FIIN-2 | Sphk Signal

Cerebral organoids reveal early cortical maldevelopment in schizophrenia— computational anatomy and genomics, role of FGFR1

Abstract
Studies of induced pluripotent stem cells (iPSCs) from schizophrenia patients and control individuals revealed that the disorder is programmed at the preneuronal stage, involves a common dysregulated mRNA transcriptome, and identiﬁed Integrative Nuclear FGFR1 Signaling a common dysregulated mechanism. We used human embryonic stemcell (hESC) and iPSC-derived cerebral organoids from four controls and three schizophrenia patients to model the ﬁrst trimester of in utero brain development. The schizophrenia organoids revealed an abnormal scattering of proliferating Ki67+ neural progenitor cells (NPCs) from the ventricular zone (VZ), throughout the intermediate (IZ) and cortical (CZ) zones. TBR1 pioneer neurons and reelin, which guides cortico-petal migration, were restricted from the schizophrenia cortex. The maturing neurons were abundantly developed in the subcortical regions, but were depleted from the schizophrenia cortex. The decreased intracortical connectivity was denoted by changes in the orientation and morphology of calretinin interneurons. In schizophrenia organoids, nuclear (n)FGFR1 was abundantly expressed by developing subcortical cells, but was depleted from the neuronal committed cells (NCCs) of the CZ. Transfection of dominant negative and constitutively active nFGFR1 caused widespread disruption of the neuro-ontogenic gene networks in hESC-derived NPCs and NCCs. The fgfr1 gene was the most prominent FGFR gene expressed in NPCs and NCCs, and blocking with PD173074 reproduced both the loss of nFGFR1 and cortical neuronal maturation in hESC cerebral organoids. We report for the ﬁrst time, progression of the cortical malformation in schizophrenia and link it to altered FGFR1 signaling. Targeting INFS may offer a preventive treatment of schizophrenia.

Introduction
Although the primary onset of schizophrenia is during adolescence to young adulthood, an early developmental origin of schizophrenia has been implied by improperly clustered immature neurons in cortical layers II, III, and V1, changes in the hippocampal nonpyramidal CA2neurons2, as well as hypoplastic midbrain dopamine neurons3–6. Such changes in brain structure most likely develop in utero during the late ﬁrst and early second trimester7–9. Additionally, disorganization of the white matter tract has been observed in schizophrenia10, sug- gesting that the disease affects not only the development and function of neurons, but also their projections. The variability in brain malformations are thought to underliethe variety of clinical ﬁndings: positive symptoms (delu- sions and hallucinations), negative symptoms (affective ﬂattening, amotivation, and anhedonia)11,12, and cognitive symptoms (disorganized speech and cognitive deﬁcits) (DSM 4th edition). Abnormal development during theﬁrst trimester is also consistent with minor physical anomalies associated with schizophrenia13.According to the neurodevelopmental hypothesis, both genetic and environmental factors broadly affect brain development and thus, contribute to the etiology of schizophrenia7. The ontogenic construction of the human cortex is thought to proceed through a series of irrever- sible cycles of neural stem and progenitor cell prolifera- tion, differentiation to neuroblasts, migration to the brain surface where early neuronal-like cells are formed, and develop into mature neurons with intercellular connec- tions and communicative pathways. Normal human brain development begins with proliferation of neurogenic neuroepithelial precursors, which act as neural stem cells and give rise to the primary progenitor cells, radial glia, that are capable of generating neurons, astrocytes, and oligodendrocytes14. In the developing vertebrate brain, the elongated bipolar radial glia are located at the apical surface of the ventricular zone (VZ) and span the width of the cortex.

These cells generate translocating radial glia, intermediate progenitor cells, and neuroblasts that migrate through the intermediate zone (IZ) to the outer cortical zone (CZ), and populate cortical layers. Via this inside-out process, the early born neurons occupy inner layers while the late born neurons migrate out toward the edge and occupy the superﬁcial cortical layers15. Disrup- tion of these processes has been hypothesized to underlie the misconstruction of the cortex and subcortical circuits observed in autism16 and schizophrenia (review-17).Coordinated transition of cells from one neurodeve- lopmental stage to another are needed to produce syn- chronized development of the cortical layers. The navigation through distinct developmental stages involves concerted regulation of thousands of genes and is likely overseen by some central guiding mechanism(s). Our recent studies have revealed one such pan-ontogenic mechanism, Integrative Nuclear FGFR1 Signaling (INFS) that controls general body and the brain devel- opment18–20. The fgfr1 gene resides at the top of the gene hierarchy that governs gastrulation, as well as the sub- sequent development of the major body axes, nervous system, muscles, and bones, by affecting downstream genes that control the cell cycle, pluripotency, and dif- ferentiation, as well as microRNAs (miRNAs)21–26. Stu- dies show that this regulation is executed by nuclear FGFR1 (nFGFR1), which integrates signals from diverse development-initiating factors, cooperates with a multi- tude of transcription factors (TFs), and targets thousands of genes encoding for mRNAs, as well as miRNAs in top ontogenic networks19,20.

