© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev170100. doi:10.1242/dev.170100

HUMAN DEVELOPMENT RESEARCH ARTICLE Characterization of the ventricular-subventricular niche during human development Amanda M. Coletti1, Deepinder Singh1, Saurabh Kumar1, Tasnuva Nuhat Shafin1, Patrick J. Briody1, Benjamin F. Babbitt1, Derek Pan1, Emily S. Norton1, Eliot C. Brown1, Kristopher T. Kahle2, Marc R. Del Bigio3 and Joanne C. Conover1,*

ABSTRACT (Bruni, 1998; Del Bigio, 1995, 2010; Roales-Buján et al., 2012). In development proceeds via a sequentially transforming mouse, formation of the epithelial ependymal cells displaces stem cell population in the ventricular- (V-SVZ). An remaining radial /stem cell somata to the subventricular zone essential, but understudied, contributor to V-SVZ stem cell niche health (SVZ). These remaining stem cells, referred to as ventricular- is the multi-ciliated ependymal epithelium, which replaces stem cells at subventricular zone (V-SVZ) stem cells, are arrayed in clusters and the ventricular surface during development. However, reorganization maintain only a thin apical process at the ventricle surface (Alvarez- of the V-SVZ stem cell niche and its relationship to ependymogenesis Buylla et al., 1998, 2001; Conover et al., 2000; Doetsch et al., 1999; has not been characterized in the human brain. Based on Kriegstein and Alvarez-Buylla, 2009; Merkle et al., 2004). Stem comprehensive comparative spatiotemporal analyses of cell apical processes surrounded by ependymal cells are referred to ‘ ’ cytoarchitectural changes along the mouse and human ventricle as pinwheels (Mirzadeh et al., 2008) and represent regenerative surface, we uncovered a distinctive stem cell retention pattern in units. Whether human V-SVZ stem cells are organized and humans as ependymal cells populate the surface of the ventricle in an maintained in similar units along the ventricle surface has not occipital-to-frontal wave. During perinatal development, ventricle- been reported. contacting stem cells are reduced. By 7 months few stem cells are After birth in humans, proliferative cells and have detected, paralleling the decline in neurogenesis. In adolescence and been observed along the lateral wall of the lateral ventricle, in the adulthood, stem cells and neurogenesis are not observed along the site of what was formerly the lateral . Perinatal lateral wall. Volume, surface area and curvature of the V-SVZ stem cells appear to be restricted in their neurogenic all significantly change during fetal development but stabilize after 1 potential and migration routes, which include three specific year, corresponding with the wave of ependymogenesis and stem cell pathways within the anterior forebrain: (1) to the in reduction. These findings reveal normal human V-SVZ development, which they distribute as within the cortical layers (arc highlighting the consequences of disease pathologies such as pathway); (2) along the medial migratory stream (MMS) to the congenital . medial prefrontal cortex; (3) along the (RMS) to the (Paredes et al., 2016a; Quiñones- KEY WORDS: Stem cell niche, Human brain development, Hinojosa et al., 2006; Sanai et al., 2011, 2004). Neurogenesis and Ependymogenesis, Ventricular-subventricular zone frontal lobe migration is robust for the first several months after birth and then declines dramatically, so that by two years of age there is INTRODUCTION little, or no, observable neurogenesis or migration (Bergmann et al., During early brain development in humans, the lining of the 2012; Paredes et al., 2016b; Quiñones-Hinojosa et al., 2006; Sanai neural tube and subsequently the (CSF)-filled et al., 2011; Wang et al., 2011, 2014). Postnatal neurogenesis in the house a pseudostratified layer of proliferative human forebrain deviates significantly from what is found in mice cells that, in the forebrain, contributes to the robust expansion of and even non-human primates (Kriegstein et al., 2006; LaMonica the . New are initially generated by et al., 2012; Lui et al., 2011). Many mammals continue to generate neuroepithelial cells, and then by descendant radial glia and outer new neurons via the V-SVZ stem cell niche throughout their radial glia via their progeny, intermediate progenitor cells (Hansen lifetime, with the newly generated neurons migrating exclusively to et al., 2010; LaMonica et al., 2012; Lui et al., 2011; Malik et al., the olfactory bulb via the RMS to function in olfaction (Alunni and 2013). Radial glia also generate a monolayer of ependymal cells that Bally-Cuif, 2016; Conover and Shook, 2011; Lledo et al., 2008; lines the ventricles (Jacquet et al., 2009; Mirzadeh et al., 2008; Peretto et al., 1999). Although the exact function of postnatal Spassky et al., 2005) and provides barrier and transport functions inhibitory neurons in the human frontal cortex is unclear, it has been between the interstitial fluid of the brain and the CSF proposed that they contribute to neurocognitive maturation and plasticity that is required in infancy (Arshad et al., 2016; Paredes et al., 2016a; Sanai et al., 2011). Disease or injury that disrupts 1Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT proliferation and differentiation of V-SVZ stem cells and migration 06269, USA. 2Department of Neurosurgery, Pediatrics, and Cellular & Molecular of their progeny may contribute to sensorimotor and neurocognitive Physiology, Yale School of Medicine, New Haven, CT 06510, USA. 3Department of Pathology, University of Manitoba, Winnipeg, R3E 3P5, Canada. deficits that are frequently seen in cerebral palsy, autism and fetal- onset hydrocephalus (Arshad et al., 2016; Paredes et al., 2016a; *Author for correspondence ( [email protected]) Sanai et al., 2011). S.K., 0000-0001-8655-9187; J.C.C., 0000-0003-0375-0141 Although the lateral ventricle neuroepithelium drives neurogenesis, overall brain development subsequently influences

Received 17 July 2018; Accepted 15 September 2018 the contour of the ventricular system and therefore the V-SVZ stem DEVELOPMENT

