveals extraordinary cellular dynamics and unexpected secondary displacement of neurons. These findings may have significance for understanding cortical development and evolution and may provide insight into the pathogenesis of cortical disorders, as well as hypoxic-ischemic lesions in preterm infants. Author contributions: A.D., Z.K., I.K., and P.R. designed research; A.D., Z.K., I.K., and P.R. performed research; A.D., Z.K., I.K., and P.R. analyzed data; and A.D., Z.K., I.K., and P.R. wrote the paper. Reviewers: H.L., University Medical Center Mainz; and Z.M., University of Oxford. The authors declare no conflict of interest. See Commentary on page 9676. 1
To whom correspondence should be addressed. Email: [email protected].
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Fig. 1. The thickness of the human SP zone is not homogeneous. (A and B) Periodic acid–Schiff-Alcian staining of human brain coronal sections at 16 and 20 PCW, showing chemoarchitectonics of the anterior (rostral) cerebral wall. Because of the high amount of extracellular matrix reactivity in the SP, this layer stands out as the most prominent in the cerebral wall. Disregarding the tapering in the most medial regions, as the brain grows it is evident that the SP becomes much thicker (from ∼2–5× at 16 PCW to ∼8–9× at 20 PCW) in densely connected associative areas (3 and d) than in basal, orbital (e.g., 2 and a, b, c) or medial, limbic (e.g., 1 and e) cortical areas. EC, external capsule; CP, cortical plate; MZ, IZ, SVZ, VZ, and PVf, are marginal, intermediate, subventricular, ventricular, and periventricular fiber-rich zones, respectively.
human forebrain tissue at comparable embryonic ages stained by histo- and immunochemical techniques to analyze development of the human SP. As shown here, our results indicate that the SP in primates has a more dynamic and complex developmental history than hitherto recognized and provides new insight into the possible role of this transient zone in the pathogenesis of congenital disorders of the cerebral cortex that range from childhood epilepsy and autism to schizophrenia (10, 14–16). Results Origin, Position, and Secondary Dispersion of SP Cells in NHPs. Thirtyone animals divided into six groups according to injection time [∼E35 (embryonic day 35), E40, E50, E60, E70, E80] were analyzed at five different postinjection periods (1 h; 3, 7, and 14 d; and ∼2.5 mo postnatal). An additional case injected at E41 and killed at E83 was added to the analysis (see Materials and Methods for further details). A total of 70,306 cells were plotted, of which 25,235 were first generation heavily labeled TdR+ cells. The results presented here are exclusively derived from first generation cells, and the description that follows focuses on cells born at ∼E40, a time when many SP (and layer 6) cells are born. Seven days after E40 cells are generated, ∼80–90% of them are already located in the PP, at ∼60–70% distance from the ventricle and, correspondingly, 40–30% distance to pia. As the growth of the cortical wall proceeds, the SP becomes established as a separate compartment within the cortical wall. The SP involves the constant arrival of newly born cells as well as ingrowth of axons from subcortical structures and other cortical areas. As time passes, these fibers not only become an obstacle for the migration of cells (still migrating in the SP) but also start dispersing some of the neurons already located in it. Areas of the SP that develop first (i.e., the lateral cortical wall), and where Duque et al.
labeled cells first accumulate, are also the areas in which cell dispersion is first observed. Dispersion of cells becomes more evident in midgestation, after the arrival of thalamocortical axons and, increasingly, a large contingent of corticocortical axons. From the original cohorts of cells, only the SP resident cells seem to be dispersed by the incoming fibers, whereas layer 6 cells remain in the CP–SP border. Further research is needed to determine the genes and mechanism by which the dichotomy between SP cells and cells committed to the CP occurs. Columns 1, 2, and 3 of Fig. 2 illustrate different dynamics of the SP 14 d post injection for three correspondingly different injection times (E40, E50, E56). Cells labeled at E40, when the SP does not yet exist, accumulate in the PP (Fig. 2C1′) by the end of the first week postinjection and are found in the newly formed SP zone (Fig. 2 A1, B1, and C1) by the end of the second week. In the lateral wall of a midrostrocaudal section (Fig. 2C1), where the SP zone is the widest (compared here to more dorsal locations), incoming fibers displace already settled SP cells toward the ventricular zone. In the rostralmost telencephalon, cells born and labeled at E50 are 14 d later found in the SP zone, where, independent of dorsolateral location, they are all lumped together in a dense band (Fig. 2A2). More caudally, cells originally settled in the dorsolateral SP appear entering the CP, that is, are displaced toward the MZ zone (Fig. 2B2), whereas cells settled in more dorsal and ventral locations at the same rostrocaudal level are not yet affected. At a further developed caudal level, the displacement of all SP cells in the dorsal, dorsolateral, and lateral positions becomes more pronounced (Fig. 2C2) than at less developed rostral levels (Fig. 2 A2 and B2). Cells born and labeled at E56, at a time when the SP zone is already formed and actively growing, migrate and enter the SP at a time when a larger number of fibers are arriving into the SP. These cells are consequently entering an increasingly more dynamic milieu, and their distributions, even 14 d postinjection at rostral levels, reflect this dynamism (Fig. 2A3). The same occurs for all later injections (i.e., ∼E60, E70, E80), independent of the time of evaluation after injection. As the SP expands, it accommodates more incoming fibers, as well as newly generated cells that transit this milieu to reach their final destinations. This is reflected by differences in cellular distributions that can be detected just 3 d after labeling (Fig. 2 B4, C4, and E). We estimate that the cellular distribution changes detected at 1 h, 3 d, and 7 d postinjection are mostly a result of migration, whereas the high expansion that can be detected 14 d (and later) postinjection, especially for injections before E50, are mostly a result of dispersion of cells that have completed their migration. In specimens injected at later times (e.g., E70>), the migratory pathways are not only initially longer but also are actively increasing. Hence, at 14 d postinjection, many cells are still migrating. Fig. 2 D–F illustrates the positions of cells as seen in the tissue. In Fig. 2 D and F, SP cells have already finished migrating and are being dispersed by incoming fibers. Also, in the more developed lateral wall in Fig. 2D, a larger number of cells have already moved into the CP, whereas in Fig. 2F, the accumulation of cells in layer 6 becomes evident by the displacement of deeper cells into the SP. In contrast, in Fig. 2E, 3 d postinjection, cells are still migrating, and many have not yet entered the SP. In addition, in Fig. 2 D–F, cogeneration of some SP and MZ cells is evident, as previously shown by others in the cat visual cortex (17). SP Expansion in Human. In human, the SP formation stage [∼12–13 postconceptional weeks (PCW)] is also known as “second CP” (5) because the changes are so distinct. Fig. 3 illustrates the dynamic expansion of the SP that is occurring at this time. The deep portion of the CP becomes gradually loose and is transformed into a new cytoarchitectonic entity, the SP. The SP can then be divided in superficial (upper SP, SPU) and deeper (lower SP, SPL) regions with a gradual merging remaining as pre-SP (Fig. 3 B and C). The border with the intermediate zone (IZ) in the midlateral and rostral cortex PNAS | August 30, 2016 | vol. 113 | no. 35 | 9893
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Fig. 2. Examples of settling and later dispersion of cells born at different gestational ages. (A–C) Coronal rostral to caudal levels. (A1–C4) Spatiotemporal characteristics of the dispersion of SP cells. (C1′) By E47, when there is no SP yet, E40 born cells have not been dispersed yet, but they are clustered in the PP. (B4, C4, and E) Three days postinjection, cells born at E52 are actively migrating through the SP. (D and F) ∼E40 labeled cells that 14 d (D) and 42 d (F) postinjection had arrived and settled in the SP are now being dispersed by incoming fibers that invade the SP. SVZ(o), subventricular zone (outer); SP(LU), subplate (lower, upper); SPGL, subpia granular layer; GE, ganglionic eminence. (A1–C4) X-axis represents the percentage of cells found in the different cortical wall compartments from VZ to SPGL. See Results for further details.