nFGFR1 binds to the promoters of genes that control the transition from proliferation to cell differentiation, as well as to the morphogens that delineate the body and CNS axes, and construct the nervous system18,19,27–29.RNA-sequencing of differentiating neuronal progenitor cells (NPCs) developed from the induced pluripotent stem cells (iPSCs) of four different schizophrenia patients and four control subjects revealed a common dysregulation of 1384 mRNAs and 18 miRNA genes, deconstruction of coordinated mRNA, and miRNA–mRNA networks, and the formation of aberrant networks29. The downregulated genes reside within pathways that block neurogenesis, e.g. Notch1 and Rest, while the upregulated genes were found in pathways that initiate neural development, including master genes, such as Ascl1, Nur77, and RXR, or genes of the Wnt signaling pathway29. ChIPseq revealed that nFGFR1 targets more than 80% of the dysregulated mRNA genes and 31% of dysregulated miRNA genes29. These results indicated an early (preneuronal) genomic etiology of schizophrenia and designated INFS as a common dysregulated mechanism in the Cannon and Keller water-shed model of schizophrenia29,30. Analyses of the differentiated neurons of the same schizophrenia and control iPSC lines revealed impaired migration in 2D cultures31, polarity32, decreased neurite numbers33, reduced synaptic maturation33–36, and decreased activity in the schizophrenia neurons35,36. These changes fore- casted a potential disruption of early brain development in schizophrenia. Therefore, in the present study we fol- lowed the neuro-ontogenic potency of these characterized iPSC lines, from the stem cell/progenitor stage through the formation of cortical layers using the 3D cerebral organoid model.Pluripotent stem cells have the potential to differentiate into various cell lines37 and to self-organize and develop organ-like structures termed “organoids.” Recently, 3D cerebral organoids have been generated, which recapitu- late key aspects of human brain development during the ﬁrst and early second trimesters of embryogenesis38. The pluripotent hESCs or iPSCs aggregate to form embryoid bodies (EBs) capable of generating all three germ layers39. EBs placed in neuroectodermal media form the neu- roectoderm germ layer, the foundation for the develop- ment of cerebral organoids. Neuroectoderm cultured in matrigel scaffolds develop a 3D cerebral organoid tissue with distinct germinal centers (rosettes) and cortical lay- ers corresponding to the human brain structure at the end of the ﬁrst trimester38,40,41.

This new technology applied to iPSCs from microcephaly patients41 and patients with autism16 revealed disruptions in early brain development and shed light on underlying cellular mechanisms involved.In the present investigation, the cerebral organoid technology applied to control and schizophrenia iPSCs29,31,33 provided for the ﬁrst time, an insight into the early stages of the telencephalic development and the role of FGFR1 in schizophrenia.These results showed an abnormal dispersion of the proliferating stem-like cells outside the VZ into the IZ and CZ, and a curtailed neuronal cortical development in the CZ of the schizophrenia cerebral organoids accompanied by turning off the expression of nFGFR1. The arrest of cortical development was reproduced in control cerebral organoids derived from hESCs, by blocking and depleting FGFR1 with its speciﬁc antagonist, PD173074. This study suggests that a reconstitution of nFGFR1 in developing cortical neurons can modify the developmental malformations.In the human embryonic stem cell (hESC) studies, line H9 (WA09) (WiCell Research Institute, Inc.) and line HUES8 (originated from Harvard University, Boston, USA) were used. The iPSC lines used in the present study (Table 1) were derived from different control individuals and from individuals diagnosed with schizophrenia at different ages33. In earlier studies these schizophrenia lines displayed similar phenotypic changes in 2D cultures as described in the section “Introduction”31,33,42. Recent RNAseq investigation showed common dysregulation of a global transcriptome in neural progenitor cells (NPCs) differentiated from all schizophrenia lines and ChIPseq showed similar changes in nFGFR1 genomic interactions in two investigated schizophrenia iPSC lines29. In the present study, we found no discernable differences between the organoids generated from control female and control male iPSC lines and between schizophrenia female and control male iPSC lines. The differences between schizophrenia and control organoids were reproduced in all control and schizophrenia iPSC lines and were statistically evaluated.

