Secondary expansion of the transient subplate zonein the developing cerebrum of human andnonhuman primatesAlvaro Duquea,b, Zeljka Krsnikc, Ivica Kostovi�cc, and Pasko Rakica,b,1

aDepartment of Neuroscience, Yale University, New Haven, CT 06510; bKavli Institute for Neuroscience, School of Medicine, Yale University, New Haven, CT06510; 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 theBoulder Committee’s list of transient embryonic zones [Bystron I,Blakemore C, Rakic P (2008) Nature Rev Neurosci 9(2):110–122]. It ishighly developed in human and nonhuman primates, but its origin,mode, and dynamics of development, resolution, and eventual extinc-tion are not well understood because human postmortem tissueoffers only static descriptive data, and mice cannot serve as anadequate experimental model for the distinct regional differencesin primates. Here, we take advantage of the large and slowly de-veloping SP in macaque monkey to examine the origin, settlingpattern, and subsequent dispersion of the SP neurons in primates.Monkey embryos exposed to the radioactive DNA replicationmarker tritiated thymidine ([3H]dT, or TdR) at early embryonic ageswere killed at different intervals postinjection to follow postmitoticcells’ positional changes. As expected in primates, most SP neuronsgenerated in the ventricular zone initially migrate radially, togetherwith prospective layer 6 neurons. Surprisingly, mostly during midg-estation, SP cells become secondarily displaced and widespread intothe expanding SP zone, which becomes particularly wide subjacentto the association cortical areas and underneath the summit of itsfolia. We found that invasion of monoamine, basal forebrain, tha-lamocortical, and corticocortical axons is mainly responsible for thisregion-dependent passive dispersion of the SP cells. Histologic andimmunohistochemical comparison with the human SP at corre-sponding fetal ages indicates that the same developmental eventsoccur in both primate species.

cerebral cortex | brain evolution | transient lamination |neuronal migration | neural stem cells

Because of its exceptionally large size, it may not be coincidencethat the subplate (SP) zone was originally discovered (1) and its

function proposed on the basis of analysis of the developing cortexin human and nonhuman primates (NHPs) (2–4). For example,examination of the embryonic cerebral wall in the macaque monkeyshowed that thalamic axons form transient synapses with SP neu-rons before entering the superjacent cerebral cortex, inspiring theterm waiting compartment (5). In addition, subsequent research inhumans and NHPs indicated that the SP is particularly large sub-jacent to the association areas, such as the prefrontal cortex, andthat a substantial number of SP cells survive and remain scatteredwithin the white matter of the adult telencephalon as interstitialneurons (3, 4). Despite intensive research in various mammalianspecies in the last 40 y (reviewed in refs. 5–11), the origin as well asevolutionary and developmental mechanisms that underlie the ex-traordinary expansion and regional diversification of the SP in hu-man 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 theprimordial preplate (PP) after its separation (split) from the mar-ginal zone (MZ) situated above by arrival of the cortical plate (CP)neurons from the proliferative ventricular zone (VZ) (13). How-ever, the “splitting hypothesis,” for example, cannot explain thelarge (5–10-fold) difference in the width of the SP subjacent to

various cytoarchitectonic areas of the human fetal cerebrum sosalient during midgestation (Fig. 1), and it also stands in contrast toexperimental data obtained in macaque monkey, using tritiatedthymidine ([3H]dT, or TdR) to label the time and place of neuronalorigin (3, 5). Part of the problem has been that most subsequentresearch on the genesis, function, and fate of SP cells was carriedout in nonprimate species, such as rodents, where the SP is smalland relatively uniform in width. Therefore, the origin, regionalevolutionary expansion, and elaboration of SP in humans remainelusive. Because of the potential significance of the SP in thepathogenesis of many developmental brain disorders (10, 14–16), itis important to experimentally resolve these conceptual matters anddiscrepancies in the scientific literature.Here we examined the origin, migration pathway, initial settling

