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Home , Gyrification, Subventricular zone, Ventricular zone

in the outer promotes enlargement and of the

Brian G. Rasha, Alvaro Duquea, Yury M. Morozova, Jon I. Arellanoa, Nicola Micalia, and Pasko Rakica,b,1

aDepartment of , Yale University, New Haven, CT 06520; and bKavli Institute for Neuroscience at Yale, Yale University, New Haven, CT 06520

Contributed by Pasko Rakic, February 8, 2019 (sent for review January 4, 2019; reviewed by Christopher Kroenke and Zoltan Molnar) The primate cerebrum is characterized by a large expansion of (presently called oRG or bRG) (20) are, at the later stages of cortical surface area, the formation of convolutions, and extraor- , the main sources of and oli- dinarily voluminous subcortical . It was recently godendrocytes (21). However, some recent studies in ferret, proposed that this expansion is primarily driven by increased monkey, and human have proposed that the oSVZ almost ex- production of superficial in the dramatically enlarged clusively produces neurons (7, 8, 22). In addition, one prominent outer subventricular zone (oSVZ). Here, we examined the devel- recent hypothesis postulated that localized areas of the oSVZ opment of the parietal cerebrum in macaque monkey and found with high numbers of progenitors give rise to gyri—but not the that, indeed, the oSVZ initially adds neurons to the superficial intervening sulci (3). These “hot spots” of by layers II and III, increasing their thickness. However, as the oSVZ oRGCs are proposed to cause a “fanning-out” of the cortex at grows in size, its output changes to production of astrocytes and specific positions, causing the cortex to bend to form a (23, , which in outnumber cerebral neurons 24). However, whether the timing of neurogenesis in primates by a factor of three. After the completion of neurogenesis around could account for such a theory, as well as whether such hot spots embryonic day (E) 90, when the cerebrum is still lissencephalic, the actually exist and correlate with specific gyri in primates, is un- + + oSVZ enlarges and contains Pax6 /Hopx outer (basal) radial glial clear (19). Here, we examined the cells produced in the oSVZ cells producing astrocytes and oligodendrocytes until after E125. toward the end of neurogenesis and beyond, in the developing Our data indicate that oSVZ gliogenesis, rather than neurogenesis, macaque monkey. We show that gliogenesis, rather than neu- correlates with rapid enlargement of the cerebrum and develop- rogenesis, is a principal function of a robust oSVZ after NEUROSCIENCE ment of convolutions, which occur concomitantly with the forma- embryonic day (E)92, which coincides with the initiation of tion of cortical connections via the underlying white matter, in gyrification between E100 and E125. addition to neuronal growth, elaboration of dendrites, and ampli- fication of neuropil in the cortex, which are primary factors in the Results formation of cerebral convolutions in primates. We examined samples of the dorsal parietal cerebral wall in the developing macaque monkey (n = 11) at E69, E70, E90, E92, | development | corticogenesis | E120, E125, E145, and E149, and postnatal day (P) 7, P65, and brain convolutions | P91. Immunohistochemistry for Pax6 demonstrated the pres- ence of the oSVZ at E70 and near disappearance by E145 (Fig. + ortical development is characterized by the orderly, se- 1 B–D). Large numbers of oSVZ Pax6 progenitors were found Cquential production of neurons followed by glia, and upon at E70 and E92, but it further expands at E125 (Fig. 1 B–D). their generation, these cell types must migrate long distances to We correlated these data with the distribution of tritiated their final destinations (1–5). The principal stem cells for excit- thymidine ([3H]dT)-labeled cells after acute injection followed atory neurons and glial cells are the radial glial cells (RGCs) by 1-h survival, which reveals the location of cells undergoing S whose bodies are situated in the (VZ) (4–6). In all mammals, including marsupials, daughter cells of RGCs give