HUMAN DEVELOPMENT RESEARCH ARTICLE

Characterization of the ventricular-subventricular stem cell nicheduring human brain developmentAmanda 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,*

ABSTRACTHuman brain development proceeds via a sequentially transformingstem cell population in the ventricular-subventricular zone (V-SVZ). Anessential, but understudied, contributor toV-SVZ stemcell niche healthis the multi-ciliated ependymal epithelium, which replaces stem cells atthe ventricular surface during development. However, reorganizationof the V-SVZ stem cell niche and its relationship to ependymogenesishas not been characterized in the human brain. Based oncomprehensive comparative spatiotemporal analyses ofcytoarchitectural changes along the mouse and human ventriclesurface, we uncovered a distinctive stem cell retention pattern inhumans as ependymal cells populate the surface of the ventricle in anoccipital-to-frontal wave. During perinatal development, ventricle-contacting stem cells are reduced. By 7 months few stem cells aredetected, paralleling the decline in neurogenesis. In adolescence andadulthood, stem cells and neurogenesis are not observed along thelateral wall. Volume, surface area and curvature of the lateral ventriclesall significantly change during fetal development but stabilize after 1year, corresponding with the wave of ependymogenesis and stem cellreduction. These findings reveal normal human V-SVZ development,highlighting the consequences of disease pathologies such ascongenital hydrocephalus.

KEY WORDS: Stem cell niche, Human brain development,Ependymogenesis, Ventricular-subventricular zone

INTRODUCTIONDuring early brain development in humans, the lining of theneural tube and subsequently the cerebrospinal fluid (CSF)-filledventricular system house a pseudostratified layer of proliferativecells that, in the forebrain, contributes to the robust expansion ofthe cerebral cortex. New neurons are initially generated byneuroepithelial cells, and then by descendant radial glia and outerradial glia via their progeny, intermediate progenitor cells (Hansenet al., 2010; LaMonica et al., 2012; Lui et al., 2011; Malik et al.,2013). Radial glia also generate a monolayer of ependymal cells thatlines the ventricles (Jacquet et al., 2009; Mirzadeh et al., 2008;Spassky et al., 2005) and provides barrier and transport functionsbetween the interstitial fluid of the brain parenchyma and the CSF

(Bruni, 1998; Del Bigio, 1995, 2010; Roales-Buján et al., 2012). Inmouse, formation of the epithelial ependymal cells displacesremaining radial glia/stem cell somata to the subventricular zone(SVZ). These remaining stem cells, referred to as ventricular-subventricular zone (V-SVZ) stem cells, are arrayed in clusters andmaintain only a thin apical process at the ventricle surface (Alvarez-Buylla et al., 1998, 2001; Conover et al., 2000; Doetsch et al., 1999;Kriegstein and Alvarez-Buylla, 2009; Merkle et al., 2004). Stemcell apical processes surrounded by ependymal cells are referred toas ‘pinwheels’ (Mirzadeh et al., 2008) and represent regenerativeunits. Whether human V-SVZ stem cells are organized andmaintained in similar units along the ventricle surface has notbeen reported.

After birth in humans, proliferative cells and neurogenesis havebeen observed along the lateral wall of the lateral ventricle, in thesite of what was formerly the lateral ganglionic eminence. PerinatalV-SVZ stem cells appear to be restricted in their neurogenicpotential and migration routes, which include three specificpathways within the anterior forebrain: (1) to the frontal lobe inwhich they distribute as interneurons within the cortical layers (arcpathway); (2) along the medial migratory stream (MMS) to themedial prefrontal cortex; (3) along the rostral migratory stream(RMS) to the olfactory bulb (Paredes et al., 2016a; Quiñones-Hinojosa et al., 2006; Sanai et al., 2011, 2004). Neurogenesis andfrontal lobe migration is robust for the first several months after birthand then declines dramatically, so that by two years of age there islittle, or no, observable neurogenesis or migration (Bergmann et al.,2012; Paredes et al., 2016b; Quiñones-Hinojosa et al., 2006; Sanaiet al., 2011; Wang et al., 2011, 2014). Postnatal neurogenesis in thehuman forebrain deviates significantly from what is found in miceand even non-human primates (Kriegstein et al., 2006; LaMonicaet al., 2012; Lui et al., 2011). Many mammals continue to generatenew neurons via the V-SVZ stem cell niche throughout theirlifetime, with the newly generated neurons migrating exclusively tothe olfactory bulb via the RMS to function in olfaction (Alunni andBally-Cuif, 2016; Conover and Shook, 2011; Lledo et al., 2008;Peretto et al., 1999). Although the exact function of postnatalinhibitory neurons in the human frontal cortex is unclear, it has beenproposed that they contribute to neurocognitive maturation andplasticity that is required in infancy (Arshad et al., 2016; Paredeset al., 2016a; Sanai et al., 2011). Disease or injury that disruptsproliferation and differentiation of V-SVZ stem cells and migrationof their progeny may contribute to sensorimotor and neurocognitivedeficits that are frequently seen in cerebral palsy, autism and fetal-onset hydrocephalus (Arshad et al., 2016; Paredes et al., 2016a;Sanai et al., 2011).

Although the lateral ventricle neuroepithelium drivesneurogenesis, overall brain development subsequently influencesthe contour of the ventricular system and therefore the V-SVZ stemReceived 17 July 2018; Accepted 15 September 2018

1Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT06269, USA. 2Department of Neurosurgery, Pediatrics, and Cellular & MolecularPhysiology, Yale School of Medicine, New Haven, CT 06510, USA. 3Department ofPathology, University of Manitoba, Winnipeg, R3E 3P5, Canada.