Plasmids and transfections are described in Supple- mental Methods.Cerebral organoids were generated from hESCs (H9 and HUES8) and from human iPSCs using a modiﬁed proto- col of Lancaster et al.40, described in the Supplementary Methods and in Supplementary Fig. 1. Eight-day hESC (H9) cerebral organoids were treated with the FGFR1 inhibitor, PD173074 (Abcam), by adding fresh media with 100 nM PD173074 or drug-free media every 2 days for10 days. On day 18, the cerebral organoids were har- vested, ﬁxed, and processed for immunocytochemistry. At day 14, cerebral organoids (controls or PD173074 treated) were treated with 100 µM BrdU for 2 h (pulse phase), washed with DMEM-F12 three times and then incubated with media (control or PD173074) for an additional 4 days (chase phase). At day 18, organoids were harvested and double immunostained with rat anti-BrdU and mouse anti-Pan-Neu antibodies, as described in the next section.For all experimental conditions (four controls and three schizophrenia iPSC lines, hESC H9 and HUES8) 4–5 organoids for each line were cryosectioned at 30 μm thickness and processed as described in the Supplemen-tary Methods. The antibodies used are listed in Supple- mentary Table 1. The control and experimental organoid sections were immunostained at the same time using identical protocols28,43,44. Each stain was performed independently four times.Quantitative analyses of histological sections and images Cell were counted using Visiopharm stereological soft- ware45. Fluorescence intensity measurements (FIM) were performed using Zen 2.0 Blue Imaging software in ran- domly selected Regions of Interest (ROIs) within organoid regions listed in the ﬁgure legends.

All images acquired were in the linear range of the camera. Images of sections from control and schizophrenia organoids were acquired under identical illumination conditions and identical camera gain, offset, and exposure times. Backgroundtracted from the images of stained sections. The total area of each ROI image was recorded, as well as the number of thresholded pixels and their integrated intensity. Each channel was converted into the eight-bit grayscale and the mean intensity was calculated. Statistical analysis was performed using t-test in MiniTab software and data is presented as mean ± Standard Error of the Mean (SEM). For detailed protocols see Supplementary Methods.Computational analyses were run on the combined sets of images and the initial results were presented to cor- responding authors without identifying the groups(schizophrenia and control). The codes used in compu- tational image analyses are provided on the following GitHub repositories:Ki67 Analysis: https://github.com/TheJaeger/CellStats- Ki16Calretinin Analysis: https://github.com/TheJaeger/ CellStats-CalretininThe code version used belongs to the master branch, any additional changes made reﬂect improvements to code readability. The code can be accessed by down- loading or cloning the repository and can be run by opening main.m in MATLAB. Additonal instructions on running the codes are provided in README.md at the root of each repository.All image ﬁles used in computational analyses can be found in the link below. Each corresponding set of images (Ki67 and calretinin) are grouped into control and schi- zophrenia sets.Images: http://www.acsu.buffalo.edu/~mks4/public_sha ring/schizophrenia2/The analysis was performed using homogenous cultures of NPCs differentiated from hESC H9, as described in ref. 29. To induce neuronal committed cells (NCCs), NPCs were treated with 20 ng ml−1 BDNF (Peprotech), 20 ng ml−1 GDNF (Peprotech), 1 mM dibutyryl-cyclic AMP (Sigma), and 200 nM ascorbic acid (Sigma)29 for two days. Plasmids and transfections of NPCs are described in Supplemental Methods. NPCs were trans- fected with control DNA or FGFR1(SP-/NLS)(TK-) and 24 h later were stimulated for 48 h with neuronal differ- entiation media induced by cAMP/BDNF/GDNF. Experiments were performed using three biological replicates of culture and transfection conditions. The RNAseq protocol was described previously19,29 and is summarized in the Supplementary Methods. The results of RNAseq have been deposited on NCBI GEO with accession code: GSE103307.Statistical analysis was performed using two-sided t-test (comparison of two groups), by one or two-way ANOVA (comparison of four or more data groups) or by two- sample Kolmogorov–Smirnov test. P < 0.05 was con- sidered signiﬁcant.The RNAseq data were statistically evaluated using Tuxedo pipeline as previously described29. The details of the sample sizes are listed in ﬁgure legends and fulﬁll the requirement of statistical signiﬁcance.