Significance rise to progenitors that lose their apical attachment to the VZ surface and populate the subventricular zone (SVZ), which is The formation of cortical convolutions of primates, including small in rodents but much larger and more complex in carnivores – humans, is one of the most important subjects in de- and primates (7 10). In many gyrencephalic mammals, the SVZ velopmental neuroscience, but the underlying mechanisms— can be divided into the inner (iSVZ) and outer (oSVZ) layers, considered mostly solved toward the end of the 20th century— which are separated by an inner fiber layer (IFL) and differ in have become controversial in the last decade. Recent studies terms of and complement of neural progenitor suggest that a zone called the outer subventricular subtypes. The oSVZ compartment becomes very prominent in zone (oSVZ) induces gyri to form directly through neuro- primates, including humans, and contains detached RGCs (11), genesis. Here, we provide evidence in macaque monkey dem- recently renamed as outer radial glia (oRG) or basal radial glia onstrating that oSVZ neurogenesis is actually complete before (bRG) (5). Knowledge about the number, types, and sequences gyrification begins and that a major function of the oSVZ is to of neurons and glia generated from cortical progenitor cells is produce the glial cells associated with the rapid expansion and essential for understanding cortical development and evolution folding of the cortical surface. as well as deciphering the mechanisms of neurodevelopmental diseases, including those that affect gyrification, e.g., lissence- Author contributions: B.G.R. designed research; B.G.R., A.D., Y.M.M., and N.M. performed phaly and (12, 13). research; J.I.A. contributed new reagents/analytic tools; B.G.R., A.D., and Y.M.M. analyzed Recently it has been postulated that the remarkable enlarge- data; B.G.R. and P.R. wrote the paper; and P.R. supervised the project. ment of the oSVZ and its addition of neurons to the superficial Reviewers: C.K., Oregon Health & Science University; and Z.M., University of Oxford. layers (III and II) is critical for the 1,000-fold expansion of the The authors declare no conflict of interest. cortical surface during evolution and the main cause of cerebral Published under the PNAS license. gyrification in carnivores, nonhuman primates, and humans (3, 1To whom correspondence should be addressed. Email: [email protected]. 14–16). However, this idea has been challenged (17–19). Actu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ally, there is compelling evidence which confirmed classic studies 1073/pnas.1822169116/-/DCSupplemental. by Retzius, Cajal, and others stating that detached fetal glial cells Published online March 20, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1822169116 PNAS | April 2, 2019 | vol. 116 | no. 14 | 7089–7094 Downloaded by guest on September 25, 2021 Fig. 1. (A) Developmental series of macaque co- ronal brain sections at the indicated fetal ages dem- onstrating the development of the oSVZ. (A)Nissl stain. (B–D) Pax6 immunostaining shows the position of the oSVZ, as well as the commencement of EGFR expression by E70 in the VZ, progressing to the oSVZ, with numerous EGFR+ cells invading the white matter and subplate by E92–E125. (D) By E125, the oSVZ is in decline. (E–H) Tritiated thymidine injections with 1-h survival demonstrate the progression of proliferative activity in the developing cortical wall. (G and H)Note that at E90, tritiated thymidine-positive cells are common in the subplate (SP) and cortical plate (CP), and by E120, tritiated thymidine positive cells are distributed almost evenly across the cortical wall, in- dicating that proliferative activity is not confined to the classical germinal zones. 2/3, cortical layers 2 and 3; 5/6, cortical layers 5 and 6; CTX, cortex; IFL, inner fiber layer (40); INS, insular cortex; iSVZ, inner SVZ; IZ, intermediate zone; LV, lateral ventricle; oSVZ, outer SVZ; STR, ; VZ, ventricular zone; WM, white matter. B–D are composite images. (Scale bars: A and E,1mm;B–D,30μm; F–H,50μm.).