*Author for correspondence ( joanne.conover@uconn.edu)

S.K., 0000-0001-8655-9187; J.C.C., 0000-0003-0375-0141

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cell niche. As the ventricles are filled with fluid and lined by apseudostratified neuroepithelium in early fetal/embryonicdevelopment, the shape of the ventricle may initially becompliant. However, late in the second trimester in humans andaround embryonic day (E)13-14 in mouse, V-SVZ stem cells (radialglia) generate a monolayer of ependymal cells that line the ventriclesurface in an occipital-to-frontal gradient (Bruni, 1998; Bruni et al.,1985; Del Bigio, 1995; Jacquet et al., 2009; Kyrousi et al., 2015;Mirzadeh et al., 2008; Paez-Gonzalez et al., 2011; Spassky et al.,2005). Ependymal cells are multi-ciliated and tightly adherent. Theyprovide several crucial functions (Johanson et al., 2011; Mirzadehet al., 2008; Paez-Gonzalez et al., 2011; Spassky et al., 2005).Motile cilia at their apical surface contribute to laminar flow at theventricle surface, and ependymal cells facilitate both barrier andtransport functions between the interstitial fluid of the brainparenchyma and the CSF of the ventricular system (Bruni, 1998;Bruni et al., 1985; Del Bigio, 1995, 2010; Spassky et al., 2005). Atthe ventricle surface, ependymal cells are generally cuboidal inshape and tightly linked by adherens and tight junction proteincomplexes (Bruni, 1998; Bruni et al., 1985; Del Bigio, 1995;Mirzadeh et al., 2008; Spassky et al., 2005). Stem cells that retain aventricle-contacting apical process also have apical adherens andtight junctions with neighboring ependymal cells and other stemcells (Jacquet et al., 2009; Mirzadeh et al., 2008; Paez-Gonzalezet al., 2011), supporting barrier and structural functions along thelateral wall.Here, we sought to investigate changes to the V-SVZ stem cell

niche over the course of human brain development and to determinethe association between ependymogenesis, stem cell number andstem cell niche organization at the ventricle surface. Based on acomprehensive spatiotemporal analysis of cytoarchitectural changesalong the ventricle surface, we found that ependymal cells wereadded to the ventricle lining of the frontal horn in a posterior-to-anterior wave beginning at ∼21 gestational weeks (gw). As moreependymal cells covered the ventricle, surface stem cell numberswere reduced and remaining stem cells were relegated to thesubependymal zone, with only an apical process contacting theventricle surface. Reduction of stem cell number corresponded todecreased neurogenesis within the SVZ. Stem cell reductioncontinued into postnatal development and no ventricle-contactingstem cells were observed in adolescent and adult lateral ventriclewall samples. Stability of the lateral ventricle volume, surface areaand curvature (concavity/convexity) occurred after 1 year andcorresponded temporally to the period of complete coverage of theventricle surface by mature ependymal cells. Together, our findingslink the timing of ependymogenesis and displacement of stem cellsalong the lateral ventricle wall with stabilization of the ventricle wallsurface conformation.

RESULTSMouse ependymogenesis proceeds caudal to rostral andstem cells persistAssessment of brain development in the mouse provides a model tocompare and contrast with human brain development. We usedserial coronal sections to generate three-dimensional (3D)reconstructions of both total brain and lateral ventricle volumes atfive discrete stages of embryonic to postnatal brain development:E13, E16, postnatal day (P)1, P7 and P30 (Fig. 1, left column). Inaddition, whole-mount preparations of the lateral and medial wall ofthe lateral ventricles were prepared for each of the five stages ofdevelopment (Doetsch et al., 1997; Mirzadeh et al., 2008; Shooket al., 2012). Changes in cell coverage along the entire extent of both

the medial and lateral walls of the lateral ventricle were examinedusing immunohistochemistry to distinguish radial glia [γ-tubulin+

basal body of single cilium, GLAST+ (also known as SLC1A3),FOXJ1−], radial glia that are transitioning to immature ependymalcells (two to five γ-tubulin+ basal bodies of cilia, FOXJ1+), matureependymal cells (multi-cilia γ-tubulin+ clusters, FOXJ1+) andneural stem cells (single cilium γ-tubulin+ basal body, GFAP+) (seeFig. S1A) (Jacquet et al., 2009; Mirzadeh et al., 2010b, 2008).

In Fig. 1, renderings of representative microscope images alongthe lateral wall detail cell organization at the ventricle surface(Fig. 1, second column, Fig. S1B). Cell type ratios (Fig. 1, thirdcolumn), the average percentages of each cell type at three locationsalong the lateral wall for each developmental time point, weredetermined based on counts of a 13,567.59 µm2 area for each rostral,middle and caudal sample (n=3 animals). Before E13, radial gliacover the surface of the entire ventricular system surface (data notshown) (Kriegstein and Alvarez-Buylla, 2009). At E13 and E16(Fig. 1A,B), immature ependymal cells, which make up ∼35% oftotal cells at the surface of the ventricle, were found primarily in thecaudal-most aspects of the lateral ventricle lateral wall. Immatureependymal cells in the middle and rostral regions comprised only∼11% and ∼7%, respectively, of the total cell number. By P1(Fig. 1C), mature ependymal cells, which are characterized by alarge tightly clustered array of multiple cilia, cover most of thecaudal wall (60%) and stem cells that are organized in the core ofpinwheel units made up the remainder (Fig. S1B). Immature andmature ependymal cells make up 34.2% of the middle lateral wall(22.1% immature ependymal cells and 8.5% mature ependymalcells), and only immature ependymal cells (20.4%) and radial glia(79.6%) line the rostral-most wall.

As the caudal-to-rostral wave of newly differentiated ependymalcells begins to cover the ventricle surface, clusters of radial glia/neural stem cells (V-SVZ stem cells) were found to retain only asmall apical process at the ventricle surface, whereas stem cellsomatas were displaced below the newly generated ependymal cellmonolayer, as previously described (Mirzadeh et al., 2008). By P7(Fig. 1D), only mature ependymal cells and clusters of stem cellapical processes, classic ‘pinwheel’ units (Mirzadeh et al., 2008),make up the caudal (58.4% mature ependymal cells, 41.6% stemcell processes) and middle (41.3% mature ependymal cells, 58.7%stem cell processes) aspect of the lateral wall. In the rostral-mostaspect of the lateral wall, radial glia (40.3%) and immatureependymal cells (5.6%) were still detected. By P30 (Fig. 1E,Fig. S1B), all regions of the lateral wall were covered with organizedpinwheel units. Cell counts at P30 indicate that the majority of cellsat the ventricle surface are mature ependymal cells (∼60%), withstem cells making up∼40% of the total cell count. However, as stemcell somatas are displaced to the SVZ, the ventricle-contactingapical process takes up only ∼10% of the ventricle surface areacompared with ependymal cells (see also Spassky et al., 2005).