1 HUMAN DEVELOPMENT Development (2018) 145, dev170100. doi:10.1242/dev.170100 cell niche. As the ventricles are filled with fluid and lined by a the medial and lateral walls of the lateral ventricle were examined pseudostratified neuroepithelium in early fetal/embryonic using immunohistochemistry to distinguish radial glia [γ-tubulin+ development, the shape of the ventricle may initially be basal body of single cilium, GLAST+ (also known as SLC1A3), compliant. However, late in the second trimester in humans and FOXJ1−], radial glia that are transitioning to immature ependymal around embryonic day (E)13-14 in mouse, V-SVZ stem cells (radial cells (two to five γ-tubulin+ basal bodies of cilia, FOXJ1+), mature glia) generate a monolayer of ependymal cells that line the ventricle ependymal cells (multi-cilia γ-tubulin+ clusters, FOXJ1+) and surface in an occipital-to-frontal gradient (Bruni, 1998; Bruni et al., neural stem cells (single cilium γ-tubulin+ basal body, GFAP+) (see 1985; Del Bigio, 1995; Jacquet et al., 2009; Kyrousi et al., 2015; Fig. S1A) (Jacquet et al., 2009; Mirzadeh et al., 2010b, 2008). Mirzadeh et al., 2008; Paez-Gonzalez et al., 2011; Spassky et al., In Fig. 1, renderings of representative microscope images along 2005). Ependymal cells are multi-ciliated and tightly adherent. They the lateral wall detail cell organization at the ventricle surface provide several crucial functions (Johanson et al., 2011; Mirzadeh (Fig. 1, second column, Fig. S1B). Cell type ratios (Fig. 1, third et al., 2008; Paez-Gonzalez et al., 2011; Spassky et al., 2005). column), the average percentages of each cell type at three locations Motile cilia at their apical surface contribute to laminar flow at the along the lateral wall for each developmental time point, were ventricle surface, and ependymal cells facilitate both barrier and determined based on counts of a 13,567.59 µm2 area for each rostral, transport functions between the interstitial fluid of the brain middle and caudal sample (n=3 animals). Before E13, radial glia parenchyma and the CSF of the ventricular system (Bruni, 1998; cover the surface of the entire ventricular system surface (data not Bruni et al., 1985; Del Bigio, 1995, 2010; Spassky et al., 2005). At shown) (Kriegstein and Alvarez-Buylla, 2009). At E13 and E16 the ventricle surface, ependymal cells are generally cuboidal in (Fig. 1A,B), immature ependymal cells, which make up ∼35% of shape and tightly linked by adherens and tight junction protein total cells at the surface of the ventricle, were found primarily in the complexes (Bruni, 1998; Bruni et al., 1985; Del Bigio, 1995; caudal-most aspects of the lateral ventricle lateral wall. Immature Mirzadeh et al., 2008; Spassky et al., 2005). Stem cells that retain a ependymal cells in the middle and rostral regions comprised only ventricle-contacting apical process also have apical adherens and ∼11% and ∼7%, respectively, of the total cell number. By P1 tight junctions with neighboring ependymal cells and other stem (Fig. 1C), mature ependymal cells, which are characterized by a cells (Jacquet et al., 2009; Mirzadeh et al., 2008; Paez-Gonzalez large tightly clustered array of multiple cilia, cover most of the et al., 2011), supporting barrier and structural functions along the caudal wall (60%) and stem cells that are organized in the core of lateral wall. pinwheel units made up the remainder (Fig. S1B). Immature and Here, we sought to investigate changes to the V-SVZ stem cell mature ependymal cells make up 34.2% of the middle lateral wall niche over the course of human brain development and to determine (22.1% immature ependymal cells and 8.5% mature ependymal the association between ependymogenesis, stem cell number and cells), and only immature ependymal cells (20.4%) and radial glia stem cell niche organization at the ventricle surface. Based on a (79.6%) line the rostral-most wall. comprehensive spatiotemporal analysis of cytoarchitectural changes As the caudal-to-rostral wave of newly differentiated ependymal along the ventricle surface, we found that ependymal cells were cells begins to cover the ventricle surface, clusters of radial glia/ added to the ventricle lining of the frontal horn in a posterior-to- neural stem cells (V-SVZ stem cells) were found to retain only a anterior wave beginning at ∼21 gestational weeks (gw). As more small apical process at the ventricle surface, whereas stem cell ependymal cells covered the ventricle, surface stem cell numbers somatas were displaced below the newly generated ependymal cell were reduced and remaining stem cells were relegated to the monolayer, as previously described (Mirzadeh et al., 2008). By P7 , with only an apical process contacting the (Fig. 1D), only mature ependymal cells and clusters of stem cell ventricle surface. Reduction of stem cell number corresponded to apical processes, classic ‘pinwheel’ units (Mirzadeh et al., 2008), decreased neurogenesis within the SVZ. Stem cell reduction make up the caudal (58.4% mature ependymal cells, 41.6% stem continued into postnatal development and no ventricle-contacting cell processes) and middle (41.3% mature ependymal cells, 58.7% stem cells were observed in adolescent and adult lateral ventricle stem cell processes) aspect of the lateral wall. In the rostral-most wall samples. Stability of the lateral ventricle volume, surface area aspect of the lateral wall, radial glia (40.3%) and immature and curvature (concavity/convexity) occurred after 1 year and ependymal cells (5.6%) were still detected. By P30 (Fig. 1E, corresponded temporally to the period of complete coverage of the Fig. S1B), all regions of the lateral wall were covered with organized ventricle surface by mature ependymal cells. Together, our findings pinwheel units. Cell counts at P30 indicate that the majority of cells link the timing of ependymogenesis and displacement of stem cells at the ventricle surface are mature ependymal cells (∼60%), with along the lateral ventricle wall with stabilization of the ventricle wall stem cells making up ∼40% of the total cell count. However, as stem surface conformation. cell somatas are displaced to the SVZ, the ventricle-contacting apical process takes up only ∼10% of the ventricle surface area RESULTS compared with ependymal cells (see also Spassky et al., 2005). Mouse ependymogenesis proceeds caudal to rostral and Ependymogenesis along the medial wall also proceeds as a stem cells persist caudal-to-rostral wave (Fig. S1C). At E13, the medial wall is Assessment of brain development in the mouse provides a model to covered by radial glia, with immature ependymal cells present only compare and contrast with human brain development. We used in the caudal-most region (not shown). By E16, differentiation of serial coronal sections to generate three-dimensional (3D) immature ependymal cells progresses rostrally along the medial wall reconstructions of both total brain and lateral ventricle volumes at and, after birth (P1), the caudal and middle regions were covered five discrete stages of embryonic to postnatal brain development: predominantly by mature multi-ciliated ependymal cells, whereas E13, E16, postnatal day (P)1, P7 and P30 (Fig. 1, left column). In the rostral region was still lined primarily with radial glia. At P30, addition, whole-mount preparations of the lateral and medial wall of the medial wall was covered by mature multi-ciliated ependymal the lateral ventricles were prepared for each of the five stages of cells: stem cells were not observed along the medial wall. Others development (Doetsch et al., 1997; Mirzadeh et al., 2008; Shook report small clusters of stem cells only along the rostral-most aspect et al., 2012). Changes in cell coverage along the entire extent of both of the medial wall in postnatal mice (Mirzadeh et al., 2008), but, as DEVELOPMENT