phase of the cell cycle. We found that the distribution of [3H]dT- neurogenesis had come to a close in dorsal parietal cortex before positive cells generally matched the position of cortical progeni- E102 (2, 25, 26). Therefore, continued presence of progenitor tors in the VZ, iSVZ, and oSVZ zones between E70 and markers such as Pax6, EGFR, and Olig2 beyond E92 in the + E92, but [3H]dT cells were also present in the intermedi- parietal cortical wall indicates gliogenesis (8, 26). ate zone (IZ), subplate (SP), and cortical plate (CP) at E69, To investigate the output of the oSVZ, we stained macaque + E90, and E120 (Fig. 1 A and E–H). We observed fewer [3H]dT tissue of different embryonic ages with various neuronal and glial cells in the periventricular iSVZ, as well as the oSVZ at E120. progenitor markers. Together with [3H]dT labeling (Fig. 1), be- + At E125, Pax6 cells were present mainly at the cortico-striatal tween E69 and E92, our analysis indicates that both neurons for boundary region, and only rarely in the nascent ependymal the superficial cortical layers, as well as some glial cells, are + zone underlying the corpus callosum (Fig. 1D). At E145, Pax6 generated simultaneously (Fig. 2). These late-generated neurons cells in the remnant germinal zones were very rare and ex- contribute to the complexity and thickness of the superficial pression was almost undetectable. cortical layers in primates, but do not add additional radial Neurogenesis is the primary output of proliferative activity in columns, which extend across all layers. During this period, the dorsal parietal VZ/SVZ at E75 (25), as shown by the postmitotic neurons move to the CP and settle in cortical col- + colabeling of the majority of cortical BrdU cells with NeuN in umns following the radial unit model (2, 21). However, we found specimens injected at E75 and killed at P91 (SI Appendix, Fig. S1 that the oSVZ also begins to produce glial cells, which migrate to + − + A and B). However, both NeuN and NeuN BrdU cortical the CP but primarily to the future white matter. We found that cells were found (SI Appendix, Fig. S1C). In monkeys injected one of the earliest astroglial precursor markers, EGFR (27–30), + with [3H]dT at E102 and killed postnatally on P65, we found costained numerous Olig2 cells exiting the germinal zones as + labeled cells of exclusively glial morphology, indicating that well as many Pax6 RGCs in the VZ and many progenitors of the

7090 | www.pnas.org/cgi/doi/10.1073/pnas.1822169116 Rash et al. Downloaded by guest on September 25, 2021 Fig. 2. The majority of EGFR+ cells exiting the ger- minal zones are Olig2+ glial precursors. Coronal sections of fetal macaque dorsal parietal cortex at E70 (A–D and F), E125 (E), E92 (G), or E145 (H), stained by immunohistochemistry for the indicated + genes. (A–D) Colabeling of migratory EGFR cells with Olig2. B–D are higher magnification images of the boxed regions in A.(E’’’’’) Most cells occupying the white matter at E125 are developing oligoden- drocytes or astrocytes. precursors are labeled by Olig2, overlapping substantially with EGFR expression (E’’’’; arrowheads). Olig2 labels some oligodendrocyte precursors that were EGFR negative (green arrowhead in E’’’’). EGFR labels some putative oligodendrocytes that have appar- ently lost Olig2 expression (arrows in E’’’’). No over- lap of EGFR or Olig2 expression with GFAP was found + (arrow in E’’’’’; H). (F)ManyEGFR cells entering the future white matter and subplate zones coexpress the OPC marker, PDGFRα.(G)GFAP+ radial fibers were still abundant at E92. (H) However, by E145, numerous + GFAP astrocytes became apparent. (I) Representative electron micrograph from E149 white matter un- derlying . While the majority of axons are nonmyelinated, some show initial steps of myeli- nation (I; red asterisks). A and F are composite images. (Scale bars: A,50μm; B–D and F,30μm; E, G,andH, NEUROSCIENCE 20 μm; I,1μm.).