Ependymogenesis along the medial wall also proceeds as acaudal-to-rostral wave (Fig. S1C). At E13, the medial wall iscovered by radial glia, with immature ependymal cells present onlyin the caudal-most region (not shown). By E16, differentiation ofimmature ependymal cells progresses rostrally along the medial walland, after birth (P1), the caudal and middle regions were coveredpredominantly by mature multi-ciliated ependymal cells, whereasthe rostral region was still lined primarily with radial glia. At P30,the medial wall was covered by mature multi-ciliated ependymalcells: stem cells were not observed along the medial wall. Othersreport small clusters of stem cells only along the rostral-most aspectof the medial wall in postnatal mice (Mirzadeh et al., 2008), but, as

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we have found, these are subsequently lost in early adulthood(Fig. S1C).Here, we highlight the conversion of neuroepithelia to an

ependymal monolayer that is interspersed with clusters of stemcells along only the lateral, not the medial, wall. These data supportearlier findings that describe the caudal-to-rostral wave ofependymogenesis along the lateral ventricle lateral wall (Mirzadehet al., 2008; Spradling et al., 2001).

Human ependymogenesis proceeds posterior to anterioralong the lateral ventricle surfaceTo characterize ependymogenesis and associated changes to theV-SVZ stem cell niche along the frontal horn of the lateralventricles in humans, we prepared whole-mount sections of thelateral wall from fetal periventricular tissue at 21 gw, 28 gw and34 gw (Fig. 2A). Wholemounts were also prepared at perinatal,adolescent and adult time points: 10 day (neonatal), 6 months, 7months, 8 years and 39 years (Fig. 2B). Human fetal, perinatal,

adolescent and adult tissues were obtained from the NationalInstitutes of Health (NIH) NeuroBioBank (University ofMaryland, MD, USA) and the University of Manitoba,Pathology Department (Winnipeg, Canada) (Table 1). Sampleswere without brain structural abnormalities or acquired lesions andwere considered normal. Wholemounts of tissue from anterior(frontal horn over the caudate nucleus head), middle (frontal hornbody near the interventricular foramen) and posterior (ventriculartrigone/body) regions were prepared for immunohistochemistry.Cell composition, based on three samples within each anterior,middle and posterior region, was based on immunocytochemicalcriteria for radial glia, immature ependymal cells, V-SVZ stemcells and mature ependymal cells (see above and Fig. S2A).Representative microscope images for each developmental timepoint and region were rendered into schematic diagrams to showcell organization from each region, as indicated on two-dimensional (2D) reconstructions of the ventricle wall(Fig. 2A,B). Cell types were quantified within a 13,567.59 µm2

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 (secondcolumn, lateral wall) highlight ependymal cell development along caudal, middle and rostral regions of the lateral ventricle wall. Wave of caudal-to-rostralependymal 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 andmature 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.

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representative area for each anterior, middle and posterior sampleand cell type ratios were indicated as pie charts.Microscopic images and cell counts revealed a posterior-to-

anterior developmental wave of ependymogenesis along the lateralwall of the frontal horn (Fig. 2A,B; Fig. S2B), similar to what wasfound in mouse (Fig. 1) (Spassky et al., 2005). At 21 gw, radial gliadominated in all regions; however, immature ependymal cells madeup 22.5% of the total cell number in the posterior region of the bodyof the frontal horn. Fewer immature ependymal cells were found inthe middle (15.2%) and anterior (12.2%) regions. By 28 gw, thenumber of immature ependymal cells increased in all regions, withthe largest percentage in the posterior region (44.2% versus 34.9%and 23.9% immature ependymal cells in the middle and anteriorregions, respectively). At 34 gw there was significant differentiationof immature ependymal cells into mature ependymal cells (36.6%

and 42.9% in anterior and middle regions, respectively) withscattered large clusters of radial glial cells (blue cells) and fewremaining immature ependymal cells (pale yellow cells). Followingbirth, at 10 days all ependymal cells along the surface were multi-ciliated mature ependymal cells, based on large clusters of basalbodies along their apical surface (73.9% and 74.2%, anterior andmiddle regions, respectively). V-SVZ stem cells made up ∼26% ofthe total cell number but retained only thin apical processes at theventricle surface, which constituted ∼15% of the surface area of anependymal cell. In striking similarity to mouse development(Mirzadeh et al., 2008; Spassky et al., 2005), the apical processesof stem cells were retained in clusters demonstrating classic‘pinwheel’ organization along the lateral wall of the humanfrontal horn (Fig. S2B). During postnatal development, matureependymal cell numbers increased as stem cell numbers declined (at

Fig. 2. Human ependymogenesis proceeds posterior to anterior along lateral ventricle wall during fetal-to-postnatal development and exhibitscharacteristic 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 indicatepercentage of radial glia, immature ependymal cells, V-SVZ stem cells andmature ependymal cells along anterior, middle and posterior regions. Scale bars: 1 cmin A (2D ventricle wall renderings); 5 cm in B (2D ventricle wall renderings); 20 µm in A,B (tissue sections).

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6 and 7 months). A reduction in stem cell numbers was observed tofollow the same posterior-to-anterior pattern, with loss occurring inthe posterior regions first. By 8 years, only mature ependymal cellswere found lining the lateral ventricle surface; no stem cell processeswere observed (Fig. 2B). This absence of stem cell apical processesalong the lateral wall was also found in adult tissue, indicating thatonly mature ependymal cells line the lateral ventricle wall inadulthood (Fig. 2B).The medial walls of the frontal horn of the lateral ventricles also

show a posterior-to-anterior wave of ependymogenesis, but V-SVZstem cells are not retained and, as a result, ependymal cellscompletely cover the medial wall (Fig. S2C). New immatureependymal cells were found scattered throughout the medial wall at21 gw, and by 34 gw the middle regions of the wall were mostlycovered by mature ependymal cells. By 10 days postpartum, anintact ependyma was found in all regions of the medial wall. We didnot detect any remaining V-SVZ stem cells in any postnatal medialwall samples.The above studies of fetal, perinatal, adolescent and adult human

lateral wall tissue demonstrate a posterior-to-anterior transition fromradial glia coverage (before 21 gw, data not shown) to a completemonolayer of ependymal cells (adolescent and adult tissue). Asependymal cells are generated (from 21 gw until perinatal timeperiods), radial glia and V-SVZ stem cell numbers (based on anapical process at the ventricle surface) decline. Pinwheel unitsinitially contain many stem cell processes, but process numberdeclines during postnatal development. Concurrent with ependymalcell maturation through postnatal development is an increase inapical surface area of ∼fivefold (Fig. S2D).