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Fig. 3. Early arrival of axons and increased size of human SP. (A) Midlateral and lateral portions of the anterior part of the cerebral wall showing axonal strata with thalamo-cortical (ThC) and basal forebrain (BF) fibers. Notice oblique entrance of fibers into the SP (arrowheads). (A–E) illustrate the formation and initial expansion of the SP with dramatic changes in cytoarchitectonics visible by different staining methods. In 1 μm Nissl-plastic stained sections (B), Nissl (C), AChE (D), and Golgi (E), the border between SPL and IZ is marked by an asterisk. This border is best seen in AChE staining (D), in which the very well defined external part of the IZ, represented by intensively-stained fibers of the external capsule (*) are clear. In Golgi (E), the change in radial orientation of the deep portion of the CP and the lost radial architecture (cells and fibers) in the SPL are easily visualized. Notice the difference in intensity of fiber labeling in A, in which more fibers (darker background) are observed midlaterally, and how intensity diminishes (lighter background) as the gradient progresses toward more dorsal regions.
is clearly visible on 1-μm thin plastic sections because of the tangentially running osmificated fibers (Fig. 3B). The most remarkable features during this period are obliquity running “wavy” fibers (Fig. 3 A and D, arrowheads and arrows). In Golgi preparations, the SP in formation, especially SPL, shows loss of radial orientation of cell bodies and processes (Fig. 3E). This phase corresponds to monkey developmental period E54–E59. After this period, the SP continues to increase in size (15–20 PCW) and is characterized by cell differentiation and increase in fiber ingrowth and enlargement of the neuropil. In NHPs, this corresponds to ∼E60–E82. Discussion The main finding in the present investigation, summarized in Fig. 4, is that after early-born neurons destined for the SP complete Duque et al.
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their migration, they become secondarily displaced downward by the arrival of axons, first from the subcortical and later from cortical origin. Because of regional differences in the magnitude of corticocortical connections in primates, the width of the SP is highly variable and is particularly large subjacent to the association areas. It should be recognized that the present findings apply to Old World Primates macaque and human, as there are considerable species-specific differences in terms of cell origin and dynamics of migration (17, 18). The present investigation was focused on the expansion of the SP zone, as this is one of the most dramatic transient events in the evolution of the human cerebral cortex. Our results demonstrate that contrary to the prevailing view, this expansion is not just a consequence of increased numbers of SP neurons in primates, but is mainly a result of their dynamic interaction with an increasing number of incoming axons. Cell dispersion was measured both toward the pia and toward the ventricle. The layer 6 neurons, which are born at the same time as the SP cells and migrate together, remain as a dense stratum and are not dispersed toward the ventricle. The results of our analysis, within the temporal limits of the injection times available, indicate that arrival of incoming fibers and growth of surrounding extracellular matrix are main contributors to the expansion of the SP and to the change in spatial distribution of cells that had already finished migrating. We believe that the connectivity requirements of the different cortical areas explain why the width of the SP is most prominent in areas subjacent to human-specific association areas. The study of such complex cellular events is, by its nature, limited in human, in which, for instance, it is not possible to date the birth of neurons. Therefore, we analyzed various immuno- and histochemical stainings to corroborate that similar events are likely to occur in our species. This extrapolation of the data is needed to elucidate the enigmatic expansion of the SP that occurs in both species and is justified because of similarities in development, including the timing, progression, and magnitude of cellular events. The addition of fibers is by no means the only event that occurs during the expansion of the SP. After an original set of neurons populates the SP, the addition of cells and formation of many synapses, among other events, add to the growth of the neuropil of this compartment (19). Hence, it would be important to determine which and how many of these later-born cells become permanent residents of the SP during its transient existence, which and how many use the SP as a waiting compartment before continuing their migration, and which remain in adults as interstitial cells (3). We did not observe evidence to support the possibility of downward active locomotion. However, the analysis of dynamic events using static data at a limited number of times is a limitation inherent to our study, and thus additional research is needed to elucidate finer details on the origin, progression, and eventual dissolution of the SP. Materials and Methods Animals, TdR Injections, and Autoradiography. Autoradiograms from 31 Rhesus monkey brains were examined. Animals were exposed to TdR as embryos and killed at various prenatal and postnatal ages. The present experimental study in developing macaque did not require the addition of a single new animal. All cases were obtained from the Rakic Collection of Nonhuman Primates at Yale University. Experimental protocols and animal care before death were conducted during the last 45 y in accordance with institutional guidelines initially at Harvard Medical School, and subsequently at Yale University. Animal breeding, dating of pregnancies, and perfusion protocols, as well as descriptions of brain processing for autoradiography, have been previously published (20–22). Briefly, all animals received a single intravenous injection of TdR (New England Nuclear; 10 mCi per kilogram of body weight). Sections were cut in the coronal plane at 8–10 μm or 30 μm, using a freezing microtome. In every case, every 10th section was stained for Nissl or toluidine blue. Having been processed ∼35–45 y ago, the mounting media on TdR sections had deteriorated. After immersion in xylenes, old coverslips were removed and new mounting media and new coverslips were used to recover the slides. The quality of the TdR autoradiography was not compromised (23).