position, and secondary dispersion of SP cells in the expandingSP zone of NHP (macaque) embryos. TdR was used as a markerof the last mitotic division of the neural stem cells and neuro-nal progenitors and positions of postmitotic cells plotted at dif-ferent postinjection intervals in regions subjacent to cortical areaswith different SP zone widths. Our major goal was to determinewhether the VZ subjacent to the cortical areas with wider SPproduces more SP neurons or whether, as hypothesized before(5), regions with wider SP (d in Fig. 1) are the result of neuronaldispersion within a larger neuropil. Our second goal was to verifywhether the SP originates by the split of the cells in the MZ, ahypothesis that was based on static human histological material(13). Our third goal was to determine experimentally whethercells bound for deep cortical layers, born at the same time as SPcells, are phenotypically different, and thus would not be affectedby downward dispersion and would remain as a distinct undis-turbed band at the CP–SP border. Finally, we used sections of

Significance

The subplate zone, a transient cellular compartment of theembryonic cerebrum, has expanded in size and complexityduring primate evolution, culminating in humans. Here, theapplication of multiple methods, including labeling time and placeof neuronal origin and subsequent changes in their positions inmacaque monkey embryos, and the use of histo- and immuno-chemistry in human fetal cerebral tissue of comparable prenatalages reveals extraordinary cellular dynamics and unexpectedsecondary displacement of neurons. These findings may havesignificance for understanding cortical development and evolutionand may provide insight into the pathogenesis of cortical dis-orders, 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 9676.1To whom correspondence should be addressed. Email: pasko.rakic@yale.edu.

9892–9897 | PNAS | August 30, 2016 | vol. 113 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1610078113

Dow

nloa

ded

by g

uest

on

July

8, 2

020

human forebrain tissue at comparable embryonic ages stained byhisto- and immunochemical techniques to analyze development ofthe human SP. As shown here, our results indicate that the SP inprimates has a more dynamic and complex developmental historythan hitherto recognized and provides new insight into the pos-sible role of this transient zone in the pathogenesis of congenitaldisorders of the cerebral cortex that range from childhood epi-lepsy and autism to schizophrenia (10, 14–16).

ResultsOrigin, Position, and Secondary Dispersion of SP Cells in NHPs. Thirty-one animals divided into six groups according to injection time[∼E35 (embryonic day 35), E40, E50, E60, E70, E80] were an-alyzed at five different postinjection periods (1 h; 3, 7, and 14 d;and ∼2.5 mo postnatal). An additional case injected at E41 andkilled at E83 was added to the analysis (see Materials andMethods 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 firstgeneration cells, and the description that follows focuses on cellsborn 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 theventricle and, correspondingly, 40–30% distance to pia. As thegrowth of the cortical wall proceeds, the SP becomes establishedas a separate compartment within the cortical wall. The SP in-volves the constant arrival of newly born cells as well as ingrowthof axons from subcortical structures and other cortical areas. Astime passes, these fibers not only become an obstacle for themigration of cells (still migrating in the SP) but also start dis-persing some of the neurons already located in it. Areas of theSP that develop first (i.e., the lateral cortical wall), and where