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Fig. 1. Ependymogenesis proceeds caudal to rostral along lateral ventricle wall during mouse brain development. (A-E) 3D reconstructions at E13 (A), E16 (B), P1 (C), P7 (D) and P30 (E) show lateral ventricles and whole-brain contours (left column). Schematics of representative microscope images (second column, lateral wall) highlight ependymal cell development along caudal, middle and rostral regions of the lateral ventricle wall. Wave of caudal-to-rostral ependymal cell formation is illustrated on 2D projections of the ventricle wall. Scale bars: 20 µm in top whole-view schematic; 1 mm in E13 and E16 2D projections; 500 µm in P1, P7 and P30 2D projections. Pie charts (third column) indicate average percentage of radial glia, immature ependymal cells, V-SVZ stem cells and mature ependymal cells along caudal, middle and rostral regions of the lateral ventricle wall (n=3) at each developmental stage. A, anterior; R, right; S, superior. we have found, these are subsequently lost in early adulthood adolescent and adult tissues were obtained from the National (Fig. S1C). Institutes of Health (NIH) NeuroBioBank (University of Here, we highlight the conversion of neuroepithelia to an Maryland, MD, USA) and the University of Manitoba, ependymal monolayer that is interspersed with clusters of stem Pathology Department (Winnipeg, Canada) (Table 1). Samples cells along only the lateral, not the medial, wall. These data support were without brain structural abnormalities or acquired lesions and earlier findings that describe the caudal-to-rostral wave of were considered normal. Wholemounts of tissue from anterior ependymogenesis along the lateral ventricle lateral wall (Mirzadeh (frontal horn over the head), middle (frontal horn et al., 2008; Spradling et al., 2001). body near the interventricular foramen) and posterior (ventricular trigone/body) regions were prepared for immunohistochemistry. Human ependymogenesis proceeds posterior to anterior Cell composition, based on three samples within each anterior, along the lateral ventricle surface middle and posterior region, was based on immunocytochemical To characterize ependymogenesis and associated changes to the criteria for radial glia, immature ependymal cells, V-SVZ stem V-SVZ stem cell niche along the frontal horn of the lateral cells and mature ependymal cells (see above and Fig. S2A). ventricles in humans, we prepared whole-mount sections of the Representative microscope images for each developmental time lateral wall from fetal periventricular tissue at 21 gw, 28 gw and point and region were rendered into schematic diagrams to show 34 gw (Fig. 2A). Wholemounts were also prepared at perinatal, cell organization from each region, as indicated on two- adolescent and adult time points: 10 day (neonatal), 6 months, 7 dimensional (2D) reconstructions of the ventricle wall 2 months, 8 years and 39 years (Fig. 2B). Human fetal, perinatal, (Fig. 2A,B). Cell types were quantified within a 13,567.59 µm DEVELOPMENT

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Fig. 2. Human ependymogenesis proceeds posterior to anterior along lateral ventricle wall during fetal-to-postnatal development and exhibits characteristic pinwheel organization. (A,B) Human fetal (A) and postnatal/adult (B) ependymal cell development was examined at 21 gw, 28 gw, 34 gw, 10 day, 6 months, 7 months, 8 years and 39 years (n=1). Representative schematics of microscope images of a 3391.90 µm2 (fetal) or 13,567.59 µm2 (postnatal) area of the lateral ventricle frontal horn are indicated by red squares on 2D ventricular surface projections (black). Pie charts below schematics indicate percentage of radial glia, immature ependymal cells, V-SVZ stem cells and mature ependymal cells along anterior, middle and posterior regions. Scale bars: 1 cm in A (2D ventricle wall renderings); 5 cm in B (2D ventricle wall renderings); 20 µm in A,B (tissue sections). representative area for each anterior, middle and posterior sample and 42.9% in anterior and middle regions, respectively) with and cell type ratios were indicated as pie charts. scattered large clusters of radial glial cells (blue cells) and few Microscopic images and cell counts revealed a posterior-to- remaining immature ependymal cells (pale yellow cells). Following anterior developmental wave of ependymogenesis along the lateral birth, at 10 days all ependymal cells along the surface were multi- wall of the frontal horn (Fig. 2A,B; Fig. S2B), similar to what was ciliated mature ependymal cells, based on large clusters of basal found in mouse (Fig. 1) (Spassky et al., 2005). At 21 gw, radial glia bodies along their apical surface (73.9% and 74.2%, anterior and dominated in all regions; however, immature ependymal cells made middle regions, respectively). V-SVZ stem cells made up ∼26% of up 22.5% of the total cell number in the posterior region of the body the total cell number but retained only thin apical processes at the of the frontal horn. Fewer immature ependymal cells were found in ventricle surface, which constituted ∼15% of the surface area of an the middle (15.2%) and anterior (12.2%) regions. By 28 gw, the ependymal cell. In striking similarity to mouse development number of immature ependymal cells increased in all regions, with (Mirzadeh et al., 2008; Spassky et al., 2005), the apical processes the largest percentage in the posterior region (44.2% versus 34.9% of stem cells were retained in clusters demonstrating classic and 23.9% immature ependymal cells in the middle and anterior ‘pinwheel’ organization along the lateral wall of the human regions, respectively). At 34 gw there was significant differentiation frontal horn (Fig. S2B). During postnatal development, mature of immature ependymal cells into mature ependymal cells (36.6% ependymal cell numbers increased as stem cell numbers declined (at DEVELOPMENT

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Table 1. Patient information, MRI scan sources and sample sex distribution for human fetal and postnatal samples Human tissue 21 gw 28 gw 34 gw 10 days 5 months 6 months 7 months 8 years 39 years

Source University of University of University of University of University of NIH NeuroBioBank NIH NeuroBioBank NIH NeuroBioBank University of Manitoba Manitoba Manitoba Manitoba Manitoba Manitoba Anatomical Right Left cerebrum Right frontal Left prefrontal Right frontal Right cerebrum Right cerebrum Anteromedial Left position cerebrum lobe cerebrum lateral lateral cortex hemisphere ventricular ventricular wall wall sections sections Sex N/A N/A N/A N/A Male Female Male Male Male Cause of death Intrapartem Intrapartem Polycystic Co-sleeping Co-sleeping Unexpected Positional Blunt force trauma Liver failure death death kidneys infant death asphyxia

MRI source Prenatal 0-1 years 1-2 years 2-3 years 3-4 years 10-11 years Total

CMIND 0 12 13 15 26 24 90 NIH 0 12 4 0 0 0 16 LONI 7 0 0 0 0 0 7 Yale School 12 0 0 0 0 0 12 of Medicine Total 19 24 17 15 26 24 125

Sex Prenatal 0-1 years 1-2 years 2-3 years 3-4 years 10-11 years Total

Male 3 13 7 5 10 16 54 Female 7 9 10 10 16 8 60 Not available 920000 11 (N/A) Total 19 24 17 15 26 24 125