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Fig. 4. Summary model of the secondary expansion of the transient SP zone. (A) E40 born SP cells are visible in the VZ 1 h after injection. By E40, there is no SP yet. (B) By E54, these early born cells accumulate in the SP, and a few Cajal-Retzius cells in the MZ become evident. (C) By E83, and later in midgestation, these cells become secondarily displaced and widespread into the expanding SP by ingrowth of the neuropil. In particular, monoamine, basal forebrain, thalamocortical, and later on a large contingent of corticocortical axons.
Microscopy, Area Sampling, and Criteria for Positive Labeling and Cell Plotting. Section outlines were drawn at 5–10×, depending on the size of the section. Very early embryonic ages, and hence small sections, were done at higher magnification. The region of interest was the midrostral to caudal telencephalon, from frontal association areas, mid-motor-somatosensory cortex, to the primary visual cortex. Because of differences in the temporal and spatial development of the subplate, in every case, cells were plotted in a minimum of three brain sections containing samples from each of these areas in the rostrocaudal axis. Plotting was also done to always cover the dorsal, dorsolateral, and lateral cortical wall and, when possible, the basal or ventral cortical wall (e.g., rostral to the ganglionic eminence). All cell plotting was done at 40×, using a Zeiss Plan-Neofluar objective lens with a 0.75 optical aperture, using a Zeiss Axioskop microscope fully motorized and interfacing to a Dell computer running StereoInvestigator (MicroBrightField Inc.). As a control, cell counts done at 100× oil immersion using a Zeiss Plan-Neofluar objective lens with a 1.3 optical aperture were done in a group of 10 sections, and the results were compared with those obtained at 40×; the difference in cell counts affected only later-generation cells (not heavily labeled), and it was less than 4% in every case. To limit the region in which cells were to be plotted, a rectangular box (500 μm width by a length as long as needed) was placed in the cortical area to be sampled. In the case of very early embryonic ages, the small sections would not accommodate more than just one box width; hence, in those cases, all cells were plotted in dorsal and lateral directions. The guide box was most useful as sections became larger. The nucleus of a TdR cell was considered positive when it contained a minimum of three discrete silver grains that would clearly outnumber any grains over a similar surface area in the background (20, 23). Regions of high
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background, where many silver grains could be observed outside of cellular profiles, were not used for counting. A heavily stained nucleus may contain ≥50 discrete silver grains. Only intensely labeled cells, with the quantity of silver grains located over the nucleus equal or more than half of the maximum grain counts in any nucleus in a given specimen, were considered born at the time of injection (20). Cell Position and Migration vs. Secondary Cell Dispersion. To distinguish between cell migration and later cell dispersion, the absolute position of each TdRlabeled cell within the cerebral wall from the ventricle to the pia in the radial direction was plotted for all injection times (∼E35, E40, E50, E60, E70, E80) and for all postinjection periods (1 h, 3 d, 7 d, 14 d, and ∼2.5 mo postnatal after injection). Although the distance from the ventricle to pia changes at different rostrocaudal, as well as dorsoventral, positions in the radial direction, this distance is 100% of the distance any one cell can travel. Hence, the position of each labeled cell was normalized to its corresponding total radial distance from ventricle to pia, and a mean population distance was calculated for dorsal, dorsolateral, lateral, and basal coordinates at different rostrocaudal levels. As cells complete migration through a milieu that continues to grow, the distances among labeled cells, and from each labeled cell to pia (and/or ventricle), continue to increase. This affects cell density measurements, but not a cell’s normalized relative distance to pia. The former would change either because of reinitiation of a migratory flux or because of other forces that affect the cell’s relative position by actively moving it. Because E40 was experimentally determined to be a time when many cells destined for the SP are born, we focused the analysis of cell dispersion on this set of cells. For this, an additional case from the collection, injected at E41 and killed at E83, was added to the analysis.
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ACKNOWLEDGMENTS. This work was supported by grants from the NIH/ National Institute on Drug Abuse (R01-DA023999), NIH/National Institute of Neurological Disorders and Stroke (R01-NS014841), NIH/National Eye Institute (R01-EY002593), and Kavli Institute for Neuroscience at Yale University (to P.R.), as well as a Croatian Science Foundation Award HRZZ (Hrvatska zaklada za znanost) (to I.K.).
1. Kostovic I, Molliver ME (1974) A new interpretation of the laminar development of cerebral cortex: Synaptogenesis in different layers of neopallium in the human fetus. Anat Rec 178:395. 2. Rakic P (1977) Prenatal development of the visual system in rhesus monkey. Philos Trans R Soc Lond B Biol Sci 278(961):245–260. 3. Kostovic I, Rakic P (1980) Cytology and time of origin of interstitial neurons in the white matter in infant and adult human and monkey telencephalon. J Neurocytol 9:2l9–242. 4. Kostovic I, Jovanov-Milosevic N (2008) Subplate zone of the human brain: Historical perspective and new concepts. Coll Antropol 32(Suppl 1):3–8. 5. Kostovic I, Rakic P (1990) Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J Comp Neurol 297(3):441–470. 6. Ghosh A, Shatz CJ (1993) A role for subplate neurons in the patterning of connections from thalamus to neocortex. Development 117(3):1031–1047. 7. Allendoerfer KL, Shatz CJ (1994) The subplate, a transient neocortical structure: Its role in the development of connections between thalamus and cortex. Annu Rev Neurosci 17:185–218. 8. Kanold PO, Luhmann HJ (2010) The subplate and early cortical circuits. Annu Rev Neurosci 33:23–48. 9. Wang WZ, et al. (2011) Comparative aspects of subplate zone studied with gene expression in sauropsids and mammals. Cereb Cortex 21(10):2187–2203. 10. Hoerder-Suabedissen A, Molnár Z (2015) Development, evolution and pathology of neocortical subplate neurons. Nat Rev Neurosci 16(3):133–146. 11. Hutsler JJ, Casanova MF (2016) Review: Cortical construction in autism spectrum disorder: Columns, connectivity and the subplate. Neuropathol Appl Neurobiol 42(2): 115–134. 12. Judaš M, Sedmak G, Kostovic I (2013) The significance of the subplate for evolution and developmental plasticity of the human brain. Front Hum Neurosci 7:423. 13. Marin-Padilla M (1978) Dual origin of the mammalian neocortex and evolution of the cortical plate. Anat Embryol (Berl) 152(2):109–126. 14. Akbarian S, et al. (1996) Maldistribution of interstitial neurons in prefrontal white matter of the brains of schizophrenic patients. Arch Gen Psychiatry 53(5):425–436.