labeled cells first accumulate, are also the areas in which celldispersion is first observed. Dispersion of cells becomes more evi-dent in midgestation, after the arrival of thalamocortical axons and,increasingly, a large contingent of corticocortical axons. From theoriginal cohorts of cells, only the SP resident cells seem to be dis-persed by the incoming fibers, whereas layer 6 cells remain in theCP–SP border. Further research is needed to determine the genesand mechanism by which the dichotomy between SP cells and cellscommitted 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 injectiontimes (E40, E50, E56). Cells labeled at E40, when the SP does notyet exist, accumulate in the PP (Fig. 2C1′) by the end of the firstweek postinjection and are found in the newly formed SP zone(Fig. 2 A1, B1, and C1) by the end of the second week. In thelateral wall of a midrostrocaudal section (Fig. 2C1), where the SPzone is the widest (compared here to more dorsal locations), in-coming fibers displace already settled SP cells toward the ven-tricular zone. In the rostralmost telencephalon, cells born andlabeled at E50 are 14 d later found in the SP zone, where, in-dependent of dorsolateral location, they are all lumped together ina dense band (Fig. 2A2). More caudally, cells originally settled inthe dorsolateral SP appear entering the CP, that is, are displacedtoward the MZ zone (Fig. 2B2), whereas cells settled in moredorsal and ventral locations at the same rostrocaudal level are notyet affected. At a further developed caudal level, the displacementof all SP cells in the dorsal, dorsolateral, and lateral positionsbecomes more pronounced (Fig. 2C2) than at less developedrostral levels (Fig. 2 A2 and B2). Cells born and labeled at E56, at atime when the SP zone is already formed and actively growing,migrate and enter the SP at a time when a larger number of fibersare arriving into the SP. These cells are consequently entering anincreasingly more dynamic milieu, and their distributions, even14 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 reachtheir final destinations. This is reflected by differences in cel-lular distributions that can be detected just 3 d after labeling(Fig. 2 B4, C4, and E). We estimate that the cellular distributionchanges detected at 1 h, 3 d, and 7 d postinjection are mostly aresult of migration, whereas the high expansion that can be detected14 d (and later) postinjection, especially for injections before E50,are mostly a result of dispersion of cells that have completed theirmigration. In specimens injected at later times (e.g., E70>), themigratory pathways are not only initially longer but also are activelyincreasing. Hence, at 14 d postinjection, many cells are still mi-grating. Fig. 2 D–F illustrates the positions of cells as seen in thetissue. In Fig. 2 D and F, SP cells have already finished migratingand are being dispersed by incoming fibers. Also, in the more de-veloped lateral wall in Fig. 2D, a larger number of cells have alreadymoved into the CP, whereas in Fig. 2F, the accumulation of cells inlayer 6 becomes evident by the displacement of deeper cells into theSP. 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 previouslyshown by others in the cat visual cortex (17).

SP Expansion in Human. In human, the SP formation stage [∼12–13postconceptional weeks (PCW)] is also known as “second CP”(5) because the changes are so distinct. Fig. 3 illustrates the dy-namic expansion of the SP that is occurring at this time. The deepportion of the CP becomes gradually loose and is transformed into anew cytoarchitectonic entity, the SP. The SP can then be divided insuperficial (upper SP, SPU) and deeper (lower SP, SPL) regions witha gradual merging remaining as pre-SP (Fig. 3 B and C). The borderwith the intermediate zone (IZ) in the midlateral and rostral cortex

SPCPIZVZSVZ

16 PCW 20 PCW

CP

SP

VZ

MZ

IZSVZPVf

*EC

a

b

c

d

e

Human Thickness of the SP(not to scale by age)

A B

a1

2

3

c2d3

1

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 at16 and 20 PCW, showing chemoarchitectonics of the anterior (rostral) ce-rebral wall. Because of the high amount of extracellular matrix reactivity inthe 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 isevident 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.

Duque et al. PNAS | August 30, 2016 | vol. 113 | no. 35 | 9893

NEU

ROSC

IENCE

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

July

8, 2

020

0 20 40 60 80 100%

SPGL

MZ

CP

SPU

SPL

IZ

SVZ

VZ

1 E40-E54

A A1 A2

Rostral

Caudal

A3

0 30 60MZPPIZ

SVZVZ E40-E47

C1’

GE

GE

4 E52-E55

2 E50-E64 3 E56-E70

C4

SPGL

MZ

CP

SPU

SPL

IZ

SVZ

VZ

SPGL

MZ

CP

SPU

SPL

IZ

SVZ

VZ

B4

incomingaxons invade SP

55E-25E45E-04E

MZCP

SPU

IZ

SVZ

VZ

SPL

SPGL

D

MZCP

SPU

IZ

SVZ

VZ

SPL

E

dorsal dorso-lateral lateral

Cells finished migrating but are beingdispersed by incoming fibers in expanding SP