Results
Cerebral organoids from hESCs were developed using a combination of two protocols (Supplementary Fig. 1a, Supplementary Methods). At day 8 the organoids formed stable cortical rosettes (Fig. 1, a1, a2) with proliferative Ki67 positive cells in the inner layer and a mixture of doublecortin positive (DCX+) neuroblasts and immatureβIII-tubulin positive neurons in the outer layers (Fig. 1, a3, a4).During the subsequent 10 days, the organoids increased in size and the number of developed rosettes increased two to three fold (Fig. 1,b1), as compared to 8-day old organoids (Fig. 1, a1). The 18-day organoids formed polarized structures with a distinguishable border that separated a forebrain-like region containing multiple rosettes from a hindbrain-like structure, which typically lacked rosettes (Fig. 1, b1), as reported previously40. In all of the 18-day organoids examined, the cortical rosettes developed three major zones distinguished by DAPI staining (Fig. 1, b2). The VZ contained a large ventricle- like lumen surrounded by compact layers of vertically aligned elongated cells. The area outside the VZ, the IZ, contained uniform, predominantly round cells. The out- ermost CZ contained horizontally aligned cortical layers (Fig. 1, b2).Structures of the female hESC H9 line-derived orga- noids, internal rosettes, and the cellular organization (Fig. 1), were reproduced using the male hESC HUES8 line (Supplementary Fig. 1b).We investigated the neurodevelopmental stages of cells in each zone by immunostaining for proliferating NPCs with the nuclear Ki67 antigen46, doublecortin (DCX)expressed by neuroblasts, and βIII-tubulin expressed byyoung, immature neurons (NCCs)45. These stainings revealed cellular organization consistent with the inside- out pattern of human neocortex development15 (Fig. 1, b3, b4 and b5). Proliferative Ki67+ cells (Fig. 1, b5, Sup- plementary Fig. 1b3) and GFAP-expressing radial glia (Supplementary Fig. 1b2) were mostly present in the VZ, similar to the developing brain in the ventricular and sub- VZs, where generation of new cells by the brain stem and progenitor cells takes place.

Few Ki67+ cells were found in the IZ and proliferating cells were not detected in the CZ (Fig. 1, b5, Supplementary Fig. 1b3). The doublecortin+ neuroblasts were present in the IZ and the CZ, and theβIII-tubulin+ NCCs were found predominantly in the CZ.Organoids derived from schizophrenia iPSCs—dispersion of proliferating cells in the cortex and blockade of cortical neuronal development. After establishing the protocol for the generation of hESC cerebral organoids, we applied this procedure tohuman iPSCs. To analyze whether early brain develop- ment may be altered in schizophrenia, we used iPSC lines reprogrammed from three schizophrenia and four control individuals, in which common dysregulated tran- scriptomes have been recently identiﬁed29. Our qualitative observations were veriﬁed by computational and statis- tical analyses of three schizophrenia and four control patients and are described below. In general, the iPSC cerebral organoids followed the developmental pattern and displayed cortical rosettes that were similar to the hESC organoids. At 5 weeks, the rosettes of both control and schizophrenia iPSC organoids had only narrow resi- dual lumens (Supplementary Fig. 2). No gross size dif- ferences were observed between control andschizophrenia iPSC organoids. However, a detailed cel- lular analysis revealed marked differences between control and schizophrenia organoids (Fig. 2). The control iPSC organoids, similar to hESC organoids, contained 2–3 layers of proliferating Ki67+ NPCs in the VZ, few Ki67+ cells in the IZ, and none or only single proliferating cells in the CZ. These layers were already observed at week 2 (Fig. 2a) and were further developed at week 5 (Fig. 2b). In contrast, in the 2-week schizophrenia organoids, only one layer of Ki67+ cells was typically present and there were no distinct palisades of proliferating cells surrounding the VZ lumens observed. Instead, the Ki67+ cells were strikingly dispersed in the IZ, as well as in the CZ (Fig. 2a).