6 and 7 months). A reduction in stem cell numbers was observed to (Table 1), we determined total brain and lateral ventricle volumes follow the same posterior-to-anterior pattern, with loss occurring in and lateral ventricle surface area at discrete developmental time the posterior regions first. By 8 years, only mature ependymal cells points (Fig. 3A,B). Semi-automated segmentation using ITK- were found lining the lateral ventricle surface; no stem cell processes SNAP (Shook et al., 2014; Snippert et al., 2010; Todd et al., 2017; were observed (Fig. 2B). This absence of stem cell apical processes Yushkevich et al., 2006) followed by 3D reconstruction of the along the lateral wall was also found in adult tissue, indicating that lateral ventricles and whole brain using 3D Slicer (Acabchuk et al., only mature ependymal cells line the lateral ventricle wall in 2015; Shook et al., 2014) revealed that the average total brain adulthood (Fig. 2B). volume increases rapidly from 15 gw to 1 year. Regression analysis The medial walls of the frontal horn of the lateral ventricles also predicts that from 19 gw to birth, the brain grows at a nearly linear show a posterior-to-anterior wave of ependymogenesis, but V-SVZ rate of 1.2×106 mm3/year (23 cm3/week) and from birth to 1 year at stem cells are not retained and, as a result, ependymal cells 5.5×105 mm3/year (11 cm3/week) (see also Kinoshita et al., 2001). completely cover the medial wall (Fig. S2C). New immature From 1 year to ∼3.5 years, brain volume growth slows to a nearly ependymal cells were found scattered throughout the medial wall at linear average rate of 1.3×105 mm3/year (2.5 cm3/week). These 21 gw, and by 34 gw the middle regions of the wall were mostly represent a 1100% increase in brain volume from 18 gw to birth, covered by mature ependymal cells. By 10 days postpartum, an 110% increase from birth to 1 year, and 16% from 1 year to 2 years intact was found in all regions of the medial wall. We did (Fig. 3A,B), similar to the findings of others (Knickmeyer et al., not detect any remaining V-SVZ stem cells in any postnatal medial 2008). In contrast, lateral ventricle volume exhibits an increase, wall samples. slight decrease and then a plateau across early postnatal The above studies of fetal, perinatal, adolescent and adult human development. Lateral ventricle volume increase occurs from lateral wall tissue demonstrate a posterior-to-anterior transition from 15 gw to ∼1.25 years. This occurs at an average rate of radial glia coverage (before 21 gw, data not shown) to a complete 8900 mm3/year (171 mm3/week) from 15 gw to birth, and monolayer of ependymal cells (adolescent and adult tissue). As 3800 mm3/year (73 mm3/week) from birth to 1.25 years. Ventricle ependymal cells are generated (from 21 gw until perinatal time volume decrease occurs at a nearly linear rate of 800 mm3/year periods), radial glia and V-SVZ stem cell numbers (based on an (15 mm3/week) from 1.25 years to 3.5 years and is significant by apical process at the ventricle surface) decline. Pinwheel units ANOVA of a simple linear regression model across this data subset initially contain many stem cell processes, but process number (P=0.032). A plateau appears to occur slightly after 3 years, declines during postnatal development. Concurrent with ependymal stabilizing at ∼8400 mm3 and there is no significant evidence to cell maturation through postnatal development is an increase in indicate that the average ventricle volume from 3-4 years differs apical surface area of ∼fivefold (Fig. S2D). from that of 10-11 years, based on an outlier-resistant two-sample Mann–Whitney nonparametric test (P=0.66). In summary, ventricle Human lateral ventricle volume and surface area changes volume increases 350% from 15 gw to birth, 86% from birth to correspond to curvature changes 1.25 years and decreases 18% from 1.25 years to 3.5 years. Certain We next examined whether the spatiotemporal posterior-to-anterior scans from the Cincinnati MR Imaging (C-MIND) database (www. progression of ependymogenesis and stem cell depletion along the cmind.research.cchmc.org) showed distinctly larger total ventricle lateral wall of the lateral ventricle were related to developmental volumes (see Fig. S3 for details). Some scans showed asymmetrical changes in lateral ventricle volume and surface area. Using ventricles, with one ventricle being larger than the other (Fig. 3B, neurologically normal prenatal T2-weighted structural magnetic marked by ‘×’ on ventricle volume and surface area graphs) or resonance imaging (MRI) scans (7T) and postnatal T1-weighted symmetric and slightly enlarged ventricles (Fig. 3B, marked by ‘O’ structural MRI scans (3T) from neurologically normal individuals on ventricle volume and surface area graphs). DEVELOPMENT

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Fig. 3. Developmental changes in human lateral ventricle volume and surface area reflected in curvature changes. (A) 3D representations show lateral view of lateral ventricle (red) and whole-brain (blue) contours across fetal (15 gw, 18 gw, 36 gw) and postnatal (3 months, 9 months, 2-3 years, 10-11 years) development. (B) Scatterplots indicate changes in brain volume, ventricle volume, ventricle to whole brain volume ratio and ventricle surface area across fetal and postnatal human development. Brain volume was modeled as a fourth degree least-squares polynomial regression curve, ventricle volume and ventricle surface area were both modeled as third degree least-squares polynomial regression curves. Symmetrically (O) and asymmetrically (×) enlarged scans are noted. Birth is depicted by a red line. (C) Curvature heat maps of lateral ventricle surface across fetal (15 gw, 18 gw, 34 gw) and postnatal (3 months, 9 months, 2-3 years, 10-11 years) development. Heat map depicts the range of curvature from concave (red) to convex (blue). A, anterior; L, left; S, superior.

Plotting the ratio of ventricle to whole brain volume ratio shows In summary, ventricle surface area increases 100% from 15 gw to that the lateral ventricles occupy ∼10% of the brain volume at birth, 62% from birth to 1.25 years and decreases 5.4% from 15 gw; this decreases rapidly to ∼2% at birth and stabilizes to ∼1% 1.25 years to 3.5 years. There is no correlation between sex and within a year (Fig. 3B). After 1 year, the brain continues to grow at a developmental age in our sample (n=114 individuals of known sex); slow rate (2.5 cm3/week), which is responsible for any systemic thus, sex was not included in our predictive models (see Materials decrease in the ventricle to whole brain volume ratio over time and Methods, Statistical analysis section). (Fig. 3B). There appears to be no significant correlation between sex Analysis of surface area changes, unlike total volume changes, and developmental age in our sample (n=114 individuals of known suggests changes in topography of the ventricle walls that may not sex); therefore, sex was not included in our predictive models (see correspond to minor volume changes (Del Bigio, 2014). Surface area Materials and Methods, Statistical analysis section). increases emphasize a growing demand for ependymal cell coverage. Lateral ventricle surface area exhibits a similar growth and We found that ventricle surface area increases during fetal plateau pattern. All expansion occurs from 15 gw to ∼1.25 years. development, plateauing at ∼1.5 years of age (Fig. 3B). Subjects The average growth from 15 gw to birth is 4700 mm2/year (90 mm2/ with enlarged ventricle volumes also show similar trends. Increasing week) and from birth to 1.25 years is 2200 mm2/year (42 mm2/ surface area results in the heightened need for ependymal cell week). Ventricle surface area decrease from 1.25 years to 3.5 years coverage and may be responsible for the steady decline in stem cell occurs at an average rate of 170 mm2/year (3.3 mm2/week). The number at the ventricle surface in postnatal development. ventricle surface area appears to plateau after 3 years, with an To characterize the changing topography associated with surface average surface area of 6800 mm2. Again, there is no significant area increases along the ventricle wall, we examined how curvature evidence to indicate that the average ventricle surface area from of the lateral ventricles changed during fetal and postnatal 3-4 years differs from that of 10-11 years, based on an outlier- development. From 15 gw to perinatal developmental stages, the resistant two-sample Mann–Whitney nonparametric test (P=0.22). curvature of the anterior horn (superior horn) of the lateral ventricles DEVELOPMENT