SPGL

B

C

B1

C1

B2

C2

Cells still migratingin an already expanding SP

500 µmTdR+ cells

CP

SPU

IZ

SVZVZ

SPL

SVZo

dorso-lateral

E41-E83

FMZ

dorsaldorsolateral

lateral

ventral

dorsal dorso-lateral lateral

Fig. 2. Examples of settling and later dispersion of cells born at different gestational ages. (A–C) Coronal rostral to caudal levels. (A1–C4) Spatiotemporalcharacteristics 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 corticalwall compartments from VZ to SPGL. See Results for further details.

9894 | www.pnas.org/cgi/doi/10.1073/pnas.1610078113 Duque et al.

Dow

nloa

ded

by g

uest

on

July

8, 2

020

is clearly visible on 1-μm thin plastic sections because of the tan-gentially running osmificated fibers (Fig. 3B). The most remarkablefeatures during this period are obliquity running “wavy” fibers (Fig.3 A and D, arrowheads and arrows). In Golgi preparations, the SPin formation, especially SPL, shows loss of radial orientation of cellbodies and processes (Fig. 3E). This phase corresponds to monkeydevelopmental period E54–E59. After this period, the SP continuesto increase in size (15–20 PCW) and is characterized by cell dif-ferentiation and increase in fiber ingrowth and enlargement of theneuropil. In NHPs, this corresponds to ∼E60–E82.

DiscussionThe main finding in the present investigation, summarized in Fig.4, is that after early-born neurons destined for the SP complete

their migration, they become secondarily displaced downward bythe arrival of axons, first from the subcortical and later fromcortical origin. Because of regional differences in the magnitudeof corticocortical connections in primates, the width of the SP ishighly variable and is particularly large subjacent to the associ-ation areas. It should be recognized that the present findingsapply to Old World Primates macaque and human, as there areconsiderable species-specific differences in terms of cell originand 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 inthe evolution of the human cerebral cortex. Our results dem-onstrate that contrary to the prevailing view, this expansion is notjust a consequence of increased numbers of SP neurons in pri-mates, but is mainly a result of their dynamic interaction with anincreasing number of incoming axons. Cell dispersion was mea-sured both toward the pia and toward the ventricle. The layer6 neurons, which are born at the same time as the SP cells andmigrate together, remain as a dense stratum and are not dispersedtoward the ventricle. The results of our analysis, within the temporallimits of the injection times available, indicate that arrival of in-coming fibers and growth of surrounding extracellular matrix aremain contributors to the expansion of the SP and to the change inspatial distribution of cells that had already finished migrating. Webelieve that the connectivity requirements of the different corticalareas explain why the width of the SP is most prominent in areassubjacent 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 birthof neurons. Therefore, we analyzed various immuno- and histo-chemical stainings to corroborate that similar events are likely tooccur in our species. This extrapolation of the data is needed toelucidate the enigmatic expansion of the SP that occurs in bothspecies and is justified because of similarities in development, in-cluding 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 neuronspopulates the SP, the addition of cells and formation of many syn-apses, among other events, add to the growth of the neuropil of thiscompartment (19). Hence, it would be important to determine whichand how many of these later-born cells become permanent residentsof the SP during its transient existence, which and how many use theSP as a waiting compartment before continuing their migration, andwhich remain in adults as interstitial cells (3). We did not observeevidence to support the possibility of downward active locomotion.However, the analysis of dynamic events using static data at a limitednumber of times is a limitation inherent to our study, and thus ad-ditional research is needed to elucidate finer details on the origin,progression, and eventual dissolution of the SP.