15. Kostovic I, Kostovic-Srzentic M, Benjak V, Jovanov-Miloševic N, Radoš M (2014) Developmental dynamics of radial vulnerability in the cerebral compartments in preterm infants and neonates. Front Neurol 5:139. 16. Kostovic I, Judaš M, Sedmak G (2011) Developmental history of the subplate zone, subplate neurons and interstitial white matter neurons: Relevance for schizophrenia. Int J Dev Neurosci 29(3):193–205. 17. Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat’s visual cortex: Cogeneration of subplate and marginal zones. J Neurosci 5(4):1062–1075. 18. Pedraza M, Hoerder-Suabedissen A, Albert-Maestro MA, Molnár Z, De Carlos JA (2014) Extracortical origin of some murine subplate cell populations. Proc Natl Acad Sci USA 111(23):8613–8618. 19. Bystron I, Blakemore C, Rakic P (2008) Development of the human cerebral cortex: Boulder Committee revisited. Nat Rev Neurosci 9(2):110–122. 20. Rakic P (1973) Kinetics of proliferation and latency between final cell division and onset of differentiation of cerebellar stellate and basket neurons. J Comp Neurol 147(4):523–546. 21. Rakic P (1976) Prenatal genesis of connections subserving ocular dominance in the rhesus monkey. Nature 26l(5560):467–47l. 22. Kornack DR, Rakic P (1998) Changes in cell-cycle kinetics during the development and evolution of primate neocortex. Proc Natl Acad Sci USA 95(3):1242–1246. 23. Duque A, Rakic P (2011) Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate. J Neurosci 31(42): 15205–15217. 24. Judaš M, et al. (2011) The Zagreb Collection of human brains: A unique, versatile, but underexploited resource for the neuroscience community. Ann N Y Acad Sci 1225(Suppl 1):E105–E130. 25. Kostovic I, Goldman-Rakic PS (1983) Transient cholinesterase staining in the mediodorsal nucleus of the thalamus and its connections in the developing human and monkey brain. J Comp Neurol 219(4):431–447. 26. Kostovic I, Judaš M, Radoš M, Hrabac P (2002) Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging. Cereb Cortex 12(5):536–544.
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previously (26). All sections were analyzed and images taken by using a high-resolution digital slide scanner NanoZoomer 2.0RS (Hamamatsu), and figures were managed in Adobe Photoshop and assembled in Adobe Illustrator.
Human Tissue. The postmortem human brains used for comparison with NHP tissue ranged in age from 7.5 to 24 PCW and are part of the large and versatile Zagreb Collection (24). Brain specimens were obtained from either medically indicated or spontaneous abortions at several clinical and pathological departments of the University of Zagreb, School of Medicine (Croatia). Patients provided informed consent and procedures were approved by the corresponding Institutional Review Boards (IRB). The fetal age was estimated on the basis of crown–rump length (in millimeters) and pregnancy records. Whole brains were fixed by immersion in 4% (40 g/L) paraformaldehyde in 0.1 M PBS at pH 7.4. Tissue blocks were either frozen or embedded in paraffin wax. Sections were cut at various thicknesses (1–30 μm). Histological Cresyl-violet (Nissl) staining was used to delineate cytoarchitectonic boundaries, and adjacent sections were processed by several histochemical methods. AChE-histochemistry (as described in ref. 25) was used for visualization of growing thalamocortical afferents and certain sagittally oriented axon strata, such as the external capsule system. Periodic acid–Schiff–Alcian blue histochemistry was used to analyze the laminar location and relative regional abundance of the extracellular matrix, as described
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Statistical Analyses. When appropriate for quantitative analysis and for comparisons, statistics were performed with Analyze-it v2.12 for Microsoft Excel. Distributions were examined to check for normalcy and homoscedasticity. Normality was assessed using the Shapiro-Wilk test. Homogeneity of variance was tested with an F-test. ANOVA was used to compare means among normally distributed populations with equal variance. Nonparametric ANOVA alternatives were performed with the Mann–Whitney and/or Kruskal-Wallis tests.
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Contributed by Pasko Rakic, June 22, 2016 (sent for review May 26, 2016; reviewed by Heiko Luhmann and Zoltan Molnar)
The subplate (SP) was the last cellular compartment added to the Boulder Committee’s list of transient embryonic zones [Bystron I, Blakemore C, Rakic P (2008) Nature Rev Neurosci 9(2):110–122]. It is highly developed in human and nonhuman primates, but its origin, mode, and dynamics of development, resolution, and eventual extinction are not well understood because human postmortem tissue offers only static descriptive data, and mice cannot serve as an adequate experimental model for the distinct regional differences in primates. Here, we take advantage of the large and slowly developing SP in macaque monkey to examine the origin, settling pattern, and subsequent dispersion of the SP neurons in primates. Monkey embryos exposed to the radioactive DNA replication marker tritiated thymidine ([3H]dT, or TdR) at early embryonic ages were killed at different intervals postinjection to follow postmitotic cells’ positional changes. As expected in primates, most SP neurons generated in the ventricular zone initially migrate radially, together with prospective layer 6 neurons. Surprisingly, mostly during midgestation, SP cells become secondarily displaced and widespread into the expanding SP zone, which becomes particularly wide subjacent to the association cortical areas and underneath the summit of its folia. We found that invasion of monoamine, basal forebrain, thalamocortical, and corticocortical axons is mainly responsible for this region-dependent passive dispersion of the SP cells. Histologic and immunohistochemical comparison with the human SP at corresponding fetal ages indicates that the same developmental events occur in both primate species.