6 HUMAN DEVELOPMENT Development (2018) 145, dev170100. doi:10.1242/dev.170100 changes significantly from convex to concave (Fig. 3C, green to region. FOXJ1+ ependymal cells were found at the ventricle surface yellow) and this concavity appears to be maintained throughout in both anterior and posterior regions. By 34 gw, SVZ postnatal development. In contrast, the temporal and posterior horn (GFAP+) possessed shorter radial processes, were still (occipital horn), although continuing to grow, remain concave from numerous and a mature ependyma demarcated the ventricle lining. fetal through postnatal development. Based on these results, the Proliferative cells were mainly found within the anterior SVZ/oSVZ posterior-to-anterior maturation of the ependymal cell monolayer at region, extending a significant distance from the ventricle lining. By the ventricle surface also corresponds to the timing of the changing 10 days postpartum, Ki67+ cells were reduced in number, and the curvature of the anterior horn from convex to concave (Fig. 3C). majority of DCX+ neuroblasts were restricted to a narrow pathway immediately subjacent and tangential to the ventricle surface. Both The V-SVZ stem cell niche and neurogenesis significantly 6-month and 7-month samples revealed few neuroblasts or decline during human brain development proliferative cells, the absence of long radial processes and a To characterize the organization of the V-SVZ stem cell niche continuous monolayer of ependymal cells making up the ventricle (frontal horn lateral wall of the lateral ventricle) across lining. We detected no Ki67+ or DCX+ cells in the 8-year-old development, we examined tissue at fetal, perinatal, postnatal, sample, with the exception of a few Ki67+ cells scattered along adolescent and adult time points. Coronal sections from anterior, blood vessels. GFAP+ astrocytes consolidated as a ribbon parallel middle and posterior regions were prepared (see Table 1) and to, but separate from, an acellular zone that lay next to the stained for GFAP (V-SVZ SCs), DCX (neuroblasts), Ki67 (cycling ependymal monolayer, as previously reported (Sanai et al., 2011). cells) and FOXJ1 [immature and mature ependymal cells, some late stage radial glia (Jacquet et al., 2009)] (Fig. 4A,B). At 21 gw, when DISCUSSION the lateral ganglionic eminence (LGE) comprises the lateral wall of Organ-specific stem cell niches can have long-lasting effects on the the lateral ventricles, immature ependymal cells (FOXJ1+) were development and function of the organ system. A vital stem cell detected primarily in the posterior region, similar to what was found niche within the developing brain is the V-SVZ. In addition to in Fig. 2A. Radial glia (GFAP+) exhibited long radial processes and supporting neurogenesis, stem cells in the V-SVZ also generate DCX+ neuroblasts were numerous throughout the SVZ and outer cells that support their own niche. An essential, but understudied, SVZ (oSVZ) regions (Hansen et al., 2010). Ki67+ proliferative cells contributor to the V-SVZ stem cell niche is the ependyma – a were more numerous in the anterior versus posterior and/or middle monolayer of multi-ciliated ependymal cells that are generated regions. At 28 gw, the long radial processes of radial glia and during mid- to late-gestation in humans. Ependymal cells anchor widespread distribution of neuroblasts were still observed, and there niche-associated stem cells and provide barrier and transport are more proliferative cells observed in the anterior versus posterior functions between the brain’s interstitial fluid and the CSF.

Fig. 4. Human V-SVZ stem cell niche and neurogenesis diminish during fetal-to-postnatal development. (A,B) Human fetal (A) and postnatal (B) LGE and V-SVZ development across 21 gw, 28 gw, 34 gw, 10 day, 7 months and 8 years along the frontal horn lateral wall of the lateral ventricle (n=1). Anterior and posterior coronal sections of the anterior horn were examined for 21 gw and 28 gw, whereas anterior and middle regions were examined for 34 gw, 10 day, 7 months and 8 years. Composite image of all four channels is positioned above separated channels for each region. Dotted white line indicates the lateral ventricle wall edge. Glial fibrillary acidic protein (GFAP, red), (DCX, blue), Ki67 (green), FOXJ1 (gray). Scale bars: 50 µm. DEVELOPMENT

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A mature ependymal cell lining is also required to support populations in different organ systems and in different species neurogenesis and migration along the lateral ventricle (Hormoz, 2013). Hormoz (2013) proposed the division of stem cell wall in mice (Paez-Gonzalez et al., 2011). population dynamics into two types, both supporting stem cell self- We have mapped the progression of ependymogenesis across the preservation and progenitor generation. (1) Stem cell population frontal horn of the lateral ventricles, along with changes in ventricle asymmetry occurs with heterogeneous proliferation rates, with some volume, surface area and curvature during human brain development stem cells dividing symmetrically to give progenitor cells (Fig. 5). Radial glia that initially line the ventricles in early fetal [‘consuming’ stem cell division (Obernier et al., 2018)] and development were sequentially replaced by ependymal cells in an neighboring slow-dividing stem cells divide symmetrically to yield occipital-to-frontal wave across the ventricle surface. Significantly, two stem cells. (2) Asymmetric stem cell division occurs when stem only the lateral wall of the frontal horn retained radial glia/stem cells cells divide to yield a stem cell (self-renewal) and a . into infancy and these stem cells maintained only an apical process at Asymmetric cell division is the predominant form of division by the ventricle surface, whereas their somata were repositioned to the radial glia in embryonic development (Kriegstein and Alvarez- SVZ. Clusters of stem cell apical processes surrounded by ependymal Buylla, 2009), but it is a process that will not support the stem cell cells, arrayed as classic ‘pinwheel’ units similar to those previously pool over extended periods of time because of stem cell exhaustion described in mouse brain (Mirzadeh et al., 2008), were found in whole- (Hormoz, 2013; Obernier et al., 2018; Shahriyari and Komarova, mount preparations of human perinatal frontal horn lateral wall tissue. 2013). Population asymmetry would allow the slow-dividing stem The number of stem cells with ventricular contact steadily declined in a cells to proliferate and purge the population of fast-dividing, older posterior-to-anterior gradient during postnatal development and no cells. This mechanism has been observed in adult V-SVZ stem cell- apical processes of stem cells were observed in whole-mount derived neurogenesis in mouse (Obernier et al., 2018) and may also preparations of adolescent and adult lateral wall samples. explain V-SVZ stem cell retention into infancy in humans. It remains In contrast to the lateral wall, the medial wall of the frontal horn of to be determined whether generation of ependymal cells is the final the lateral ventricles, which also shows a similar posterior-to- division product (through symmetric division) of stem cells with anterior ependymogenesis progression, does not retain stem cells ventricle contact. A similar ‘disposable stem cell’ model was proposed with ventricle contact after birth. Instead, an uninterrupted wall of by Encinas et al. (2011) to explain age-related loss of mouse ependymal cells eventually covers the entire medial wall. hippocampal neural stem cells and the appearance of new astrocytes. Our findings related to ependymogenesis in humans during the The extent of ependymogenesis is likely driven by alterations to fetal-to-postnatal transition mimics what has been demonstrated the contours of the ventricle surface. In an attempt to correlate the previously in mice [this study and others (Jacquet et al., 2009; spatiotemporal development of the ependymal lining with changing Kyrousi et al., 2015; Mirzadeh et al., 2010b, 2008; Paez-Gonzalez conformation of the ventricle wall, we assessed lateral ventricle et al., 2011; Spassky et al., 2005)]. However, in contrast to human, volume and surface area changes over the course of fetal and ventricle-contacting stem cells persist along the lateral wall postnatal development. Using MRI data from our human sources, throughout adulthood in mouse and other mammals (Alunni and we plotted volumes and surface area from 15 gw to 11 years. Rapid Bally-Cuif, 2016; Lledo et al., 2008; Peretto et al., 1999; Conover increases in total brain volumes and increases in lateral ventricle and Shook, 2011) and although stem cell numbers decline over the volumes were found from 15 gw until ∼1 year. After 1 year, the course of aging, stem cells with ventricle contact are still found in total brain volume rate of increase slows but continues at a linear elderly mice (Capilla-Gonzalez et al., 2015; Conover and Shook, rate. In contrast, lateral ventricle volume exhibits an increase, slight 2011; Conover and Todd, 2017; Luo et al., 2006; Maslov et al., decrease, and then a plateau across early postnatal development; the 2004; Shook et al., 2012). ventricle volume at 3 years is not significantly different from that The life-long retention of V-SVZ stem cells and accompanying seen at 11 years. Others have reported similar trends for brain and neurogenesis found in adult mice and other mammals differs from lateral ventricle increases from the second trimester to birth the significant diminution of V-SVZ stem cells and neurogenesis (Kinoshita et al., 2001; Sakai et al., 2012) to 2 years of age that we, and others (Arshad et al., 2016; Hansen et al., 2010; Malik (Knickmeyer et al., 2008; Leigh, 2004). To extend earlier studies, et al., 2013; Paredes et al., 2016a,b; Sanai et al., 2011; Wang et al., we found that early in the second trimester (15 gw) the lateral 2011, 2014), have observed by the time of adolescence in humans. ventricles occupy ∼10% of the total brain volume, 2% of the total This discrepancy has recently garnered special attention and driven brain volume at birth and 1% of the total brain volume at 1 year. theoretical models to explain retention or loss of stem cell Lateral ventricle surface area showed similar gestational increases