Materials and MethodsAnimals, TdR Injections, and Autoradiography. Autoradiograms from 31 Rhesusmonkey brains were examined. Animals were exposed to TdR as embryos andkilled at various prenatal and postnatal ages. The present experimental study indevelopingmacaquedid not require the addition of a single newanimal. All caseswere obtained from the Rakic Collection of Nonhuman Primates at YaleUniversity. Experimental protocols and animal care before death were con-ducted during the last 45 y in accordancewith institutional guidelines initially atHarvardMedical School, and subsequently at Yale University. Animal breeding,dating of pregnancies, and perfusion protocols, as well as descriptions of brainprocessing for autoradiography, have been previously published (20–22).Briefly, all animals received a single intravenous injection of TdR (New EnglandNuclear; 10 mCi per kilogram of body weight). Sections were cut in the coronalplane at 8–10 μm or 30 μm, using a freezing microtome. In every case, every10th section was stained for Nissl or toluidine blue. Having been processed∼35–45 y ago, the mounting media on TdR sections had deteriorated. Afterimmersion in xylenes, old coverslips were removed and new mounting mediaand new coverslips were used to recover the slides. The quality of the TdRautoradiography was not compromised (23).

CPSPU

SPL

VZIZ

INT. C

BF

ThC

12-13 PCW

A

MZCP

SPu

SPl

VZ

SVZ

IZ

B DC E

* * * *

AChE

Nissl AChENissl-plastic Golgi

Fig. 3. Early arrival of axons and increased size of human SP. (A) Midlateraland lateral portions of the anterior part of the cerebral wall showing axonalstrata with thalamo-cortical (ThC) and basal forebrain (BF) fibers. Notice obli-que entrance of fibers into the SP (arrowheads). (A–E) illustrate the formationand initial expansion of the SP with dramatic changes in cytoarchitectonicsvisible 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 markedby an asterisk. This border is best seen in AChE staining (D), in which the verywell defined external part of the IZ, represented by intensively-stained fibersof the external capsule (*) are clear. In Golgi (E), the change in radial orien-tation of the deep portion of the CP and the lost radial architecture (cells andfibers) in the SPL are easily visualized. Notice the difference in intensity of fiberlabeling in A, in which more fibers (darker background) are observed mid-laterally, and how intensity diminishes (lighter background) as the gradientprogresses toward more dorsal regions.

Duque et al. PNAS | August 30, 2016 | vol. 113 | no. 35 | 9895

NEU

ROSC

IENCE

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

July

8, 2

020

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 highermagnification. The region of interest was the midrostral to caudal telen-cephalon, from frontal association areas, mid-motor-somatosensory cortex,to the primary visual cortex. Because of differences in the temporal andspatial development of the subplate, in every case, cells were plotted in aminimum of three brain sections containing samples from each of theseareas in the rostrocaudal axis. Plotting was also done to always cover thedorsal, dorsolateral, and lateral cortical wall and, when possible, the basal orventral cortical wall (e.g., rostral to the ganglionic eminence).

All cell plotting was done at 40×, using a Zeiss Plan-Neofluar objective lenswith a 0.75 optical aperture, using a Zeiss Axioskop microscope fully motorizedand interfacing to a Dell computer running StereoInvestigator (MicroBright-Field Inc.). As a control, cell counts done at 100× oil immersion using a ZeissPlan-Neofluar objective lens with a 1.3 optical aperture were done in a groupof 10 sections, and the results were compared with those obtained at 40×; thedifference in cell counts affected only later-generation cells (not heavily la-beled), and it was less than 4% in every case. To limit the region in which cellswere to be plotted, a rectangular box (500 μm width by a length as long asneeded) was placed in the cortical area to be sampled. In the case of very earlyembryonic ages, the small sections would not accommodate more than just onebox width; hence, in those cases, all cells were plotted in dorsal and lateraldirections. The guide box was most useful as sections became larger.

The nucleus of a TdR cell was considered positive when it contained aminimum of three discrete silver grains that would clearly outnumber anygrains over a similar surface area in the background (20, 23). Regions of high

background, where many silver grains could be observed outside of cellularprofiles, were not used for counting. A heavily stained nucleus may contain≥50 discrete silver grains. Only intensely labeled cells, with the quantity ofsilver grains located over the nucleus equal or more than half of the maxi-mum grain counts in any nucleus in a given specimen, were considered bornat the time of injection (20).