Human lateral ventricle volume and surface area changescorrespond to curvature changesWe next examined whether the spatiotemporal posterior-to-anteriorprogression of ependymogenesis and stem cell depletion along thelateral wall of the lateral ventricle were related to developmentalchanges in lateral ventricle volume and surface area. Usingneurologically normal prenatal T2-weighted structural magneticresonance imaging (MRI) scans (7T) and postnatal T1-weightedstructural MRI scans (3T) from neurologically normal individuals

(Table 1), we determined total brain and lateral ventricle volumesand lateral ventricle surface area at discrete developmental timepoints (Fig. 3A,B). Semi-automated segmentation using ITK-SNAP (Shook et al., 2014; Snippert et al., 2010; Todd et al., 2017;Yushkevich et al., 2006) followed by 3D reconstruction of thelateral ventricles and whole brain using 3D Slicer (Acabchuk et al.,2015; Shook et al., 2014) revealed that the average total brainvolume increases rapidly from 15 gw to 1 year. Regression analysispredicts that from 19 gw to birth, the brain grows at a nearly linearrate of 1.2×106 mm3/year (23 cm3/week) and from birth to 1 year at5.5×105 mm3/year (11 cm3/week) (see also Kinoshita et al., 2001).From 1 year to ∼3.5 years, brain volume growth slows to a nearlylinear average rate of 1.3×105 mm3/year (2.5 cm3/week). Theserepresent a 1100% increase in brain volume from 18 gw to birth,110% increase from birth to 1 year, and 16% from 1 year to 2 years(Fig. 3A,B), similar to the findings of others (Knickmeyer et al.,2008). In contrast, lateral ventricle volume exhibits an increase,slight decrease and then a plateau across early postnataldevelopment. Lateral ventricle volume increase occurs from15 gw to ∼1.25 years. This occurs at an average rate of8900 mm3/year (171 mm3/week) from 15 gw to birth, and3800 mm3/year (73 mm3/week) from birth to 1.25 years. Ventriclevolume decrease occurs at a nearly linear rate of 800 mm3/year(15 mm3/week) from 1.25 years to 3.5 years and is significant byANOVA of a simple linear regression model across this data subset(P=0.032). A plateau appears to occur slightly after 3 years,stabilizing at ∼8400 mm3 and there is no significant evidence toindicate that the average ventricle volume from 3-4 years differsfrom that of 10-11 years, based on an outlier-resistant two-sampleMann–Whitney nonparametric test (P=0.66). In summary, ventriclevolume increases 350% from 15 gw to birth, 86% from birth to1.25 years and decreases 18% from 1.25 years to 3.5 years. Certainscans from the Cincinnati MR Imaging (C-MIND) database (www.cmind.research.cchmc.org) showed distinctly larger total ventriclevolumes (see Fig. S3 for details). Some scans showed asymmetricalventricles, with one ventricle being larger than the other (Fig. 3B,marked by ‘×’ on ventricle volume and surface area graphs) orsymmetric and slightly enlarged ventricles (Fig. 3B, marked by ‘O’on ventricle volume and surface area graphs).

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 ofManitoba

University ofManitoba

University ofManitoba

University ofManitoba

University ofManitoba

NIH NeuroBioBank NIH NeuroBioBank NIH NeuroBioBank University ofManitoba

Anatomicalposition

Right cerebrum Left cerebrum Right frontalcerebrum

Left prefrontallobe

Right frontalcerebrum

Right cerebrumlateralventricularwall sections

Right cerebrumlateralventricular wallsections

Anteromedialcortex

Lefthemisphere

Sex N/A N/A N/A N/A Male Female Male Male MaleCause of death Intrapartem

deathIntrapartem

deathPolycystic

kidneysCo-sleeping Co-sleeping Unexpected

infant deathPositional

asphyxiaBlunt force trauma Liver failure

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 90NIH 0 12 4 0 0 0 16LONI 7 0 0 0 0 0 7Yale School

of Medicine12 0 0 0 0 0 12

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 54Female 7 9 10 10 16 8 60Not available

(N/A)9 2 0 0 0 0 11

Total 19 24 17 15 26 24 125

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Plotting the ratio of ventricle to whole brain volume ratio showsthat the lateral ventricles occupy ∼10% of the brain volume at15 gw; this decreases rapidly to ∼2% at birth and stabilizes to ∼1%within a year (Fig. 3B). After 1 year, the brain continues to grow at aslow rate (2.5 cm3/week), which is responsible for any systemicdecrease in the ventricle to whole brain volume ratio over time(Fig. 3B). There appears to be no significant correlation between sexand developmental age in our sample (n=114 individuals of knownsex); therefore, sex was not included in our predictive models (seeMaterials and Methods, Statistical analysis section).Lateral ventricle surface area exhibits a similar growth and

plateau pattern. All expansion occurs from 15 gw to ∼1.25 years.The average growth from 15 gw to birth is 4700 mm2/year (90 mm2/week) and from birth to 1.25 years is 2200 mm2/year (42 mm2/week). Ventricle surface area decrease from 1.25 years to 3.5 yearsoccurs at an average rate of 170 mm2/year (3.3 mm2/week). Theventricle surface area appears to plateau after 3 years, with anaverage surface area of 6800 mm2. Again, there is no significantevidence to indicate that the average ventricle surface area from3-4 years differs from that of 10-11 years, based on an outlier-resistant two-sample Mann–Whitney nonparametric test (P=0.22).

In summary, ventricle surface area increases 100% from 15 gw tobirth, 62% from birth to 1.25 years and decreases 5.4% from1.25 years to 3.5 years. There is no correlation between sex anddevelopmental age in our sample (n=114 individuals of known sex);thus, sex was not included in our predictive models (see Materialsand Methods, Statistical analysis section).

Analysis of surface area changes, unlike total volume changes,suggests changes in topography of the ventricle walls that may notcorrespond to minor volume changes (Del Bigio, 2014). Surface areaincreases emphasize a growing demand for ependymal cell coverage.We found that ventricle surface area increases during fetaldevelopment, plateauing at ∼1.5 years of age (Fig. 3B). Subjectswith enlarged ventricle volumes also show similar trends. Increasingsurface area results in the heightened need for ependymal cellcoverage and may be responsible for the steady decline in stem cellnumber at the ventricle surface in postnatal development.

To characterize the changing topography associated with surfacearea increases along the ventricle wall, we examined how curvatureof the lateral ventricles changed during fetal and postnataldevelopment. From 15 gw to perinatal developmental stages, thecurvature of the anterior horn (superior horn) of the lateral ventricles

Fig. 3. Developmental changes in human lateral ventricle volume and surface area reflected in curvature changes. (A) 3D representations show lateralview 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 andpostnatal human development. Brain volume was modeled as a fourth degree least-squares polynomial regression curve, ventricle volume and ventriclesurface areawere bothmodeled 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 (3months, 9months, 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.