+ + SVZ at E70, E92, and E125 (Figs. 1–3), but EGFR did not P91. Thus, Olig2 /EGFR putative OPCs are likely grossly di- colocalize with the neuronal markers, Tbr1 or βIII-tubulin (SI minished or are no longer being produced by P7 and have dif- + Appendix,Fig.S2). Furthermore, at E70, many migrating EGFR ferentiated into more mature states with accumulation of cells coexpressed the oligodendrocyte precursor cell (OPC) in their cytoplasmic processes enveloping the axons. marker, PDGFRα (Fig. 2F). Together, these data strongly indicate To determine whether oRGCs give rise to glial cells, we that many cells maintaining EGFR expression after their exit from coimmunostained sections for the oRG marker, Hopx (31), and the VZ/SVZ have glial fates, many of which are likely OPCs. At EGFR at E70, E125, and E145. At E70, Hopx labeled numerous, E70 and through E145, Olig2/EGFR double-positive cells were found streaming out of the dorsal parietal oSVZ radially into the IZ, SP, and CP, and many of the displayed migratory morphology, with orientation parallel to developing large axonal tracts such as lateral longitudinal bundle and the corpus callosum (Figs. 1C and + 2D). Migratory EGFR cells positioned outside the germinal zones, roughly bipolar and displaying a characteristic leading + + process, were Olig2 (Fig. 2E), whereas more mature EGFR − cells with more elaborated processes at E125 were typically Olig2 (Fig. 2E). This suggests that, as putative OPCs cease migrating and mature, they lose Olig2 expression but retain EGFR for some time. EGFR was detected in extremely rare cells at P7, and not at all at P91 (SI Appendix,Fig.S1), indicating that EGFR expression in cells emanating from the germinal zones is a characteristic feature of developing, but not mature, oligodendrocytes. + GFAP radial fibers of RGCs were visible throughout the + cortical wall at E92, but GFAP astrocytes were difficult to distinguish among the many RGC fibers at this age (Fig. 2G). In + contrast, at E125 and E145, GFAP astrocytes were numerous in the SP, white matter, and oSVZ (Fig. 2H). In our material, Fig. 3. Differentiation of EGFR+ putative oligodendrocytes from Pax6+ cells. + EGFR glial precursors outside the germinal zones did not Coronal sections of macaque dorsal parietal cerebral wall were stained by colocalize with the robust GFAP expression in putative astro- immunohistochemistry for Pax6 (green) and EGFR (red); DAPI is blue. (A, D, + cytes, indicating that they are likely to be developing oligoden- and G) By E70, many EGFR cells in the VZ express high levels of Pax6 and drocytes (Fig. 2 E and H). At E149, using electron microscopy, some also in the oSVZ, but few reside in the SP. (B, E,andH) By E92, EGFR we detected the formation of myelin among initially myelin-free expression is found in more cells in the oSVZ and many costain for Pax6, while many are now visible in the SP. (C, F, and I) The VZ is mostly dissipated immature oligodendrocytes in the white matter underlying cin- + by E125, with a large reduction in the number of Pax6 cells in the oSVZ; gulate cortex (Fig. 2I), representing the earliest detection of + + EGFR cells begin to differentiate into putative early oligodendrocytes in the myelination in the developing monkey. Although Olig2 OPCs oSVZ, IZ, and SP, predominantly with nonmigratory morphology, with were observed invading the CP by E70 and were abundant at elaborate cellular processes, and primarily located in nascent white matter. E125 and E145, Olig2 expression was not detected at P7 and (E) Ependymal zone. (Scale bar: 50 μm.)

Rashetal. PNAS | April 2, 2019 | vol. 116 | no. 14 | 7091 Downloaded by guest on September 25, 2021 Fig. 4. oRG generate putative oligodendrocytes in macaque dorsal parietal cortex. Coronal sections of developing macaque cerebral wall at the indicated ages, stained via immunohistochemistry for the in- dicated genes. Hopx marks the VZ/SVZ as well as oRG within the oSVZ, and large numbers of these cells were double labeled for EGFR. (A and B) At E70, most EGFR+ cells entering the white matter/SP were Ki67+. Some EGFR+ cells in the oSVZ were Hopx+ (C), − + while others were Hopx (D). By E125, many Hopx + cells in the oSVZ (E) were EGFR and displayed dif- ferentiating morphology characteristic of glia (F). + After exit from the germinal zones, EGFR putative oligodendrocyte precursors were either negative for Hopx or expressed low levels. (E) At E125, the lateral tip of the lateral ventricle showed the largest con- + centration of Hopx cells. A and E are composite images. (Scale bars: A and E,100μm; B, D, and F, 20 μm.)