Fig. 5. Summary of human ependymogenesis across fetal-postnatal development. Human ependymogenesis proceeds posterior to anterior along the lateral wall of the frontal horn of the lateral ventricle. At 21 gw, the majority of the lateral wall surface is covered by radial glial cells, as immature ependymal cells are forming in the posterior region. The wall is convex at this time. At 34 gw, a mixture of radial glial cells, immature ependymal cells and mature ependymal cells are found along the length of the wall and the anterior-most region is now concave. By 7 months, the lateral wall is composed of mature ependymal cells and stem cell clusters that are arranged in a pinwheel unit organization. Stem cells are replaced in a posterior-to-anterior manner. The concavity of the anterior horn is clearly evident. By 8 years, the lateral wall is comprised entirely of mature ependymal cells, with no pinwheels present. The conformation of the ventricle surface is stabilized. 3D representations of the lateral ventricle are shown (black), with a dotted red line marking the end of the frontal horn of the lateral ventricle, above the trigone region. Scale bars: 2.5 cm. DEVELOPMENT

8 HUMAN DEVELOPMENT Development (2018) 145, dev170100. doi:10.1242/dev.170100 from 15 gw until birth, showing a slight decrease after 1 year and surface. Disruption, hypoproliferation or hyperproliferation of then reaching a plateau by 2-3 years of age. Volume and surface area the ependymal layer is implicated in a variety of psychiatric, are general measurements that do not reveal topographical changes neurodegenerative and neurodevelopmental conditions and is at the ventricle surface [e.g. volume may increase through understudied. The above studies define normal development of the displacement (convexity) of the ventricle walls, whereas surface V-SVZ stem cell niche and associated ependymal lining along the area remains unchanged (Del Bigio, 2014)]. To evaluate lateral ventricles and provide a developmental platform from which conformational changes along the ventricle surface, we used to assess disruption of this vital stem cell niche in diseases that result curvature analysis to reveal patterns of concavity and convexity in infant or hydrocephalus. along the extra-ventricle surface. These patterns are not outwardly apparent from MRI scan segments; however, they do become more MATERIALS AND METHODS obvious from 3D reconstructions. From 15 gw to late gestation Animals (36 gw), the anterior horn exhibits significant conformational Male CD-1 mice (Mus musculus) (Charles River Laboratories, Wilmington, changes from convex to concave. Concavity of the anterior horn MA, USA) at E13, E16, P1, P7 and P30 (adult) were used. Housing, then stabilized through postnatal periods. The occipital horns grow handling, care and processing of the animals were carried out in accordance and elongate extensively in late gestation, but remain concave from with regulations approved by the Institutional Animal Care and Use 36 gw through postnatal development. In the lateral ventricle frontal Committee of the University of Connecticut. horn, we found that the eventual coverage of the lateral ventricle wall with mature ependymal cells corresponded to the period when Mouse brain tissue immunohistochemistry ventricle volume, surface area and curvature stabilized (after ∼1.5 All antibodies used in this study were used previously and validated by our years). The mirroring of conformational stabilization and group and others; the expression patterns were as expected and referenced. ependymogenesis suggests that deposition of ependymal cells For coronal sections, P7 and P30 mice were anesthetized with isoflurane, then transcardially perfused with 0.9% saline followed by 4% along the ventricle walls likely contributes to the lateral ventricle paraformaldehyde (PFA). The extracted were fixed overnight in 4% wall stability. PFA at 4°C. E13, E16, and P1 mice were anesthetized with isoflurane, heads Ependymogenesis in ventricle regions other than the lateral wall were removed and fixed overnight in 4% PFA at 4°C. After removing the corresponds to completion of neurogenesis. V-SVZ neurogenesis skin, embryonic and P1 brains were removed from the skull using a Leica along the frontal horn lateral wall in the postnatal human anterior MZ95 stereomicroscope. All brains were washed for 3×10 min in PBS forebrain populates the ‘arc’ pathway to the frontal cortex, and the before dissection and vibratome sectioning for 3D reconstructions. MMS and RMS pathways (Hansen et al., 2010; Paredes et al., Lateral ventricle wall wholemounts were prepared as described (Mirzadeh 2016a; Sanai et al., 2011, 2004). We documented changes to the V- et al., 2008). Following isofluorane anesthesia and saline perfusion, P7 and SVZ and oSVZ in coronal sections along the lateral ventricle wall P30 brains were extracted and whole-mount preparations were placed in that showed substantial proliferation and neurogenesis during PFA with 1% Triton X-100 overnight. E13, E16 and P1 mice were gestation, followed by decreased proliferation and neurogenesis anesthetized with isofluorane, heads were extracted and placed in 4% PFA with 1% Triton X-100 overnight and wholemounts were then prepared after birth, in full support of the findings of others (Bergmann et al., (Mirzadeh et al., 2010a). Wholemounts were immunostained with the 2012; Paredes et al., 2016b; Quiñones-Hinojosa et al., 2006; Sanai following primary antibodies: mouse monoclonal anti-β-catenin (1:250; BD et al., 2011; Wang et al., 2011, 2014). In addition, we observed a Biosciences, #610154), rabbit polyclonal anti-β-catenin (1:100; Cell reorganization of migratory neuroblasts to a narrow tangential Signaling Technology, #9562), rabbit polyclonal anti-γ-tubulin (1:500; pathway subjacent to the ependymal lining from birth until 7 months Sigma-Aldrich, #T5192), rabbit polyclonal anti-GLAST (1:200; Abcam, of age. By 8 years no proliferative cells or migratory neuroblasts #ab416), goat polyclonal anti-GFAP (1:250; Abcam, #ab53554), rat were detected, instead a periventricular acellular gap region and monoclonal anti-GFAP (1:250; Invitrogen, #13-0300) and mouse associated ribbon was detected, as previously described in monoclonal anti-FOXJ1 (1:250; Invitrogen, #14-9965-80). Alexa Fluor the adult human brain (Sanai et al., 2011, 2004). A recent study dye-conjugated polyclonal secondary antibodies (1:500, Invitrogen) were provided strong evidence that human hippocampal used: donkey anti-mouse 488 (#21202), donkey anti-mouse 546 (#A10036), donkey anti-rabbit (#A21206), donkey anti-goat-647(#A21447) and donkey neurogenesis declines rapidly during the first year of life and persists anti-rat 647 (#A18744). Blocking solutions contained 1% Triton X-100. at low levels into early adolescence, but does not continue into Validations of all commercial antibodies are available from manufacturer’s adulthood (Sorrells et al., 2018). This challenges previous reports datasheets. Lateral wall tissue wholemounts were coverslipped with Aqua- (Dennis et al., 2016; Eriksson et al., 1998; Knoth et al., 2010; Poly/Mount (Polyscience) and imaged on a Leica TCS SP8 confocal laser Spradling et al., 2001) and a new study (Boldrini et al., 2018) that scan microscope (Leica Microsystems). find daily production of new neurons in adulthood (∼700/dentate gyrus/day). Although our study and others provide evidence that Mouse lateral ventricle reconstruction and analysis robust, or even moderate, levels of neurogenesis along the lateral To generate 3D reconstructions of the mouse brain and ventricles, coronal ventricle surface do not continue into adulthood in the human brain, sections from E13 (42 µm), E16, P1, P7 and P30 (all 50 µm) brains were it remains unresolved, and highly controversial, whether this applies sectioned on a vibratome (VT-1000S, Leica). Mouse brain tissue sections to other forms of adult human neurogenesis. were stained with β-catenin overnight (rabbit polyclonal anti-β-catenin, 1:100; Cell Signaling Technology, #9562), secondary antibody for 1 h Concluding remarks (donkey anti-rabbit 546, 1:500; Invitrogen, #A10040), nuclear stain DAPI Full ependymal cell coverage of the ventricle walls supports both (300 mM; Molecular Probes, #D-1306) for 10 min and imaged on a Zeiss Axio Imager M2 microscope with ApoTome (Carl Zeiss MicroImaging) barrier and transport functions between the interstitial fluid and CSF with a Hamamatsu Photonics ORCA-R2 digital camera (C10600). and aids in maintaining brain . Diseases resulting in Alternating coronal sections were imaged, and the contours of the lateral ventriculomegaly, such as fetal-onset hydrocephalus, an abnormal ventricle walls and surface of the brain were traced to generate 3D enlargement of the lateral ventricles, places an extraordinary demand reconstructions, as described (Acabchuk et al., 2015). Volume and surface on the stem cell population to provide both crucial neurogenic area analysis were performed using StereoInvestigator and Neurolucida functions and adequate ependymal cell coverage at the ventricle Explorer software (MBF Bioscience). DEVELOPMENT