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changes significantly from convex to concave (Fig. 3C, green toyellow) and this concavity appears to be maintained throughoutpostnatal development. In contrast, the temporal and posterior horn(occipital horn), although continuing to grow, remain concave fromfetal through postnatal development. Based on these results, theposterior-to-anterior maturation of the ependymal cell monolayer atthe ventricle surface also corresponds to the timing of the changingcurvature of the anterior horn from convex to concave (Fig. 3C).

The V-SVZ stem cell niche and neurogenesis significantlydecline during human brain developmentTo characterize the organization of the V-SVZ stem cell niche(frontal horn lateral wall of the lateral ventricle) acrossdevelopment, we examined tissue at fetal, perinatal, postnatal,adolescent and adult time points. Coronal sections from anterior,middle and posterior regions were prepared (see Table 1) andstained for GFAP (V-SVZ SCs), DCX (neuroblasts), Ki67 (cyclingcells) and FOXJ1 [immature and mature ependymal cells, some latestage radial glia (Jacquet et al., 2009)] (Fig. 4A,B). At 21 gw, whenthe lateral ganglionic eminence (LGE) comprises the lateral wall ofthe lateral ventricles, immature ependymal cells (FOXJ1+) weredetected primarily in the posterior region, similar to what was foundin Fig. 2A. Radial glia (GFAP+) exhibited long radial processes andDCX+ neuroblasts were numerous throughout the SVZ and outerSVZ (oSVZ) regions (Hansen et al., 2010). Ki67+ proliferative cellswere more numerous in the anterior versus posterior and/or middleregions. At 28 gw, the long radial processes of radial glia andwidespread distribution of neuroblasts were still observed, and thereare more proliferative cells observed in the anterior versus posterior

region. FOXJ1+ ependymal cells were found at the ventricle surfacein both anterior and posterior regions. By 34 gw, SVZ astrocytes(GFAP+) possessed shorter radial processes, neuroblasts were stillnumerous and a mature ependyma demarcated the ventricle lining.Proliferative cells were mainly found within the anterior SVZ/oSVZregion, extending a significant distance from the ventricle lining. By10 days postpartum, Ki67+ cells were reduced in number, and themajority of DCX+ neuroblasts were restricted to a narrow pathwayimmediately subjacent and tangential to the ventricle surface. Both6-month and 7-month samples revealed few neuroblasts orproliferative cells, the absence of long radial processes and acontinuous monolayer of ependymal cells making up the ventriclelining. We detected no Ki67+ or DCX+ cells in the 8-year-oldsample, with the exception of a few Ki67+ cells scattered alongblood vessels. GFAP+ astrocytes consolidated as a ribbon parallelto, but separate from, an acellular zone that lay next to theependymal monolayer, as previously reported (Sanai et al., 2011).

DISCUSSIONOrgan-specific stem cell niches can have long-lasting effects on thedevelopment and function of the organ system. A vital stem cellniche within the developing brain is the V-SVZ. In addition tosupporting neurogenesis, stem cells in the V-SVZ also generatecells that support their own niche. An essential, but understudied,contributor to the V-SVZ stem cell niche is the ependyma – amonolayer of multi-ciliated ependymal cells that are generatedduring mid- to late-gestation in humans. Ependymal cells anchorniche-associated stem cells and provide barrier and transportfunctions 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 andV-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 andposterior 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, 7months and 8 years. Composite image of all four channels is positioned above separated channels for each region. Dotted white line indicates the lateral ventriclewall edge. Glial fibrillary acidic protein (GFAP, red), doublecortin (DCX, blue), Ki67 (green), FOXJ1 (gray). Scale bars: 50 µm.

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A mature ependymal cell lining is also required to supportneurogenesis and neuroblast migration along the lateral ventriclewall in mice (Paez-Gonzalez et al., 2011).We have mapped the progression of ependymogenesis across the

frontal horn of the lateral ventricles, along with changes in ventriclevolume, surface area and curvature during human brain development(Fig. 5). Radial glia that initially line the ventricles in early fetaldevelopment were sequentially replaced by ependymal cells in anoccipital-to-frontal wave across the ventricle surface. Significantly,only the lateral wall of the frontal horn retained radial glia/stem cellsinto infancy and these stem cells maintained only an apical process atthe ventricle surface, whereas their somata were repositioned to theSVZ. Clusters of stem cell apical processes surrounded by ependymalcells, arrayed as classic ‘pinwheel’ units similar to those previouslydescribed inmouse brain (Mirzadeh et al., 2008), were found inwhole-mount preparations of human perinatal frontal horn lateral wall tissue.The number of stem cells with ventricular contact steadily declined in aposterior-to-anterior gradient during postnatal development and noapical processes of stem cells were observed in whole-mountpreparations of adolescent and adult lateral wall samples.In contrast to the lateral wall, the medial wall of the frontal horn of

the lateral ventricles, which also shows a similar posterior-to-anterior ependymogenesis progression, does not retain stem cellswith ventricle contact after birth. Instead, an uninterrupted wall ofependymal cells eventually covers the entire medial wall.Our findings related to ependymogenesis in humans during the

fetal-to-postnatal transition mimics what has been demonstratedpreviously in mice [this study and others (Jacquet et al., 2009;Kyrousi et al., 2015; Mirzadeh et al., 2010b, 2008; Paez-Gonzalezet al., 2011; Spassky et al., 2005)]. However, in contrast to human,ventricle-contacting stem cells persist along the lateral wallthroughout adulthood in mouse and other mammals (Alunni andBally-Cuif, 2016; Lledo et al., 2008; Peretto et al., 1999; Conoverand Shook, 2011) and although stem cell numbers decline over thecourse of aging, stem cells with ventricle contact are still found inelderly mice (Capilla-Gonzalez et al., 2015; Conover and Shook,2011; Conover and Todd, 2017; Luo et al., 2006; Maslov et al.,2004; Shook et al., 2012).The life-long retention of V-SVZ stem cells and accompanying

neurogenesis found in adult mice and other mammals differs fromthe significant diminution of V-SVZ stem cells and neurogenesisthat we, and others (Arshad et al., 2016; Hansen et al., 2010; Maliket al., 2013; Paredes et al., 2016a,b; Sanai et al., 2011; Wang et al.,2011, 2014), have observed by the time of adolescence in humans.This discrepancy has recently garnered special attention and driventheoretical models to explain retention or loss of stem cell