+ radial process-bearing, Ki67 progenitors in the oSVZ, although oligodendrocytes were dispersed in massive numbers, populating + + not all Hopx oRGCs were Ki67 (Fig. 4 A, C, and D). We found the entirety of the IZ, which is transforming into large volumes + + that some Hopx oRG were EGFR in the oSVZ (Fig. 4C), of subcortical white matter (SI Appendix,Fig.S3). Immature − + + although most were EGFR (Fig. 4 A, C, and D). By E125, many EGFR /Olig2 cells within the white matter and SP were usually + + − + Hopx oRGCs had lost their basal radial fiber, were EGFR /Ki67 , Ki67 at E70, and many also at E125, indicating that they continue and had acquired a more elaborated, nonmigratory morphology to proliferate outside the original germinal zones, likely corre- characteristic of differentiating glia (Fig. 4F), indicating that sponding to many [3H]dT-labeled cells present in the cortical wall + + + HOPX /EGFR cells in the oSVZ may directly differentiate into (Fig. 1). Thus, these cells, together with GFAP astrocytes, + oligodendrocytes without dividing further. Some EGFR cells appeared to occupy a large portion of the cerebral white matter, + were also Ki67 in the large white matter tracts, subplate, and the which is in primates much greater in volume than the cortex and iSVZ and oSVZ at E70 and E125, indicating continued pro- becomes populated by glial cells that are more numerous than + duction of oligodendrocytic fates by early EGFR OPCs (Fig. 4 A, cortical neurons by E125–E145 (Fig. 5 and SI Appendix,Fig.S3). + + − B,andD). Since many HOPX /EGFR cells were Ki67 ,thedata appear to support both direct astroglial differentiation from Discussion oRGCs as well as indirect differentiation via proliferating OPCs. Our findings demonstrate that the large oSVZ of the dorsal + − By E145, a few Hopx /Ki67 cells were found in the peri- parietal cerebrum of macaque monkey becomes primarily glio- ventricular remnant iSVZ, enriched at the corticoseptal bound- genic after E92—before the onset of gyrification—and is a major ary region, which displayed long radial fibers oriented latero- source of the astrocytes and oligodendrocytes that will out- ventrally toward the temporal lobe. This is consistent with con- number cerebral neurons by about threefold in macaque and tinued, but spatially localized and possibly cortical area-specific, human (32, 33). Although oRGCs have previously been charac- gliogenesis of generally nonproliferative oRGCs. Thus, pro- terized as almost exclusively neurogenic (7), our data in macaque duction of oligodendrocytes, while initially occurring relatively demonstrate a gradual transition of this zone from neurogenesis to evenly across the tangential dimension at E70–E92, appears to oligodendrogenesis between E70 and E92, a period when upper be more robust in lateral cortical regions than in dorsal/medial layer II and III neurons are being generated (25, 26). This period of regions in macaque at late fetal stages around E125–E145, overlapping neurogenesis and gliogenesis underscores the initial co- during the period of initial gyrification. At E145, putative existence of heterogeneous groups of neural progenitor cells—with

7092 | www.pnas.org/cgi/doi/10.1073/pnas.1822169116 Rash et al. Downloaded by guest on September 25, 2021 + − EGFR RGCs may be able to give rise to EGFR superficial + neurons as well as to EGFR glia. However, since our [3H]dT data show that after about E92 there is no addition of neurons + (25), our present data indicate that EGFR progenitors in the dorsal parietal VZ, iSVZ, and oSVZ at that fetal age produce exclusively glial cells. The finding that oRG can generate glial cells is not unexpected, as RGCs have previously been shown to transform into astrocytes, and oRG are quite similar to RGCs of + the VZ (11, 21). We found that GFAP differentiation began in the dorsal parietal monkey cortex between E92 and E125, although at this time we are not able to determine whether cortical astrocytes predominantly originate from VZ or oSVZ progenitors, as this will require future fate mapping studies. The demise of the oSVZ as a discernible layer by E145, principally due to heavy inundation by transgressing axons, is a logical conclusion to the late cell fates produced by oRG. Namely, the final glial fates leave oRG progeny contained within their mi- gratory target tissue (white matter). Thus, our data fill a gap in our understanding of the development of VZ and oSVZ pro- genitors and more precisely demonstrate the time-course and developmental role of the -oligodendrocyte-astrocyte fate progression in cortical progenitor dynamics. We found that some of these putative OPCs are proliferative even after leaving the germinal zones, likely continuing to gen- erate additional glia. Indeed, areal differences in production of oligodendrocytes, combined with their targeted migration to specific cortical white matter areas with high densities of pro-

