Gliogenesis in the outer subventricular zone promotes enlargement and gyrification of the primate cerebrum Brian G. Rasha, Alvaro Duquea, Yury M. Morozova, Jon I. Arellanoa, Nicola Micalia, and Pasko Rakica,b,1 aDepartment of Neuroscience, 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- prenatal development, the main sources of astrocytes and oli- dinarily voluminous subcortical white matter. 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 neurons 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 neurogenesis 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 gyrus (23, oligodendrocytes, which in primates 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, cerebral cortex | brain development | corticogenesis | E120, E125, E145, and E149, and postnatal day (P) 7, P65, and brain convolutions | glia 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 ventricular zone (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 gene expression and complement of neural progenitor suggest that a stem cell 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 polymicrogyria (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, striatum; 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).