populations in different organ systems and in different species(Hormoz, 2013). Hormoz (2013) proposed the division of stem cellpopulation dynamics into two types, both supporting stem cell self-preservation and progenitor generation. (1) Stem cell populationasymmetry occurs with heterogeneous proliferation rates, with somestem cells dividing symmetrically to give progenitor cells[‘consuming’ stem cell division (Obernier et al., 2018)] andneighboring slow-dividing stem cells divide symmetrically to yieldtwo stem cells. (2) Asymmetric stem cell division occurs when stemcells divide to yield a stem cell (self-renewal) and a progenitor cell.Asymmetric cell division is the predominant form of division byradial glia in embryonic development (Kriegstein and Alvarez-Buylla, 2009), but it is a process that will not support the stem cellpool over extended periods of time because of stem cell exhaustion(Hormoz, 2013; Obernier et al., 2018; Shahriyari and Komarova,2013). Population asymmetry would allow the slow-dividing stemcells to proliferate and purge the population of fast-dividing, oldercells. This mechanism has been observed in adult V-SVZ stem cell-derived neurogenesis in mouse (Obernier et al., 2018) and may alsoexplain V-SVZ stem cell retention into infancy in humans. It remainsto be determined whether generation of ependymal cells is the finaldivision product (through symmetric division) of stem cells withventricle contact.A similar ‘disposable stemcell’modelwas proposedby Encinas et al. (2011) to explain age-related loss of mousehippocampal neural stem cells and the appearance of new astrocytes.

The extent of ependymogenesis is likely driven by alterations tothe contours of the ventricle surface. In an attempt to correlate thespatiotemporal development of the ependymal lining with changingconformation of the ventricle wall, we assessed lateral ventriclevolume and surface area changes over the course of fetal andpostnatal development. Using MRI data from our human sources,we plotted volumes and surface area from 15 gw to 11 years. Rapidincreases in total brain volumes and increases in lateral ventriclevolumes were found from 15 gw until ∼1 year. After 1 year, thetotal brain volume rate of increase slows but continues at a linearrate. In contrast, lateral ventricle volume exhibits an increase, slightdecrease, and then a plateau across early postnatal development; theventricle volume at 3 years is not significantly different from thatseen at 11 years. Others have reported similar trends for brain andlateral ventricle increases from the second trimester to birth(Kinoshita et al., 2001; Sakai et al., 2012) to 2 years of age(Knickmeyer et al., 2008; Leigh, 2004). To extend earlier studies,we found that early in the second trimester (15 gw) the lateralventricles occupy ∼10% of the total brain volume, 2% of the totalbrain volume at birth and 1% of the total brain volume at 1 year.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 thelateral wall of the frontal horn of the lateral ventricle. At 21 gw, themajority of the lateral wall surface is covered by radial glial cells, as immature ependymal cells areforming 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 ependymalcells 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 andstem 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 isclearly evident. By 8 years, the lateral wall is comprised entirely of mature ependymal cells, with no pinwheels present. The conformation of the ventriclesurface 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.

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from 15 gw until birth, showing a slight decrease after 1 year andthen reaching a plateau by 2-3 years of age. Volume and surface areaare general measurements that do not reveal topographical changesat the ventricle surface [e.g. volume may increase throughdisplacement (convexity) of the ventricle walls, whereas surfacearea remains unchanged (Del Bigio, 2014)]. To evaluateconformational changes along the ventricle surface, we usedcurvature analysis to reveal patterns of concavity and convexityalong the extra-ventricle surface. These patterns are not outwardlyapparent from MRI scan segments; however, they do become moreobvious from 3D reconstructions. From 15 gw to late gestation(36 gw), the anterior horn exhibits significant conformationalchanges from convex to concave. Concavity of the anterior hornthen stabilized through postnatal periods. The occipital horns growand elongate extensively in late gestation, but remain concave from36 gw through postnatal development. In the lateral ventricle frontalhorn, we found that the eventual coverage of the lateral ventriclewall with mature ependymal cells corresponded to the period whenventricle volume, surface area and curvature stabilized (after ∼1.5years). The mirroring of conformational stabilization andependymogenesis suggests that deposition of ependymal cellsalong the ventricle walls likely contributes to the lateral ventriclewall stability.Ependymogenesis in ventricle regions other than the lateral wall

corresponds to completion of neurogenesis. V-SVZ neurogenesisalong the frontal horn lateral wall in the postnatal human anteriorforebrain populates the ‘arc’ pathway to the frontal cortex, and theMMS and RMS pathways (Hansen et al., 2010; Paredes et al.,2016a; Sanai et al., 2011, 2004). We documented changes to the V-SVZ and oSVZ in coronal sections along the lateral ventricle wallthat showed substantial proliferation and neurogenesis duringgestation, followed by decreased proliferation and neurogenesisafter birth, in full support of the findings of others (Bergmann et al.,2012; Paredes et al., 2016b; Quiñones-Hinojosa et al., 2006; Sanaiet al., 2011; Wang et al., 2011, 2014). In addition, we observed areorganization of migratory neuroblasts to a narrow tangentialpathway subjacent to the ependymal lining from birth until 7 monthsof age. By 8 years no proliferative cells or migratory neuroblastswere detected, instead a periventricular acellular gap region andassociated astrocyte ribbon was detected, as previously described inthe adult human brain (Sanai et al., 2011, 2004). A recent studyprovided strong evidence that human hippocampal dentate gyrusneurogenesis declines rapidly during the first year of life and persistsat low levels into early adolescence, but does not continue intoadulthood (Sorrells et al., 2018). This challenges previous reports(Dennis et al., 2016; Eriksson et al., 1998; Knoth et al., 2010;Spradling et al., 2001) and a new study (Boldrini et al., 2018) thatfind daily production of new neurons in adulthood (∼700/dentategyrus/day). Although our study and others provide evidence thatrobust, or even moderate, levels of neurogenesis along the lateralventricle surface do not continue into adulthood in the human brain,it remains unresolved, and highly controversial, whether this appliesto other forms of adult human neurogenesis.