Staining with a monoclonal Pan-Neu antibody, which reacts with key somatic, nuclear, dendritic, and axonal proteins of the pan-neuronal architecture, revealeddifferentiated Pan-Neu+ neurons concentrated in the CZ of the control iPSC organoids, forming a distinct cortical layer at 2 weeks (Fig. 2a) and more pronounced at 5 weeks(Fig. 2b). These mature neurons formed a dense network of long processes parallel, as well as perpendicular to the cortical surface. At 2 weeks, the schizophrenia iPSC organoids had noticeably fewer Pan-Neu neurons and Pan-Neu positive dendrites in the CZ (Fig. 2a, Supple- mentary Fig. 2b). Instead, the schizophrenia organoids displayed differentiated Pan-Neu+ neurons deep within the IZ and VZ regions. These mature subcortical neurons were found already at 2 weeks in the schizophrenia organoids, at the time when no such neurons were observed in the control organoids (Fig. 2a, Supplementary Fig. 2b). At 5 weeks, the basal cortical Pan-Neu positive neurons in schizophrenia organoids displayed dense short processes, different from the network of the long pro- cesses formed in the cortex of the control organoids (Fig. 2b). Overall, density of the Pan-Neu ﬁbers in schi- zophrenia appeared reduced. This decrease was veriﬁed by measuring the Pan-Neu ﬂuorescence intensity in the basal CZ, as compared to the IZ. In three patients (compared to three control individuals), we found a sig- niﬁcant reduction in Pan-Neu ﬂuorescence in the basal CZ and no signiﬁcant change in the IZ (Fig. 2c, d).The impaired development of the schizophrenia cortical neurons was accompanied by a visible dispersion of the proliferating NPCs (Fig. 2a, b). To quantitatively asses these changes, we analyzed images of the organoids form three controls and three patients (Fig. 3). Computer-based cell identiﬁcation and automated counting showed a sig- niﬁcant increase in the density of Ki67+ cells/area of the schizophrenia ROIs (Fig. 3b). For each ROI (example in Fig. 3, c1), we generated Minimum Spanning Tree graphs (Fig. 3, c2) (Supplementary Methods). This analysis revealed signiﬁcant increases in the distances between cells, i.e., NPC dispersion, in the schizophrenia organoids. Two-Way ANOVA demonstrated a highly signiﬁcant relation between disease and cell migration (Fig. 3, c3).To quantitatively assess changes involving the Ki67+NPCs, we analyzed images of the organoids of three controls and three patients. Computer-based cellidentiﬁcation and counting showed a signiﬁcant increase in the density of Ki67+ cells/area in the analyzed ROIs (Fig. 2b).

For each ROI, Minimum Spanning Tree graphs were generated (Fig. 2, c1, c2), which showed signiﬁcant increases in the distances between cells, i.e., increased NPC dispersion, in the schizophrenia organoids (Fig. 2, c3). Two-Way ANOVA demonstrated a highly signiﬁcant relation between disease and cell migration.Together, our observations showed proliferation and migration of the schizophrenia NPCs, premature devel- opment of neurons in the subcortical region, and impaired neuronal development in the forming cortex of the schizophrenia organoids. The antibody against autism-linked transcription factor T-Box Brain 1 (TBR1), identiﬁes developing neuroblasts of the subplate (SP) and cortical plate (CP), which provide the ﬁrst pioneer neurons of the developing cerebral cor- tex47. TBR1 is necessary for neuronal differentiation of NPCs and is a potential master regulator in autism spectrum disorders48. At 5 weeks of control iPSC orga- noid development, cells expressing nuclear TBR1 were distributed throughout the entire CZ and IZ (Fig. 4, a1). In contrast, in schizophrenia organoids, TBR1+ cells were absent from the upper cortical region, while cells expressing high levels of TBR1 were found concentrated predominantly in the cortical plate and IZ. The loss of TBR1 expression from the upper cortical layers was documented in all the three schizophrenia organoids, as compared to three controls. Quantitative stereological counting showed 32.2 + 2.0% of DAPI-stained nuclei were positive for TBR1 in organoids from control subjects. This number was signiﬁcantly reduced in the schizophrenia organoids to 17.1 + 1.4 (p < 0.001) (Fig. 4, a2, Supple- mentary Fig. 5c). Thus, impaired development of the cortical neurons is associated with an overexpression of TBR1 in the cortical plate and the absence of the super- ﬁcial pioneer TBR1+ neuroblasts.We next analyzed expression of reelin, a secreted gly- cosylated protein deposited by the neurons of the mar- ginal zone and other neurons in the developing cortex.