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ecause of its exceptionally large size, it may not be coincidence that the subplate (SP) zone was originally discovered (1) and its function proposed on the basis of analysis of the developing cortex in human and nonhuman primates (NHPs) (2–4). For example, examination of the embryonic cerebral wall in the macaque monkey showed that thalamic axons form transient synapses with SP neurons before entering the superjacent cerebral cortex, inspiring the term waiting compartment (5). In addition, subsequent research in humans and NHPs indicated that the SP is particularly large subjacent to the association areas, such as the prefrontal cortex, and that a substantial number of SP cells survive and remain scattered within the white matter of the adult telencephalon as interstitial neurons (3, 4). Despite intensive research in various mammalian species in the last 40 y (reviewed in refs. 5–11), the origin as well as evolutionary and developmental mechanisms that underlie the extraordinary expansion and regional diversification of the SP in human are not well understood (10, 12). A generally accepted hypothesis, based on studies of the human fetal brain, is that the SP evolves from the deeper stratum of the primordial preplate (PP) after its separation (split) from the marginal zone (MZ) situated above by arrival of the cortical plate (CP) neurons from the proliferative ventricular zone (VZ) (13). However, the “splitting hypothesis,” for example, cannot explain the large (5–10-fold) difference in the width of the SP subjacent to 9892–9897 | PNAS | August 30, 2016 | vol. 113 | no. 35
various cytoarchitectonic areas of the human fetal cerebrum so salient during midgestation (Fig. 1), and it also stands in contrast to experimental data obtained in macaque monkey, using tritiated thymidine ([3H]dT, or TdR) to label the time and place of neuronal origin (3, 5). Part of the problem has been that most subsequent research on the genesis, function, and fate of SP cells was carried out in nonprimate species, such as rodents, where the SP is small and relatively uniform in width. Therefore, the origin, regional evolutionary expansion, and elaboration of SP in humans remain elusive. Because of the potential significance of the SP in the pathogenesis of many developmental brain disorders (10, 14–16), it is important to experimentally resolve these conceptual matters and discrepancies in the scientific literature. Here we examined the origin, migration pathway, initial settling position, and secondary dispersion of SP cells in the expanding SP zone of NHP (macaque) embryos. TdR was used as a marker of the last mitotic division of the neural stem cells and neuronal progenitors and positions of postmitotic cells plotted at different postinjection intervals in regions subjacent to cortical areas with different SP zone widths. Our major goal was to determine whether the VZ subjacent to the cortical areas with wider SP produces more SP neurons or whether, as hypothesized before (5), regions with wider SP (d in Fig. 1) are the result of neuronal dispersion within a larger neuropil. Our second goal was to verify whether the SP originates by the split of the cells in the MZ, a hypothesis that was based on static human histological material (13). Our third goal was to determine experimentally whether cells bound for deep cortical layers, born at the same time as SP cells, are phenotypically different, and thus would not be affected by downward dispersion and would remain as a distinct undisturbed band at the CP–SP border. Finally, we used sections of Significance The subplate zone, a transient cellular compartment of the embryonic cerebrum, has expanded in size and complexity during primate evolution, culminating in humans. Here, the application of multiple methods, including labeling time and place of neuronal origin and subsequent changes in their positions in macaque monkey embryos, and the use of histo- and immunochemistry in human fetal cerebral tissue of comparable prenatal ages reveals extraordinary cellular dynamics and unexpected secondary displacement of neurons. These findings may have significance for understanding cortical development and evolution and may provide insight into the pathogenesis of cortical disorders, as well as hypoxic-ischemic lesions in preterm infants. Author contributions: A.D., Z.K., I.K., and P.R. designed research; A.D., Z.K., I.K., and P.R. performed research; A.D., Z.K., I.K., and P.R. analyzed data; and A.D., Z.K., I.K., and P.R. wrote the paper. Reviewers: H.L., University Medical Center Mainz; and Z.M., University of Oxford. The authors declare no conflict of interest. See Commentary on page 9676. 1
To whom correspondence should be addressed. Email: [email protected].
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Fig. 1. The thickness of the human SP zone is not homogeneous. (A and B) Periodic acid–Schiff-Alcian staining of human brain coronal sections at 16 and 20 PCW, showing chemoarchitectonics of the anterior (rostral) cerebral wall. Because of the high amount of extracellular matrix reactivity in the SP, this layer stands out as the most prominent in the cerebral wall. Disregarding the tapering in the most medial regions, as the brain grows it is evident that the SP becomes much thicker (from ∼2–5× at 16 PCW to ∼8–9× at 20 PCW) in densely connected associative areas (3 and d) than in basal, orbital (e.g., 2 and a, b, c) or medial, limbic (e.g., 1 and e) cortical areas. EC, external capsule; CP, cortical plate; MZ, IZ, SVZ, VZ, and PVf, are marginal, intermediate, subventricular, ventricular, and periventricular fiber-rich zones, respectively.
human forebrain tissue at comparable embryonic ages stained by histo- and immunochemical techniques to analyze development of the human SP. As shown here, our results indicate that the SP in primates has a more dynamic and complex developmental history than hitherto recognized and provides new insight into the possible role of this transient zone in the pathogenesis of congenital disorders of the cerebral cortex that range from childhood epilepsy and autism to schizophrenia (10, 14–16). Results Origin, Position, and Secondary Dispersion of SP Cells in NHPs. Thirtyone animals divided into six groups according to injection time [∼E35 (embryonic day 35), E40, E50, E60, E70, E80] were analyzed at five different postinjection periods (1 h; 3, 7, and 14 d; and ∼2.5 mo postnatal). An additional case injected at E41 and killed at E83 was added to the analysis (see Materials and Methods for further details). A total of 70,306 cells were plotted, of which 25,235 were first generation heavily labeled TdR+ cells. The results presented here are exclusively derived from first generation cells, and the description that follows focuses on cells born at ∼E40, a time when many SP (and layer 6) cells are born. Seven days after E40 cells are generated, ∼80–90% of them are already located in the PP, at ∼60–70% distance from the ventricle and, correspondingly, 40–30% distance to pia. As the growth of the cortical wall proceeds, the SP becomes established as a separate compartment within the cortical wall. The SP involves the constant arrival of newly born cells as well as ingrowth of axons from subcortical structures and other cortical areas. As time passes, these fibers not only become an obstacle for the migration of cells (still migrating in the SP) but also start dispersing some of the neurons already located in it. Areas of the SP that develop first (i.e., the lateral cortical wall), and where Duque et al.