Cell Position and Migration vs. Secondary Cell Dispersion. To distinguish be-tween cell migration and later cell dispersion, the absolute position of each TdR-labeled cell within the cerebral wall from the ventricle to the pia in the radialdirection was plotted for all injection times (∼E35, E40, E50, E60, E70, E80) andfor all postinjection periods (1 h, 3 d, 7 d, 14 d, and ∼2.5 mo postnatal afterinjection). Although the distance from the ventricle to pia changes at differentrostrocaudal, as well as dorsoventral, positions in the radial direction, this dis-tance is 100% of the distance any one cell can travel. Hence, the position of eachlabeled cell was normalized to its corresponding total radial distance fromventricle to pia, and a mean population distance was calculated for dorsal,dorsolateral, lateral, and basal coordinates at different rostrocaudal levels. Ascells complete migration through a milieu that continues to grow, the distancesamong labeled cells, and from each labeled cell to pia (and/or ventricle), con-tinue to increase. This affects cell density measurements, but not a cell’s nor-malized relative distance to pia. The former would change either because ofreinitiation of a migratory flux or because of other forces that affect the cell’srelative position by actively moving it. Because E40 was experimentally de-termined to be a time when many cells destined for the SP are born, we focusedthe analysis of cell dispersion on this set of cells. For this, an additional case fromthe collection, injected at E41 and killed at E83, was added to the analysis.

VZSVZ

IZ

SP

CP

MZ

VZ

SVZ

IZ

MZCP

E40-E40.1

E40-E54

E40-E83

Downward spreadof SP cells born at E40

A B C

VZ

PP

SVZSP

Incoming axons frommonoamine centers, basal forebrain,thalamus, and cortex

IZ

Cells born at E40

L6cells

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 noSP 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.

9896 | www.pnas.org/cgi/doi/10.1073/pnas.1610078113 Duque et al.

Dow

nloa

ded

by g

uest

on

July

8, 2

020

Human Tissue. The postmortem human brains used for comparison with NHPtissue ranged in age from 7.5 to 24 PCW and are part of the large andversatile Zagreb Collection (24). Brain specimens were obtained from eithermedically indicated or spontaneous abortions at several clinical and path-ological departments of the University of Zagreb, School of Medicine(Croatia). Patients provided informed consent and procedures were ap-proved by the corresponding Institutional Review Boards (IRB). The fetalage was estimated on the basis of crown–rump length (in millimeters) andpregnancy 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 frozenor embedded in paraffin wax. Sections were cut at various thicknesses (1–30 μm).Histological Cresyl-violet (Nissl) staining was used to delineate cytoarchi-tectonic boundaries, and adjacent sections were processed by several his-tochemical methods. AChE-histochemistry (as described in ref. 25) wasused for visualization of growing thalamocortical afferents and certainsagittally oriented axon strata, such as the external capsule system. Periodicacid–Schiff–Alcian blue histochemistry was used to analyze the laminar locationand relative regional abundance of the extracellular matrix, as described

previously (26). All sections were analyzed and images taken by using ahigh-resolution digital slide scanner NanoZoomer 2.0RS (Hamamatsu), andfigures were managed in Adobe Photoshop and assembled in Adobe Illustrator.

Statistical Analyses. When appropriate for quantitative analysis and for com-parisons, 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 variancewas testedwith an F-test. ANOVAwas used to compare means among normallydistributed populations with equal variance. Nonparametric ANOVA alterna-tives were performed with the Mann–Whitney and/or Kruskal-Wallis tests.

ACKNOWLEDGMENTS. This work was supported by grants from the NIH/National Institute on Drug Abuse (R01-DA023999), NIH/National Institute ofNeurological Disorders and Stroke (R01-NS014841), NIH/National Eye In-stitute (R01-EY002593), and Kavli Institute for Neuroscience at Yale Univer-sity (to P.R.), as well as a Croatian Science Foundation Award HRZZ (Hrvatskazaklada za znanost) (to I.K.).