Extracellular reelin supplies essential migratory cues to the waves of the developing cortico-petal neuroblasts and immature neurons and for the formation of the cortical layers49. In 2-week control organoids, reelin was not expressed, but at 5 weeks, reelin expression was detected in cells dispersed within the CZ, including its marginal layer, as well as in the IZ (Fig. 4b). Quantitative analysis of reelin immunoﬂuorescence intensity in organoids from three patients and three controls showed a consistent depletion (−40%, p < 0.001) of reelin in the schizophrenia cortex (Fig. 4b, Supplementary Fig. 5b). A small reelin decrease in the IZ was observed in some organoids, but did not attain statistical signiﬁcance (Fig. 4b, Supple- mentary Fig. 5b).Calretinin is a cytoplasmic protein abundantly expres- sed by cortical and retinal interneurons50. At 5 weeks, the calretinin interneurons were abundant in the CZ (Fig. 4c), and less abundant in the IZ in control organoids (Fig. 4e). The density of the calretinin neurons in schizophrenia organoids appeared somewhat reduced, but this change was not signiﬁcant (Supplementary Fig. 3a and b). Also, the length of calretinin neurons, including processes, was not signiﬁcantly different between the control and schi- zophrenia organoids (Fig. 3, c1, c2). Calretinin inter- neurons are known to form horizontal connections between cortical neurons and cortical ﬁelds51. Consistent with this function, calretinin+ processes ran pre- dominantly parallel or near parallel to the cortical surface of the control organoids. In contrast, in schizophrenia organoids, this preferential horizontal directionality was lost. Thus, the formation of connections between cortical ﬁelds by the calretinin interneurons was diminished in the developing schizophrenia cortex.Given the role of cytoplasmic FGFR1 in cell prolifera- tion and nFGFR1 in neuronal differentiation and devel- opment, we analyzed the protein expression of FGFR1 in 2 and in 5-week-old iPSC organoids (Fig. 5).

At 2 weeks, strong FGFR1 expression and high density of FGFR1+ cells, were detected in the VZ of controls, as well as schizophrenia iPSC organoids (Fig. 5a, Supplementary Fig. 4, a4 and a5). Both control and schizophrenia orga- noids displayed a less dense population of FGFR1 expressing cells in the IZ. The CZ cells of control orga- noids expressed FGFR1 (Fig. 5a), with many cells pre- dominantly expressing FGFR1 associated with prominent nuclear speckles (Supplementary Fig. 4, a2). These speckles were previously shown to represent sites of RNA co-transcriptional processing and nFGFR1 interaction with the common transcriptional co-activator, CREB- binding protein (CBP)43.In schizophrenia organoids, FGFR1 expression in the CZ was greatly reduced, as compared to control iPSC organoids at 2 weeks (Fig. 5a; Supplementary Fig. 4, a4 and a5), as well as at 5 weeks (Fig. 5b, Supplementary Fig. 4b and c). Few or no FGFR1-expressing cells were detected in the cortex of the schizophrenia organoids in investigated iPSC lines. In three patients and three con- trols, using multiple sections and ROIs, we counted the percent of DAPI-stained nuclei that were positive for nFGFR1. The number of nFGFR1-expressing cells was reduced from 37% in control organoids to 6.9% in the schizophrenia organoids (p < 0.0001) (Fig. 5c; Supple- mentary Fig. 5d).Supplementary Videos 2a and b further illustrate the expression of FGFR1 in subcortical cells and the loss of FGFR1 in cortical cells of schizophrenia organoids.Thus, the maldevelopment of the cortex in schizo- phrenia iPSC organoids was accompanied by a depletion of FGFR1 in the cortical cells.Speciﬁcally blocking nFGFR1 signaling with a nuclear dominant negative FGFR1(SP-/NLS)(TK-) in hESCs or in human or mouse NPCs blocked their neuronal differ- entiation and development. On the other hand, transfec- tion of a nuclear constitutively active FGFR1(SP-/NLS) was sufﬁcient to induce neuron development, sometimes producing unusually large neurons28,52.