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Human brain tissue immunohistochemistry constant and erosion size were manually modified as necessary with each Postmortem human brain tissue (whole hemispheres and regional portions MRI scan. Manual edits using the edit mask function of Brain Suite were of the lateral ventricle wall) ranging from 21 gw to 39 years of age were made following automatic skull stripping as needed. obtained from the NIH NeuroBioBank (University of Maryland, MD, USA) Curvature was analyzed in Meshmixer software (www.meshmixer.com). and the University of Manitoba, Pathology Department (Winnipeg, 3D OBJ files created from lateral ventricle segmentations were imported into Canada). Tissues were acquired under protocol H2011-212, approved by Meshmixer. Mean curvature was calculated and generated on the 3D model the University of Manitoba Health Research Ethics Board. All tissue was as a heat map using the mesh query analysis function. Meshmixer archival and came de-identified. Hemispheres from 21 gw, 28 gw, 34 gw, automatically calculates the mean curvatures based on a system of 10 day and 39 year, and V-SVZ sections from 5 and 7 months were triangles (faces) and vertices on the 3D model, providing curvature values examined (Table 1). For lateral ventricle whole-mount preparations, several at each vertex of the model. sections from the anterior (frontal horn over the caudate nucleus head), middle (frontal horn near the interventricular foramen) and posterior Statistical analysis (frontal horn) lateral ventricle surfaces were dissected. Lateral ventricle wall Data are reported as mean±s.e.m. Statistical analysis was performed in wholemounts were coverslipped with AquaPoly/Mount and imaged on GraphPad Prism software (www.graphpad.com). One-way ANOVA with Leica TCS SP8 confocal laser scan microscope. Lateral ventricle Bonferroni’s multiple comparisons post-test was used. All statistical wholemounts were imaged using a 100×/1.4 HC PL APO oil immersion regression analysis was performed in SAS 9.1. The minimum level of objective lens, taken at scan zoom 1 (13,567.59 µm2 field area) or scan zoom significance for all tests was P<0.05. All regression models were fitted using 2 (3391.90 µm2 field area). The following antibodies were used: rabbit least-squares polynomial regression and analyzed with sequential-addition polyclonal anti-β-catenin (1:100; Cell Signaling Technology, #9562), rabbit (type-I sum of squares) partial F-tests of terms with increasing degree. All polyclonal anti-γ-tubulin (1:500; Sigma-Aldrich, #T5192); rabbit terms in our models, including the intercept term, are significant based on polyclonal anti-GLAST (1:200; Abcam, #ab416), goat polyclonal anti- type-III sum of squares, partial F-tests. The following regression models GFAP (1:250; Abcam, #ab53554), rat monoclonal anti-GFAP (1:250; were obtained, with ‘x’ denoting developmental age in years, brain and Invitrogen, #13-0300) and mouse monoclonal anti-FOXJ1 (1:250; ventricle volume in mm3, and ventricle surface area in mm2. Invitrogen, #14-9965-80). Alexa Fluor dye-conjugated polyclonal secondary antibodies (1:500, Invitrogen) were used: donkey anti-mouse Brain Volume : Y^ ¼ 484954 þ 927627x–497701x2 þ 133771x3 –13428x4 488 (#21202), donkey anti-rabbit 546 (#A10040), donkey anti-goat-647 Ventricle Volume : Y^ ¼ 5500:9 þ 7233:2x–3300:4x2 þ 420:45x3 (#A21447) and donkey anti-rat 647 (#A18744). Validations of all commercial antibodies are available from manufacturer’s datasheets. Ventricle Surface Area : Y^ ¼ 4428:9 þ 3901x 1620:5x2 þ 199:36x3 Representative areas (13,567.59 µm2) were imaged and cell types were identified based on basal body number (radial glia and V-SVZ stem cells, 1 basal body; immature ependymal cells, 2-5 basal bodies; mature ependymal Acknowledgements cells, an array of many basal bodies) and immunostaining criteria. We gratefully acknowledge the NIH NeuroBioBank and the University of Manitoba, Pathology Department, for providing human tissue. We also thank and gratefully Schematics were generated by outlining an identified cell and assigning a acknowledge the LONI database, the University of Southern California, C-MIND, ‘ ’ fill color. NIH NIMH Data Archive and Dr Dustin Scheinost (Yale School of Medicine) for Regions corresponding to the LGE in fetal tissue and the lateral wall of providing MRI data. This manuscript reflects the views of the authors and may the lateral ventricle in postnatal tissue were dissected and sectioned not reflect the opinions or views of the NIH or of the Submitters submitting original coronally at 100 mm thickness. Immunohistochemistry was performed as data to NDAR. described (Todd et al., 2017) using the following antibodies: goat polyclonal anti-GFAP (1:250; Abcam, #ab53554), mouse monoclonal anti-FOXJ1 Competing interests (1:250; Invitrogen, #14-9965-80), guinea