labeled cells first accumulate, are also the areas in which cell dispersion is first observed. Dispersion of cells becomes more evident in midgestation, after the arrival of thalamocortical axons and, increasingly, a large contingent of corticocortical axons. From the original cohorts of cells, only the SP resident cells seem to be dispersed by the incoming fibers, whereas layer 6 cells remain in the CP–SP border. Further research is needed to determine the genes and mechanism by which the dichotomy between SP cells and cells committed to the CP occurs. Columns 1, 2, and 3 of Fig. 2 illustrate different dynamics of the SP 14 d post injection for three correspondingly different injection times (E40, E50, E56). Cells labeled at E40, when the SP does not yet exist, accumulate in the PP (Fig. 2C1′) by the end of the first week postinjection and are found in the newly formed SP zone (Fig. 2 A1, B1, and C1) by the end of the second week. In the lateral wall of a midrostrocaudal section (Fig. 2C1), where the SP zone is the widest (compared here to more dorsal locations), incoming fibers displace already settled SP cells toward the ventricular zone. In the rostralmost telencephalon, cells born and labeled at E50 are 14 d later found in the SP zone, where, independent of dorsolateral location, they are all lumped together in a dense band (Fig. 2A2). More caudally, cells originally settled in the dorsolateral SP appear entering the CP, that is, are displaced toward the MZ zone (Fig. 2B2), whereas cells settled in more dorsal and ventral locations at the same rostrocaudal level are not yet affected. At a further developed caudal level, the displacement of all SP cells in the dorsal, dorsolateral, and lateral positions becomes more pronounced (Fig. 2C2) than at less developed rostral levels (Fig. 2 A2 and B2). Cells born and labeled at E56, at a time when the SP zone is already formed and actively growing, migrate and enter the SP at a time when a larger number of fibers are arriving into the SP. These cells are consequently entering an increasingly more dynamic milieu, and their distributions, even 14 d postinjection at rostral levels, reflect this dynamism (Fig. 2A3). The same occurs for all later injections (i.e., ∼E60, E70, E80), independent of the time of evaluation after injection. As the SP expands, it accommodates more incoming fibers, as well as newly generated cells that transit this milieu to reach their final destinations. This is reflected by differences in cellular distributions that can be detected just 3 d after labeling (Fig. 2 B4, C4, and E). We estimate that the cellular distribution changes detected at 1 h, 3 d, and 7 d postinjection are mostly a result of migration, whereas the high expansion that can be detected 14 d (and later) postinjection, especially for injections before E50, are mostly a result of dispersion of cells that have completed their migration. In specimens injected at later times (e.g., E70>), the migratory pathways are not only initially longer but also are actively increasing. Hence, at 14 d postinjection, many cells are still migrating. Fig. 2 D–F illustrates the positions of cells as seen in the tissue. In Fig. 2 D and F, SP cells have already finished migrating and are being dispersed by incoming fibers. Also, in the more developed lateral wall in Fig. 2D, a larger number of cells have already moved into the CP, whereas in Fig. 2F, the accumulation of cells in layer 6 becomes evident by the displacement of deeper cells into the SP. In contrast, in Fig. 2E, 3 d postinjection, cells are still migrating, and many have not yet entered the SP. In addition, in Fig. 2 D–F, cogeneration of some SP and MZ cells is evident, as previously shown by others in the cat visual cortex (17). SP Expansion in Human. In human, the SP formation stage [∼12–13 postconceptional weeks (PCW)] is also known as “second CP” (5) because the changes are so distinct. Fig. 3 illustrates the dynamic expansion of the SP that is occurring at this time. The deep portion of the CP becomes gradually loose and is transformed into a new cytoarchitectonic entity, the SP. The SP can then be divided in superficial (upper SP, SPU) and deeper (lower SP, SPL) regions with a gradual merging remaining as pre-SP (Fig. 3 B and C). The border with the intermediate zone (IZ) in the midlateral and rostral cortex PNAS | August 30, 2016 | vol. 113 | no. 35 | 9893
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Fig. 2. Examples of settling and later dispersion of cells born at different gestational ages. (A–C) Coronal rostral to caudal levels. (A1–C4) Spatiotemporal characteristics of the dispersion of SP cells. (C1′) By E47, when there is no SP yet, E40 born cells have not been dispersed yet, but they are clustered in the PP. (B4, C4, and E) Three days postinjection, cells born at E52 are actively migrating through the SP. (D and F) ∼E40 labeled cells that 14 d (D) and 42 d (F) postinjection had arrived and settled in the SP are now being dispersed by incoming fibers that invade the SP. SVZ(o), subventricular zone (outer); SP(LU), subplate (lower, upper); SPGL, subpia granular layer; GE, ganglionic eminence. (A1–C4) X-axis represents the percentage of cells found in the different cortical wall compartments from VZ to SPGL. See Results for further details.
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Fig. 3. Early arrival of axons and increased size of human SP. (A) Midlateral and lateral portions of the anterior part of the cerebral wall showing axonal strata with thalamo-cortical (ThC) and basal forebrain (BF) fibers. Notice oblique entrance of fibers into the SP (arrowheads). (A–E) illustrate the formation and initial expansion of the SP with dramatic changes in cytoarchitectonics visible by different staining methods. In 1 μm Nissl-plastic stained sections (B), Nissl (C), AChE (D), and Golgi (E), the border between SPL and IZ is marked by an asterisk. This border is best seen in AChE staining (D), in which the very well defined external part of the IZ, represented by intensively-stained fibers of the external capsule (*) are clear. In Golgi (E), the change in radial orientation of the deep portion of the CP and the lost radial architecture (cells and fibers) in the SPL are easily visualized. Notice the difference in intensity of fiber labeling in A, in which more fibers (darker background) are observed midlaterally, and how intensity diminishes (lighter background) as the gradient progresses toward more dorsal regions.
is clearly visible on 1-μm thin plastic sections because of the tangentially running osmificated fibers (Fig. 3B). The most remarkable features during this period are obliquity running “wavy” fibers (Fig. 3 A and D, arrowheads and arrows). In Golgi preparations, the SP in formation, especially SPL, shows loss of radial orientation of cell bodies and processes (Fig. 3E). This phase corresponds to monkey developmental period E54–E59. After this period, the SP continues to increase in size (15–20 PCW) and is characterized by cell differentiation and increase in fiber ingrowth and enlargement of the neuropil. In NHPs, this corresponds to ∼E60–E82. Discussion The main finding in the present investigation, summarized in Fig. 4, is that after early-born neurons destined for the SP complete Duque et al.