Together these loss and gain of function experiments demonstrated the importance of nFGFR1 homeostasis in neuronal devel- opment. Similar observations, albeit limited by the efﬁ- ciency of in vivo transfection, were made upon nFGFR1 and nuclear FGF2 transfections of mouse brain stem cells53,54.We next inquired whether downregulation of nFGFR1 in differentiating cortical neurons (NCCs) and its robust expression in subcortical NPCs (Fig. 5, Supplementary Fig. 4; overexpression in schizophrenia iPSC-derived NPCs was shown in ref. 55) could affect neuro- ontogenic gene activities. Toward this goal, we analyzed the transcriptomes of homogenous 2D cultures of NPCs derived from H9 hESCs and of their NCC progeny. NCCs were induced by treatment (48 h) of NPCs with cAMP, BDNF, and GDNF29 (see Supplementary Materials and Methods). Loss of nFGFR1 function was instated by transfection of the dominant negative nFGFR1(SP-/NLS) (TK-). Twenty-four hours after transfection, cells were maintained in NPC medium or treated with the differ- entiating factors, AMP/BDNF/GDNF, for an additional 48 h. To model excessive nFGFR1 signaling, NPC cultures were transfected with constitutively active nFGFR1(SP-/NLS) and treated with AMP/BDNF/GDNF. Parallel con- trols were transfected with a β-galactosidase-expressing plasmid.Biological triplicates of each condition were used to isolate RNA and were analyzed independently by RNA- seq using an Illumina HiSeq2500 instrument. The raw data were analyzed by the Tuxedo pipeline and aligned to the UCSC genome hg38build. The expression levels of over 24,331 genes were assessed and considered sig- niﬁcant (16,137 genes) when expressed in all samples (Supplementary Table S2). The remaining 8194 genes were not detected in all samples and were eliminated from further analysis due to their near-threshold expression. Atotal of 4704 genes showed expression levels that differed signiﬁcantly (FC ≥ ± 1.5 and q-value > 0.05) between nondifferentiated NPCs and their NCC progeny inducedby differentiating factors (cAMP/BDNF/GDNF) (Supple- mentary Table S3).

Three hundred and thirty-two genes were differentially expressed between NPCs and NPCs+ FGFR1(SP-/NLS(TK-) (Supplementary Table S4). Expression of 861 genes differed signiﬁcantly in NCCspre-transfected with FGFR1(SP-/NLS)(TK-), as compared to β-galactosidase (Supplementary Table S5). In NCCs, 440 genes were affected by transfection of active nFGFR1(SP-/NLS).Gene ontology (GO) analysis revealed many develop- mental categories overrepresented by the genes that were differentially expressed in nondifferentiated NPCs and NCCs (Table 2). The overrepresented categories included development of the nervous system, of the brain and its parts, cell pluripotency, proliferation, neuronal differ- entiation, axonal guidance and growth, synapseformation, glial development, and neuronal apoptosis. The same categories, with the exception of glial devel- opment, were overrepresented by genes affected by FGFR1(SP-/NLS)(TK-) in NPCs and/or NCCs. Thus, nFGFR1 appears to speciﬁcally control neuronal NPC development. Overexpression of active nFGFR1(SP-/ NLS), along with the cAMP/BDNF/GDNF stimulation, affected genes of brain development, cell proliferation, neurogenesis, neuronal differentiation, and apoptosis. Thus, both insufﬁcient and excessive nFGFR1 signaling disrupt neuro-ontogenic gene programs.The ontological gene categories affected by cAMP/ BDNF/GDNF, FGFR1(SP-/NLS)(TK-) and/or FGFR1(SP-/NLS) included also general multicellular organism development, morphogenesis, organ, cardiovascular, and limb development, cell–cell signaling, retinoic acid, and 2nd messenger signal transduction (Table 2). These ﬁndings match the widespread gene targeting by nFGFR119,29 and its proposed pan-ontogenic function20. Also, consistent with the established transcriptional functions of nFGFR118, genes which were affected by FGFR1(SP-NLS)(TK-) and FGFR1(SP-/NLS) over-represented the functional category of RNA PolII- mediated transcription (Table 2).The ingenuity pathway analysis (IPA) (a proprietary curation of pathways by Qiagen that estimates gene overrepresentation within speciﬁc pathways), revealed FGFR1(SP-/NLS)(TK-) dysregulation of many genes in NPCs and/or NCCs involved in pathways controlling neuronal and brain development, which were regulated during the cAMP/BDNF/GDNF-induced NCC differ-entiation (Table 3). These pathways included Wnt/β catenin signaling, axonal growth and guidance, ephrin, G-protein signaling, cAMP signaling, PKA signaling, CREB signaling, ERB tyrosine kinase signaling, growth hormone receptor signaling, interleukin signaling, prolactin, etc. (Table 3).