pig anti-doublecortin (DCX) The authors declare no competing or financial interests. (1:1000, EMD Millipore, #AB2253) and rabbit anti-Ki67 (1:1000; Novocastra, #6013874). Alexa Fluor dye-conjugated polyclonal Author contributions secondary antibodies (1:500) were used: donkey anti-mouse 405 (Abcam, Conceptualization: D.S., S.K., T.N.S., M.R.D.B., J.C.C.; Methodology: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., D.P., E.S.N., E.C.B., J.C.C.; Software: D.S., S.K.; #ab175658), donkey anti-mouse 488 (Invitrogen, #21202), donkey anti- Validation: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., D.P., E.S.N., E.C.B., J.C.C.; rabbit 546 (Invitrogen, #A10040), donkey anti-goat-647 (Invitrogen, Formal analysis: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., D.P., E.S.N.; #A21447) and donkey anti-rat 647 (Invitrogen, #A18744). Validations of Investigation: A.M.C., D.S., S.K., T.N.S., P.J.B.; Resources: A.M.C., D.S., S.K., all commercial antibodies are available from manufacturer’s datasheets. K.T.K., M.R.D.B., J.C.C.; Data curation: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., Coronal sections were imaged on a Leica TCS SP8 confocal laser scan D.P., E.C.B.; Writing - original draft: A.M.C., D.S., S.K., K.T.K., M.R.D.B., J.C.C.; microscope using a 40×/1.3 HC PL APO oil immersion objective lens. Writing - review & editing: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., K.T.K., M.R.D.B., J.C.C.; Visualization: A.M.C., D.S., S.K., P.J.B., B.F.B., D.P., E.S.N., Human MRI lateral ventricle reconstruction and analysis E.C.B., J.C.C.; Supervision: K.T.K., M.R.D.B., J.C.C.; Project administration: J.C.C.; Funding acquisition: J.C.C. De-identified archival MRI scans ranging from 15 gw-10 years were used in this study. T1 or T2 structural scans were obtained from the C-MIND Funding database with a 3T MRI scanner, the NIH National Institute of Mental This research was funded by the National Institutes of Health (NS090092 and Health (NIMH) data archive (https://ndar.nih.gov/), Laboratory of Neuro NS098091 to J.C.C.; instrument grant S10ODO16435), the Hydrocephalus Imaging (LONI) database of the University of Southern California (www. Association (to J.C.C.), the University of Connecticut Institute for Brain and Cognitive loni.usc.edu, 7T MRI scanner) and Yale School of Medicine (3T MRI Sciences (to A.M.C., D.S., B.F.B., S.K. and J.C.C.). Dr Del Bigio holds the Canada scanner) (Table 1). Lateral ventricle and whole-brain semi-automated Research Chair in Developmental Neuropathology. Deposited in PMC for release segmentation and volume/surface area analyses were completed using ITK- after 12 months. SNAP and 3D Slicer software as previously described (Acabchuk et al., 2015; Todd et al., 2017). To determine accurate whole-brain volume and Data availability surface area measurements, skulls were digitally removed from MRI scans Data used in the preparation of this article were obtained from the following studies: using Brain Suite software (www.brainsuite.org). De-skulled masks of MRI (1) Brain Suite, LONI software (NIH-NINDS R01 NS074980, NIH-NIBIB R01 EB002010, NIH-NIBIB P41-EB015922). (2) The C-MIND Data Repository created scans were automatically generated using the skull stripping tool under the by the C-MIND study of Normal Brain Development (CMINDS data set version cortical surface extraction sequence function of Brain Suite. Brain surface 1.720). This is a multisite, longitudinal study of typically developing children from extractor mechanism was set to trim brain stem and and dilate the ages newborn through young adulthood conducted by Cincinnati Children’s Hospital final mask. Extractor settings that were kept constant include five automated Medical Center and UCLA and supported by the National Institute of Child Health iterations, three diffusion iterations and a diffusion constant of 25. Edge and Human Development (Contract HHSN275200900018C). A listing of the DEVELOPMENT

10 HUMAN DEVELOPMENT Development (2018) 145, dev170100. doi:10.1242/dev.170100 participating sites and a complete listing of the study investigators can be found at ependymal cells and a subset of astrocytes in the postnatal brain. Development https://research.cchmc.org/c-mind. (3) The NIH-supported National Database for 136, 4021-4031. Autism Research (NDAR). NDAR is a collaborative informatics system created by Johanson, C., Stopa, E., McMillan, P., Roth, D., Funk, J. and Krinke, G. (2011). the National Institutes of Health to provide a national resource to support and The distributional nexus of to cerebrospinal fluid, ependyma and accelerate research in autism. brain: toxicologic/pathologic phenomena, periventricular destabilization, and lesion spread. Toxicol. Pathol. 39, 186-212. Kinoshita, Y., Okudera, T., Tsuru, E. and Yokota, A. (2001). 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