jection axons entering or exiting the cortical plate (36), could NEUROSCIENCE feature prominently in the mechanical mechanisms directing certain gyral and sulcal formations in primates, and warrants further study (37). For example, development of the longitudinal axonal bundle of the cingulum is associated with specific for- mation of the cingulate gyrus. Likewise, massive thalamic input to the somatosensory cortex contrasts with nearby motor cortex axons traveling the opposite direction to subcortical targets, and association areas do not send or receive axons to/from sub- cortical regions. The result is a spatio-temporally patterned growth of individual, area-specific axon tracts, which are asso- ciated with accumulation of oligodendrocytes. Surgical manipu- lations including enucleation of the eyes creates changes in white + + Fig. 5. EGFR and GFAP glial cells populate the cerebral white matter by matter ingrowth to the visual cortex, leading to massive changes E125–145. (A–C) The early cortical layer 6 and SP are marked by Tbr1 expres- in the magnitude and pattern of gyrification (2). sion. (D) Tbr1 expression dissipated by about E145. Early gliogenesis at E70–E92 While a certain amount of neuronal production and surface area is characterized by OPCs invading the white matter and subplate with imma- undoubtedly must be achieved as a prerequisite for gyrification, the ture migratory morphology (A and B), whereas large numbers of more dif- insufficiency of oRG neurogenesis in the oSVZ to produce cortical ferentiated EGFR+ (C–E)andGFAP+ (E) glia are visible at E125–E145. The + + convolutions in the common marmoset monkey indicates that other developing cortex contained a few EGFR and GFAP glia, but staining was μ factors are at work (18, 38). Similarly, the appearance of an oSVZ generally very faint. All locants are composite images. (Scale bar: 100 m.) in a large-brained rodent, the agouti, again does not generally correlate with gyral development in that species, since a robust some RGCs neurogenic and others gliogenic (21). Accordingly, oSVZ is present across the tangential cortical dimension, but only we found that the timing of onset of EGFR expression in RGCs one is visible at the dorsal extremity of the cortex (38), and correlates with initial gliogenesis, supporting the view that MAPK- therefore any potential role of oSVZ neurogenesis in cortical folding is debatable in multiple species. related growth factor signaling, initially provided by other pathways Are the production of glia by the oSVZ and associated differences such as fibroblast growth factor (FGF) signaling (34), continues in in white matter development important for gyrification? It appears cortical RGCs via EGF signaling to promote their proliferation that gyrification requires a certain number of axons filling the during the gliogenic phase (27). + + developing white matter, attracting large numbers of glial cells The emergence of EGFR /Olig2 cells by E70 indicates that that are produced by the oSVZ. Thus, while the simple presence increasing numbers of RGCs and oSVZ precursors, principally of a large oSVZ is not sufficient to induce gyrification, the con- oRGCs, evolve to produce only glial fates after E92. In agree- tribution of oSVZ gliogenesis to gyral development may be com- ment with our findings, high EGFR expression has been repor- mensurate with the level of white matter axonal production, ted to be an early oligodendrocyte fate determinant in cortical amplifying its volume. RGCs of the mouse (27), and increasing numbers of both VZ + In conclusion, the expansion of the cerebral cortex and its in- RGCs and oRG have been found to be Olig2 at the terminal timately linked process of gyrification are known to involve many stage of neurogenesis in monkey and human (27, 35). mechanical factors, including neuronal growth, elaboration of It is likely that the antibody to EGFR can label both neuronal dendrites, and general amplification of cortical neuropil (4, 39), and glial progenitors in the germinal zones. Indeed, we cannot and our data do not support a decisive role for neuronal pro- exclude the possibility that between E70 and E90, some of the duction in the oSVZ in the folding of the macaque cerebrum.