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their migration, they become secondarily displaced downward by the arrival of axons, first from the subcortical and later from cortical origin. Because of regional differences in the magnitude of corticocortical connections in primates, the width of the SP is highly variable and is particularly large subjacent to the association areas. It should be recognized that the present findings apply to Old World Primates macaque and human, as there are considerable species-specific differences in terms of cell origin and dynamics of migration (17, 18). The present investigation was focused on the expansion of the SP zone, as this is one of the most dramatic transient events in the evolution of the human cerebral cortex. Our results demonstrate that contrary to the prevailing view, this expansion is not just a consequence of increased numbers of SP neurons in primates, but is mainly a result of their dynamic interaction with an increasing number of incoming axons. Cell dispersion was measured both toward the pia and toward the ventricle. The layer 6 neurons, which are born at the same time as the SP cells and migrate together, remain as a dense stratum and are not dispersed toward the ventricle. The results of our analysis, within the temporal limits of the injection times available, indicate that arrival of incoming fibers and growth of surrounding extracellular matrix are main contributors to the expansion of the SP and to the change in spatial distribution of cells that had already finished migrating. We believe that the connectivity requirements of the different cortical areas explain why the width of the SP is most prominent in areas subjacent to human-specific association areas. The study of such complex cellular events is, by its nature, limited in human, in which, for instance, it is not possible to date the birth of neurons. Therefore, we analyzed various immuno- and histochemical stainings to corroborate that similar events are likely to occur in our species. This extrapolation of the data is needed to elucidate the enigmatic expansion of the SP that occurs in both species and is justified because of similarities in development, including the timing, progression, and magnitude of cellular events. The addition of fibers is by no means the only event that occurs during the expansion of the SP. After an original set of neurons populates the SP, the addition of cells and formation of many synapses, among other events, add to the growth of the neuropil of this compartment (19). Hence, it would be important to determine which and how many of these later-born cells become permanent residents of the SP during its transient existence, which and how many use the SP as a waiting compartment before continuing their migration, and which remain in adults as interstitial cells (3). We did not observe evidence to support the possibility of downward active locomotion. However, the analysis of dynamic events using static data at a limited number of times is a limitation inherent to our study, and thus additional research is needed to elucidate finer details on the origin, progression, and eventual dissolution of the SP. Materials and Methods Animals, TdR Injections, and Autoradiography. Autoradiograms from 31 Rhesus monkey brains were examined. Animals were exposed to TdR as embryos and killed at various prenatal and postnatal ages. The present experimental study in developing macaque did not require the addition of a single new animal. All cases were obtained from the Rakic Collection of Nonhuman Primates at Yale University. Experimental protocols and animal care before death were conducted during the last 45 y in accordance with institutional guidelines initially at Harvard Medical School, and subsequently at Yale University. Animal breeding, dating of pregnancies, and perfusion protocols, as well as descriptions of brain processing for autoradiography, have been previously published (20–22). Briefly, all animals received a single intravenous injection of TdR (New England Nuclear; 10 mCi per kilogram of body weight). Sections were cut in the coronal plane at 8–10 μm or 30 μm, using a freezing microtome. In every case, every 10th section was stained for Nissl or toluidine blue. Having been processed ∼35–45 y ago, the mounting media on TdR sections had deteriorated. After immersion in xylenes, old coverslips were removed and new mounting media and new coverslips were used to recover the slides. The quality of the TdR autoradiography was not compromised (23).
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Fig. 4. Summary model of the secondary expansion of the transient SP zone. (A) E40 born SP cells are visible in the VZ 1 h after injection. By E40, there is no SP yet. (B) By E54, these early born cells accumulate in the SP, and a few Cajal-Retzius cells in the MZ become evident. (C) By E83, and later in midgestation, these cells become secondarily displaced and widespread into the expanding SP by ingrowth of the neuropil. In particular, monoamine, basal forebrain, thalamocortical, and later on a large contingent of corticocortical axons.
Microscopy, Area Sampling, and Criteria for Positive Labeling and Cell Plotting. Section outlines were drawn at 5–10×, depending on the size of the section. Very early embryonic ages, and hence small sections, were done at higher magnification. The region of interest was the midrostral to caudal telencephalon, from frontal association areas, mid-motor-somatosensory cortex, to the primary visual cortex. Because of differences in the temporal and spatial development of the subplate, in every case, cells were plotted in a minimum of three brain sections containing samples from each of these areas in the rostrocaudal axis. Plotting was also done to always cover the dorsal, dorsolateral, and lateral cortical wall and, when possible, the basal or ventral cortical wall (e.g., rostral to the ganglionic eminence). All cell plotting was done at 40×, using a Zeiss Plan-Neofluar objective lens with a 0.75 optical aperture, using a Zeiss Axioskop microscope fully motorized and interfacing to a Dell computer running StereoInvestigator (MicroBrightField Inc.). As a control, cell counts done at 100× oil immersion using a Zeiss Plan-Neofluar objective lens with a 1.3 optical aperture were done in a group of 10 sections, and the results were compared with those obtained at 40×; the difference in cell counts affected only later-generation cells (not heavily labeled), and it was less than 4% in every case. To limit the region in which cells were to be plotted, a rectangular box (500 μm width by a length as long as needed) was placed in the cortical area to be sampled. In the case of very early embryonic ages, the small sections would not accommodate more than just one box width; hence, in those cases, all cells were plotted in dorsal and lateral directions. The guide box was most useful as sections became larger. The nucleus of a TdR cell was considered positive when it contained a minimum of three discrete silver grains that would clearly outnumber any grains over a similar surface area in the background (20, 23). Regions of high
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background, where many silver grains could be observed outside of cellular profiles, were not used for counting. A heavily stained nucleus may contain ≥50 discrete silver grains. Only intensely labeled cells, with the quantity of silver grains located over the nucleus equal or more than half of the maximum grain counts in any nucleus in a given specimen, were considered born at the time of injection (20). Cell Position and Migration vs. Secondary Cell Dispersion. To distinguish between cell migration and later cell dispersion, the absolute position of each TdRlabeled cell within the cerebral wall from the ventricle to the pia in the radial direction was plotted for all injection times (∼E35, E40, E50, E60, E70, E80) and for all postinjection periods (1 h, 3 d, 7 d, 14 d, and ∼2.5 mo postnatal after injection). Although the distance from the ventricle to pia changes at different rostrocaudal, as well as dorsoventral, positions in the radial direction, this distance is 100% of the distance any one cell can travel. Hence, the position of each labeled cell was normalized to its corresponding total radial distance from ventricle to pia, and a mean population distance was calculated for dorsal, dorsolateral, lateral, and basal coordinates at different rostrocaudal levels. As cells complete migration through a milieu that continues to grow, the distances among labeled cells, and from each labeled cell to pia (and/or ventricle), continue to increase. This affects cell density measurements, but not a cell’s normalized relative distance to pia. The former would change either because of reinitiation of a migratory flux or because of other forces that affect the cell’s relative position by actively moving it. Because E40 was experimentally determined to be a time when many cells destined for the SP are born, we focused the analysis of cell dispersion on this set of cells. For this, an additional case from the collection, injected at E41 and killed at E83, was added to the analysis.
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ACKNOWLEDGMENTS. This work was supported by grants from the NIH/ National Institute on Drug Abuse (R01-DA023999), NIH/National Institute of Neurological Disorders and Stroke (R01-NS014841), NIH/National Eye Institute (R01-EY002593), and Kavli Institute for Neuroscience at Yale University (to P.R.), as well as a Croatian Science Foundation Award HRZZ (Hrvatska zaklada za znanost) (to I.K.).