Concluding remarksFull ependymal cell coverage of the ventricle walls supports bothbarrier and transport functions between the interstitial fluid and CSFand aids in maintaining brain homeostasis. Diseases resulting inventriculomegaly, such as fetal-onset hydrocephalus, an abnormalenlargement of the lateral ventricles, places an extraordinary demandon the stem cell population to provide both crucial neurogenicfunctions and adequate ependymal cell coverage at the ventricle

surface. Disruption, hypoproliferation or hyperproliferation ofthe ependymal layer is implicated in a variety of psychiatric,neurodegenerative and neurodevelopmental conditions and isunderstudied. The above studies define normal development of theV-SVZ stem cell niche and associated ependymal lining along thelateral ventricles and provide a developmental platform from whichto assess disruption of this vital stem cell niche in diseases that resultin infant ventriculomegaly or hydrocephalus.

MATERIALS AND METHODSAnimalsMale CD-1 mice (Mus musculus) (Charles River Laboratories, Wilmington,MA, USA) at E13, E16, P1, P7 and P30 (adult) were used. Housing,handling, care and processing of the animals were carried out in accordancewith regulations approved by the Institutional Animal Care and UseCommittee of the University of Connecticut.

Mouse brain tissue immunohistochemistryAll antibodies used in this study were used previously and validated by ourgroup and others; the expression patterns were as expected and referenced.For coronal sections, P7 and P30 mice were anesthetized withisoflurane, then transcardially perfused with 0.9% saline followed by 4%paraformaldehyde (PFA). The extracted brains were fixed overnight in 4%PFA at 4°C. E13, E16, and P1micewere anesthetized with isoflurane, headswere removed and fixed overnight in 4% PFA at 4°C. After removing theskin, embryonic and P1 brains were removed from the skull using a LeicaMZ95 stereomicroscope. All brains were washed for 3×10 min in PBSbefore dissection and vibratome sectioning for 3D reconstructions.

Lateral ventricle wall wholemounts were prepared as described (Mirzadehet al., 2008). Following isofluorane anesthesia and saline perfusion, P7 andP30 brains were extracted and whole-mount preparations were placed inPFA with 1% Triton X-100 overnight. E13, E16 and P1 mice wereanesthetized with isofluorane, heads were extracted and placed in 4% PFAwith 1% Triton X-100 overnight and wholemounts were then prepared(Mirzadeh et al., 2010a). Wholemounts were immunostained with thefollowing primary antibodies: mouse monoclonal anti-β-catenin (1:250; BDBiosciences, #610154), rabbit polyclonal anti-β-catenin (1:100; CellSignaling Technology, #9562), rabbit polyclonal anti-γ-tubulin (1:500;Sigma-Aldrich, #T5192), rabbit polyclonal anti-GLAST (1:200; Abcam,#ab416), goat polyclonal anti-GFAP (1:250; Abcam, #ab53554), ratmonoclonal anti-GFAP (1:250; Invitrogen, #13-0300) and mousemonoclonal anti-FOXJ1 (1:250; Invitrogen, #14-9965-80). Alexa Fluordye-conjugated polyclonal secondary antibodies (1:500, Invitrogen) wereused: donkey anti-mouse 488 (#21202), donkey anti-mouse 546 (#A10036),donkey anti-rabbit (#A21206), donkey anti-goat-647(#A21447) and donkeyanti-rat 647 (#A18744). Blocking solutions contained 1% Triton X-100.Validations of all commercial antibodies are available from manufacturer’sdatasheets. Lateral wall tissue wholemounts were coverslipped with Aqua-Poly/Mount (Polyscience) and imaged on a Leica TCS SP8 confocal laserscan microscope (Leica Microsystems).

Mouse lateral ventricle reconstruction and analysisTo generate 3D reconstructions of the mouse brain and ventricles, coronalsections from E13 (42 µm), E16, P1, P7 and P30 (all 50 µm) brains weresectioned on a vibratome (VT-1000S, Leica). Mouse brain tissue sectionswere stained with β-catenin overnight (rabbit polyclonal anti-β-catenin,1:100; Cell Signaling Technology, #9562), secondary antibody for 1 h(donkey anti-rabbit 546, 1:500; Invitrogen, #A10040), nuclear stain DAPI(300 mM; Molecular Probes, #D-1306) for 10 min and imaged on a ZeissAxio Imager M2 microscope with ApoTome (Carl Zeiss MicroImaging)with a Hamamatsu Photonics ORCA-R2 digital camera (C10600).Alternating coronal sections were imaged, and the contours of the lateralventricle walls and surface of the brain were traced to generate 3Dreconstructions, as described (Acabchuk et al., 2015). Volume and surfacearea analysis were performed using StereoInvestigator and NeurolucidaExplorer software (MBF Bioscience).

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Human brain tissue immunohistochemistryPostmortem human brain tissue (whole hemispheres and regional portionsof the lateral ventricle wall) ranging from 21 gw to 39 years of age wereobtained from the NIH NeuroBioBank (University of Maryland, MD, USA)and the University of Manitoba, Pathology Department (Winnipeg,Canada). Tissues were acquired under protocol H2011-212, approved bythe University of Manitoba Health Research Ethics Board. All tissue wasarchival and came de-identified. Hemispheres from 21 gw, 28 gw, 34 gw,10 day and 39 year, and V-SVZ sections from 5 and 7 months wereexamined (Table 1). For lateral ventricle whole-mount preparations, severalsections from the anterior (frontal horn over the caudate nucleus head),middle (frontal horn near the interventricular foramen) and posterior(frontal horn) lateral ventricle surfaces were dissected. Lateral ventricle wallwholemounts were coverslipped with AquaPoly/Mount and imaged onLeica TCS SP8 confocal laser scan microscope. Lateral ventriclewholemounts were imaged using a 100×/1.4 HC PL APO oil immersionobjective lens, taken at scan zoom 1 (13,567.59 µm2 field area) or scan zoom2 (3391.90 µm2 field area). The following antibodies were used: rabbitpolyclonal anti-β-catenin (1:100; Cell Signaling Technology, #9562), rabbitpolyclonal anti-γ-tubulin (1:500; Sigma-Aldrich, #T5192); rabbitpolyclonal anti-GLAST (1:200; Abcam, #ab416), goat polyclonal anti-GFAP (1:250; Abcam, #ab53554), rat monoclonal anti-GFAP (1:250;Invitrogen, #13-0300) and mouse monoclonal anti-FOXJ1 (1:250;Invitrogen, #14-9965-80). Alexa Fluor dye-conjugated polyclonalsecondary antibodies (1:500, Invitrogen) were used: donkey anti-mouse488 (#21202), donkey anti-rabbit 546 (#A10040), donkey anti-goat-647(#A21447) and donkey anti-rat 647 (#A18744). Validations of allcommercial antibodies are available from manufacturer’s datasheets.Representative areas (13,567.59 µm2) were imaged and cell types wereidentified based on basal body number (radial glia and V-SVZ stem cells, 1basal body; immature ependymal cells, 2-5 basal bodies; mature ependymalcells, an array of many basal bodies) and immunostaining criteria.Schematics were generated by outlining an identified cell and assigning a‘fill’ color.