Rashetal. PNAS | April 2, 2019 | vol. 116 | no. 14 | 7093 Downloaded by guest on September 25, 2021 Instead, given the almost completely lissencephalic form of the Coherent Chameleon titanium sapphire 2-photon laser. [3H]dT cases human cerebrum at 25 gestational weeks, as well as that of the were part of the MacBrainResource, and no additional animals were macaque monkey at the time of cessation of neurogenesis in pa- killed for those data. BrdU injections were given at 50 mg/kg. Stereo- 3 + rietal cortex by about E92, our data indicate that robust oSVZ logical counts of [ H]dT cells utilized a StereoInvestigator system. gliogenesis is needed to support the rapid and patterned growth of Electron microscopy utilized standard protocols including embedding in cortical connections in the cerebral white matter, which in ma- Epon-Araldite mixture, with thin sections prepared on a Reichert ul- caque represents a prominent factor in determining the magnitude tramicrotome and imaged with a JEM 1010 electron microscope. Full methods information, including all antibodies and dilutions, is available and arrangement of cortical convolutions. online in SI Appendix. Materials and Methods ACKNOWLEDGMENTS. We thank Mariamma Pappy for laboratory help including All animal work was performed in accordance with Yale Institutional the restoration of [3H]dT-labeled macaque brain specimens, Albert Ayoub for col- Animal Care and Use Committee guidelines. Timed-pregnant fetal and lecting fetal monkey brain specimens, YARC for macaque breedings, and Yale postnatal monkey were obtained in-house at the Yale Animal veterinary clinical services for surgery services related to fetal monkey C-sections. Resource Center (YARC). Brains were immerse fixed in formaldehyde and We thank the MacBrainResource (https://medicine.yale.edu/neuroscience/macbrain/) processed into cryosections according to standard methods. Fluores- for access to archived macaque tissue specimens. We thank the Kavli In- cence immunohistochemistry utilized a standard citrate-based antigen stitute for Neuroscience at Yale and National Institutes of Health (NIH retrieval step, with imaging on a Zeiss LSM 510 confocal microscope with Grants R01 MH113257, R01 DA02399, and R01 EY002593) for funding.

1. Rakic P (1972) Mode of cell migration to the superficial layers of fetal monkey neo- 21. Levitt P, Cooper ML, Rakic P (1981) Coexistence of neuronal and glial precursor cells in cortex. J Comp Neurol 145:61–83. the cerebral ventricular zone of the fetal monkey: An ultrastructural immunoperox- 2. Rakic P (1988) Specification of cerebral cortical areas. Science 241:170–176. idase analysis. J Neurosci 1:27–39. 3. Kriegstein A, Noctor S, Martínez-Cerdeño V (2006) Patterns of neural stem and pro- 22. Reillo I, Borrell V (2012) Germinal zones in the developing cerebral cortex of ferret: genitor cell division may underlie evolutionary cortical expansion. Nat Rev Neurosci 7: Ontogeny, cell cycle kinetics, and diversity of progenitors. Cereb Cortex 22: 883–890. 2039–2054. 4. Rakic P (2009) Evolution of the : A perspective from developmental biology. 23. Zilles K, Palomero-Gallagher N, Amunts K (2013) Development of cortical folding Nat Rev Neurosci 10:724–735. during evolution and ontogeny. Trends Neurosci 36:275–284. 5. Dehay C, Kennedy H (2007) Cell-cycle control and cortical development. Nat Rev 24. Reillo I, de Juan Romero C, García-Cabezas MA, Borrell V (2011) A role for in- Neurosci 8:438–450. termediate radial glia in the tangential expansion of the mammalian cerebral cortex. 6. Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons Cereb Cortex 21:1674–1694. derived from radial glial cells establish radial units in neocortex. Nature 409:714–720. 25. Rakic P (1974) Neurons in rhesus monkey visual cortex: Systematic relation between – 7. Hansen DV, Lui JH, Parker PR, Kriegstein AR (2010) Neurogenic radial glia in the outer time of origin and eventual disposition. Science 183:425 427. subventricular zone of human neocortex. Nature 464:554–561. 