1. Kostovic I, Molliver ME (1974) A new interpretation of the laminar development of cerebral cortex: Synaptogenesis in different layers of neopallium in the human fetus. Anat Rec 178:395. 2. Rakic P (1977) Prenatal development of the visual system in rhesus monkey. Philos Trans R Soc Lond B Biol Sci 278(961):245–260. 3. Kostovic I, Rakic P (1980) Cytology and time of origin of interstitial neurons in the white matter in infant and adult human and monkey telencephalon. J Neurocytol 9:2l9–242. 4. Kostovic I, Jovanov-Milosevic N (2008) Subplate zone of the human brain: Historical perspective and new concepts. Coll Antropol 32(Suppl 1):3–8. 5. Kostovic I, Rakic P (1990) Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J Comp Neurol 297(3):441–470. 6. Ghosh A, Shatz CJ (1993) A role for subplate neurons in the patterning of connections from thalamus to neocortex. Development 117(3):1031–1047. 7. Allendoerfer KL, Shatz CJ (1994) The subplate, a transient neocortical structure: Its role in the development of connections between thalamus and cortex. Annu Rev Neurosci 17:185–218. 8. Kanold PO, Luhmann HJ (2010) The subplate and early cortical circuits. Annu Rev Neurosci 33:23–48. 9. Wang WZ, et al. (2011) Comparative aspects of subplate zone studied with gene expression in sauropsids and mammals. Cereb Cortex 21(10):2187–2203. 10. Hoerder-Suabedissen A, Molnár Z (2015) Development, evolution and pathology of neocortical subplate neurons. Nat Rev Neurosci 16(3):133–146. 11. Hutsler JJ, Casanova MF (2016) Review: Cortical construction in autism spectrum disorder: Columns, connectivity and the subplate. Neuropathol Appl Neurobiol 42(2): 115–134. 12. Judaš M, Sedmak G, Kostovic I (2013) The significance of the subplate for evolution and developmental plasticity of the human brain. Front Hum Neurosci 7:423. 13. Marin-Padilla M (1978) Dual origin of the mammalian neocortex and evolution of the cortical plate. Anat Embryol (Berl) 152(2):109–126. 14. Akbarian S, et al. (1996) Maldistribution of interstitial neurons in prefrontal white matter of the brains of schizophrenic patients. Arch Gen Psychiatry 53(5):425–436.
15. Kostovic I, Kostovic-Srzentic M, Benjak V, Jovanov-Miloševic N, Radoš M (2014) Developmental dynamics of radial vulnerability in the cerebral compartments in preterm infants and neonates. Front Neurol 5:139. 16. Kostovic I, Judaš M, Sedmak G (2011) Developmental history of the subplate zone, subplate neurons and interstitial white matter neurons: Relevance for schizophrenia. Int J Dev Neurosci 29(3):193–205. 17. Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat’s visual cortex: Cogeneration of subplate and marginal zones. J Neurosci 5(4):1062–1075. 18. Pedraza M, Hoerder-Suabedissen A, Albert-Maestro MA, Molnár Z, De Carlos JA (2014) Extracortical origin of some murine subplate cell populations. Proc Natl Acad Sci USA 111(23):8613–8618. 19. Bystron I, Blakemore C, Rakic P (2008) Development of the human cerebral cortex: Boulder Committee revisited. Nat Rev Neurosci 9(2):110–122. 20. Rakic P (1973) Kinetics of proliferation and latency between final cell division and onset of differentiation of cerebellar stellate and basket neurons. J Comp Neurol 147(4):523–546. 21. Rakic P (1976) Prenatal genesis of connections subserving ocular dominance in the rhesus monkey. Nature 26l(5560):467–47l. 22. Kornack DR, Rakic P (1998) Changes in cell-cycle kinetics during the development and evolution of primate neocortex. Proc Natl Acad Sci USA 95(3):1242–1246. 23. Duque A, Rakic P (2011) Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate. J Neurosci 31(42): 15205–15217. 24. Judaš M, et al. (2011) The Zagreb Collection of human brains: A unique, versatile, but underexploited resource for the neuroscience community. Ann N Y Acad Sci 1225(Suppl 1):E105–E130. 25. Kostovic I, Goldman-Rakic PS (1983) Transient cholinesterase staining in the mediodorsal nucleus of the thalamus and its connections in the developing human and monkey brain. J Comp Neurol 219(4):431–447. 26. Kostovic I, Judaš M, Radoš M, Hrabac P (2002) Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging. Cereb Cortex 12(5):536–544.
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previously (26). All sections were analyzed and images taken by using a high-resolution digital slide scanner NanoZoomer 2.0RS (Hamamatsu), and figures were managed in Adobe Photoshop and assembled in Adobe Illustrator.
Human Tissue. The postmortem human brains used for comparison with NHP tissue ranged in age from 7.5 to 24 PCW and are part of the large and versatile Zagreb Collection (24). Brain specimens were obtained from either medically indicated or spontaneous abortions at several clinical and pathological departments of the University of Zagreb, School of Medicine (Croatia). Patients provided informed consent and procedures were approved by the corresponding Institutional Review Boards (IRB). The fetal age was estimated on the basis of crown–rump length (in millimeters) and pregnancy records. Whole brains were fixed by immersion in 4% (40 g/L) paraformaldehyde in 0.1 M PBS at pH 7.4. Tissue blocks were either frozen or embedded in paraffin wax. Sections were cut at various thicknesses (1–30 μm). Histological Cresyl-violet (Nissl) staining was used to delineate cytoarchitectonic boundaries, and adjacent sections were processed by several histochemical methods. AChE-histochemistry (as described in ref. 25) was used for visualization of growing thalamocortical afferents and certain sagittally oriented axon strata, such as the external capsule system. Periodic acid–Schiff–Alcian blue histochemistry was used to analyze the laminar location and relative regional abundance of the extracellular matrix, as described
NEUROSCIENCE
Statistical Analyses. When appropriate for quantitative analysis and for comparisons, statistics were performed with Analyze-it v2.12 for Microsoft Excel. Distributions were examined to check for normalcy and homoscedasticity. Normality was assessed using the Shapiro-Wilk test. Homogeneity of variance was tested with an F-test. ANOVA was used to compare means among normally distributed populations with equal variance. Nonparametric ANOVA alternatives were performed with the Mann–Whitney and/or Kruskal-Wallis tests.
Duque et al.
PNAS | August 30, 2016 | vol. 113 | no. 35 | 9897