1. Kostovi�c I, Molliver ME (1974) A new interpretation of the laminar development ofcerebral 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. PhilosTrans R Soc Lond B Biol Sci 278(961):245–260.

3. Kostovi�c I, Rakic P (1980) Cytology and time of origin of interstitial neurons in thewhite matter in infant and adult human and monkey telencephalon. J Neurocytol9:2l9–242.

4. Kostovi�c I, Jovanov-Milosevi�c N (2008) Subplate zone of the human brain: Historicalperspective and new concepts. Coll Antropol 32(Suppl 1):3–8.

5. Kostovi�c I, Rakic P (1990) Developmental history of the transient subplate zone in thevisual and somatosensory cortex of the macaque monkey and human brain. J CompNeurol 297(3):441–470.

6. Ghosh A, Shatz CJ (1993) A role for subplate neurons in the patterning of connectionsfrom thalamus to neocortex. Development 117(3):1031–1047.

7. Allendoerfer KL, Shatz CJ (1994) The subplate, a transient neocortical structure: Itsrole in the development of connections between thalamus and cortex. Annu RevNeurosci 17:185–218.

8. Kanold PO, Luhmann HJ (2010) The subplate and early cortical circuits. Annu RevNeurosci 33:23–48.

9. Wang WZ, et al. (2011) Comparative aspects of subplate zone studied with geneexpression in sauropsids and mammals. Cereb Cortex 21(10):2187–2203.

10. Hoerder-Suabedissen A, Molnár Z (2015) Development, evolution and pathology ofneocortical subplate neurons. Nat Rev Neurosci 16(3):133–146.

11. Hutsler JJ, Casanova MF (2016) Review: Cortical construction in autism spectrumdisorder: Columns, connectivity and the subplate. Neuropathol Appl Neurobiol 42(2):115–134.

12. Judaš M, Sedmak G, Kostovi�c I (2013) The significance of the subplate for evolutionand 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 thecortical plate. Anat Embryol (Berl) 152(2):109–126.

14. Akbarian S, et al. (1996) Maldistribution of interstitial neurons in prefrontal whitematter of the brains of schizophrenic patients. Arch Gen Psychiatry 53(5):425–436.

15. Kostovi�c I, Kostovi�c-Srzenti�c M, Benjak V, Jovanov-Miloševi�c N, Radoš M (2014) De-velopmental dynamics of radial vulnerability in the cerebral compartments in preterminfants and neonates. Front Neurol 5:139.

16. Kostovi�c 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 visualcortex: 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 AcadSci 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 andonset of differentiation of cerebellar stellate and basket neurons. J Comp Neurol147(4):523–546.

21. Rakic P (1976) Prenatal genesis of connections subserving ocular dominance in therhesus monkey. Nature 26l(5560):467–47l.

22. Kornack DR, Rakic P (1998) Changes in cell-cycle kinetics during the development andevolution of primate neocortex. Proc Natl Acad Sci USA 95(3):1242–1246.

23. Duque A, Rakic P (2011) Different effects of bromodeoxyuridine and [3H]thymidineincorporation 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 Sci1225(Suppl 1):E105–E130.

25. Kostovi�c I, Goldman-Rakic PS (1983) Transient cholinesterase staining in the medi-odorsal nucleus of the thalamus and its connections in the developing human andmonkey brain. J Comp Neurol 219(4):431–447.

26. Kostovi�c I, Judaš M, Radoš M, Hrabac P (2002) Laminar organization of the humanfetal cerebrum revealed by histochemical markers and magnetic resonance imaging.Cereb Cortex 12(5):536–544.

Duque et al. PNAS | August 30, 2016 | vol. 113 | no. 35 | 9897

NEU

ROSC

IENCE

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

July

8, 2

020