Regions corresponding to the LGE in fetal tissue and the lateral wall ofthe lateral ventricle in postnatal tissue were dissected and sectionedcoronally at 100 mm thickness. Immunohistochemistry was performed asdescribed (Todd et al., 2017) using the following antibodies: goat polyclonalanti-GFAP (1:250; Abcam, #ab53554), mouse monoclonal anti-FOXJ1(1:250; Invitrogen, #14-9965-80), guinea pig anti-doublecortin (DCX)(1:1000, EMD Millipore, #AB2253) and rabbit anti-Ki67 (1:1000;Novocastra, #6013874). Alexa Fluor dye-conjugated polyclonalsecondary antibodies (1:500) were used: donkey anti-mouse 405 (Abcam,#ab175658), donkey anti-mouse 488 (Invitrogen, #21202), donkey anti-rabbit 546 (Invitrogen, #A10040), donkey anti-goat-647 (Invitrogen,#A21447) and donkey anti-rat 647 (Invitrogen, #A18744). Validations ofall commercial antibodies are available from manufacturer’s datasheets.Coronal sections were imaged on a Leica TCS SP8 confocal laser scanmicroscope using a 40×/1.3 HC PL APO oil immersion objective lens.

Human MRI lateral ventricle reconstruction and analysisDe-identified archival MRI scans ranging from 15 gw-10 years were used inthis study. T1 or T2 structural scans were obtained from the C-MINDdatabase with a 3T MRI scanner, the NIH National Institute of MentalHealth (NIMH) data archive (https://ndar.nih.gov/), Laboratory of NeuroImaging (LONI) database of the University of Southern California (www.loni.usc.edu, 7T MRI scanner) and Yale School of Medicine (3T MRIscanner) (Table 1). Lateral ventricle and whole-brain semi-automatedsegmentation and volume/surface area analyses were completed using ITK-SNAP and 3D Slicer software as previously described (Acabchuk et al.,2015; Todd et al., 2017). To determine accurate whole-brain volume andsurface area measurements, skulls were digitally removed from MRI scansusing Brain Suite software (www.brainsuite.org). De-skulled masks of MRIscans were automatically generated using the skull stripping tool under thecortical surface extraction sequence function of Brain Suite. Brain surfaceextractor mechanism was set to trim brain stem and spinal cord and dilate thefinal mask. Extractor settings that were kept constant include five automatediterations, three diffusion iterations and a diffusion constant of 25. Edge

constant and erosion size were manually modified as necessary with eachMRI scan. Manual edits using the edit mask function of Brain Suite weremade following automatic skull stripping as needed.

Curvature was analyzed in Meshmixer software (www.meshmixer.com).3DOBJ files created from lateral ventricle segmentations were imported intoMeshmixer. Mean curvature was calculated and generated on the 3D modelas a heat map using the mesh query analysis function. Meshmixerautomatically calculates the mean curvatures based on a system oftriangles (faces) and vertices on the 3D model, providing curvature valuesat each vertex of the model.

Statistical analysisData are reported as mean±s.e.m. Statistical analysis was performed inGraphPad Prism software (www.graphpad.com). One-way ANOVA withBonferroni’s multiple comparisons post-test was used. All statisticalregression analysis was performed in SAS 9.1. The minimum level ofsignificance for all tests was P<0.05. All regression models were fitted usingleast-squares polynomial regression and analyzed with sequential-addition(type-I sum of squares) partial F-tests of terms with increasing degree. Allterms in our models, including the intercept term, are significant based ontype-III sum of squares, partial F-tests. The following regression modelswere obtained, with ‘x’ denoting developmental age in years, brain andventricle volume in mm3, and ventricle surface area in mm2.

Brain Volume : Y ¼ 484954þ 927627x–497701x2 þ 133771x3–13428x4

Ventricle Volume : Y ¼ 5500:9þ 7233:2x–3300:4x2 þ 420:45x3

Ventricle Surface Area : Y ¼ 4428:9þ 3901x� 1620:5x2 þ 199:36x3

AcknowledgementsWe gratefully acknowledge the NIH NeuroBioBank and the University of Manitoba,Pathology Department, for providing human tissue. We also thank and gratefullyacknowledge the LONI database, the University of Southern California, C-MIND,NIH NIMH Data Archive and Dr Dustin Scheinost (Yale School of Medicine) forproviding MRI data. This manuscript reflects the views of the authors and maynot reflect the opinions or views of the NIH or of the Submitters submitting originaldata to NDAR.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: 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.;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.;Formal analysis: A.M.C., D.S., S.K., T.N.S., P.J.B., B.F.B., D.P., E.S.N.;Investigation: A.M.C., D.S., S.K., T.N.S., P.J.B.; Resources: A.M.C., D.S., S.K.,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.,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.;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.,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.

FundingThis research was funded by the National Institutes of Health (NS090092 andNS098091 to J.C.C.; instrument grant S10ODO16435), the HydrocephalusAssociation (to J.C.C.), the University of Connecticut Institute for Brain and CognitiveSciences (to A.M.C., D.S., B.F.B., S.K. and J.C.C.). Dr Del Bigio holds the CanadaResearch Chair in Developmental Neuropathology. Deposited in PMC for releaseafter 12 months.

Data availabilityData used in the preparation of this article were obtained from the following studies:(1) Brain Suite, LONI software (NIH-NINDS R01 NS074980, NIH-NIBIB R01EB002010, NIH-NIBIB P41-EB015922). (2) The C-MIND Data Repository createdby the C-MIND study of Normal Brain Development (CMINDS data set version1.720). This is a multisite, longitudinal study of typically developing children fromages newborn through young adulthood conducted by Cincinnati Children’s HospitalMedical Center and UCLA and supported by the National Institute of Child Healthand Human Development (Contract HHSN275200900018C). A listing of the

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participating sites and a complete listing of the study investigators can be found athttps://research.cchmc.org/c-mind. (3) The NIH-supported National Database forAutism Research (NDAR). NDAR is a collaborative informatics system created bythe National Institutes of Health to provide a national resource to support andaccelerate research in autism.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.170100.supplemental

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