26. Rakic P (1995) A small step for the cell, a giant leap for mankind: A hypothesis of – 8. Martínez-Cerdeño V, et al. (2012) Comparative analysis of the subventricular zone in neocortical expansion during evolution. Trends Neurosci 18:383 388. rat, ferret and macaque: Evidence for an outer subventricular zone in rodents. PLoS 27. Sun Y, Goderie SK, Temple S (2005) Asymmetric distribution of EGFR during – One 7:e30178. mitosis generates diverse CNS progenitor cells. Neuron 45:873 886. 9. Molnár Z, et al. (2014) Evolution and development of the mammalian cerebral cortex. 28. Aguirre A, Dupree JL, Mangin JM, Gallo V (2007) A functional role for EGFR signaling in myelination and remyelination. Nat Neurosci 10:990–1002. Brain Behav Evol 83:126–139. 29. Aguirre A, Gallo V (2007) Reduced EGFR signaling in progenitor cells of the adult 10. Cheung AF, et al. (2010) The subventricular zone is the developmental milestone of a subventricular zone attenuates oligodendrogenesis after demyelination. Neuron Glia 6-layered neocortex: Comparisons in metatherian and eutherian mammals. Cereb Biol 3:209–220. Cortex 20:1071–1081. 30. Sugiarto S, et al. (2011) Asymmetry-defective oligodendrocyte progenitors are glioma 11. Schmechel DE, Rakic P (1979) A golgi study of radial glial cells in developing monkey precursors. Cancer Cell 20:328–340. telencephalon: Morphogenesis and transformation into astrocytes. Anat Embryol 31. Pollen AA, et al. (2015) Molecular identity of human outer radial glia during cortical (Berl) 156:115–152. development. Cell 163:55–67. 12. Gleeson JG, et al. (1998) , a brain-specific gene mutated in human X- 32. Azevedo FA, et al. (2009) Equal numbers of neuronal and nonneuronal cells make the linked and double cortex syndrome, encodes a putative signaling pro- an isometrically scaled-up primate brain. J Comp Neurol 513:532–541. – tein. Cell 92:63 72. 33. Gabi M, et al. (2010) Cellular scaling rules for the brains of an extended number of 13. Bae BI, et al. (2014) Evolutionarily dynamic alternative splicing of GPR56 regulates primate species. Brain Behav Evol 76:32–44. – regional cerebral . Science 343:764 768. 34. Rash BG, Lim HD, Breunig JJ, Vaccarino FM (2011) FGF signaling expands embryonic 14. Namba T, Huttner WB (2017) Neural progenitor cells and their role in the develop- cortical surface area by regulating Notch-dependent neurogenesis. J Neurosci 31: ment and evolutionary expansion of the neocortex. Wiley Interdiscip Rev Dev Biol 6: 15604–15617. e256. 35. Mo Z, Zecevic N (2009) Human fetal radial glia cells generate oligodendrocytes – 15. Borrell V (2018) How cells fold the cerebral cortex. J Neurosci 38:776 783. in vitro. Glia 57:490–498. 16. Stahl R, et al. (2013) Trnp1 regulates expansion and folding of the mammalian ce- 36. Price DJ, et al. (2006) The development of cortical connections. Eur J Neurosci 23: rebral cortex by control of radial glial fate. Cell 153:535–549. 910–920. 17. Hevner RF, Haydar TF (2012) The (not necessarily) convoluted role of basal radial glia 37. Van Essen DC (1997) A tension-based theory of morphogenesis and compact wiring in in cortical neurogenesis. Cereb Cortex 22:465–468. the . Nature 385:313–318. 18. Kelava I, et al. (2012) Abundant occurrence of basal radial glia in the subventricular 38. García-Moreno F, Vasistha NA, Trevia N, Bourne JA, Molnár Z (2012) Compartmen- zone of embryonic neocortex of a lissencephalic primate, the common marmoset talization of cerebral cortical germinal zones in a lissencephalic primate and gyr- Callithrix jacchus. Cereb Cortex 22:469–481. encephalic rodent. Cereb Cortex 22:482–492. 19. Kroenke CD, Bayly PV (2018) How forces fold the cerebral cortex. J Neurosci 38: 39. Welker W (1990) Why does cerebral cortex fissure and fold? Cerebral Cortex, eds 767–775. Jones EG, Peters A (Springer, Boston), Vol 8B, pp 3–136. 20. Bystron I, Blakemore C, Rakic P (2008) Development of the human cerebral cortex: 40. Molnár Z, Clowry G (2012) Cerebral cortical development in rodents and primates. Boulder committee revisited. Nat Rev Neurosci 9:110–122. Prog Brain Res 195:45–70.

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