In addition, inhibition with FGFR1(SP-/NLS) (TK-) revealed nFGFR1 regulation of additional develop- mental pathways, which were not affected by cAMP/ BDNF/GDNF: Notch, Wnt/Ca++, neurotrophins/TRK, ERK/MAPK, reelin, ephrin, glucocorticoid receptors,GDNF and PDGF, as well as cell cycle and p53 controlling pathways. Overexpression of constitutively active nFGFR1 (SP-/NLS) in NCCs affected genes in Notch, Ca++, GH, EGFR, CXCR4, pluripotency, and glucocorticoid signal- ing, neurotrophin/Trk receptors, NGF, Erk/MAPK sig- naling, ephrin signaling, FGF signaling, p53, and RAR signaling. Blocking nFGFR1 signaling with FGFR1(SP-/ NLS)(TK-) and/or activation with FGFR1(SP-/NLS) affected genes in pathways involved in neuronal functions, such as GABA receptor signaling, calcium, melatonin, LTP, and LTD (Table 3). Thus, loss of nFGFR1 and excessive nFGFR1 signaling lead directly to dysregulation of major developmental pathways.Graphs of selected nFGFR1-dependent pathways and their affected genes are shown in Supplementary Fig. 6.Genome function is organized into highly coordinated and dynamically changing networks of genes. To detect such networks among the FGFR1-controlled genes, we performed a pairwise correlation29 of all differentially regulated genes. Among 4704 genes that were regulated during NPC differentiation to NCCs, we detected groups of genes that displayed high-positive correlation (+0.9 to+1.0; changing activities in the same direction) and genes which showed high-negative correlation (−0.9 to −1.0; changing activities in opposite direction) (Supplementary Fig. 7a). In each category, the 200 most connected genes from the differentiated NCCs were analyzed using circular network graphs. A common feature of both positive and negative networks was that their top 200 coordinate genes were highly interconnected in NCCs (Supplementary Fig. 7b, c).

These networks formed during NCC differ- entiation as they showed little or no connectivity in NPCs. We then analyzed network formation by 861 NCC genes, which were affected by FGFR1(SP-/NLS)(TK-) (Fig. 6). Signiﬁcantly, the networks formed by the top 200 positively and 200 negatively correlated genes in controlβ-galactosidase transfected NCCs were deconstructed in the NCCs transfected with FGFR1(SP-/NLS)(TK-)(Fig. 7). Genes forming these nFGFR1-dependent positive and negative correlative networks represented several of the neuro-ontogenic categories (Fig. 6).FGFR inhibitor, PD173074, models loss of nFGFR1 and cortical maldevelopment in hESC cerebral organoidsGiven the broad neuro-ontogenic functions of nFGFR1 in NPCs and NCCs, we next asked if the loss of nFGFR1 function could arrest the ongoing cortical development in control hESC organoids, similar to as observed in cortical cells of the schizophrenia organoids. We used PD173074, which speciﬁcally inhibits FGFRs (R1 > R2 > R3 > R4)56. Since fgfr1 was the highest expressed fgfr gene in NPCs and NCCs (Supplementary Table 2; 1.0FGFR1 > 0.16FGFR2> 0.26FGFR3 > 0.008FGFR4), the effects of PD173074 wouldlikely reﬂect the FGFR1 blockade. After the initial 8 days of cortical development, structurally stable hESC cerebral organoids were incubated with 100 nM PD173074 or indrug-free medium for an additional 10 days, during the ongoing CZ expansion.In general, many changes induced by PD173074 treated hESC H9 organoids were similar as in the schizophrenia iPSC organoids. Expression of the cortical TBR1 marker of early pioneer neuroblasts, was abolished by PD173074 (Supplementary Fig. 8a, b). Consistent with these ﬁndings, the highly abundant doublecortin+ neuroblasts (Fig. 7a) and the calretinin+ interneurons (Fig. 7b) in the IZ and CZ of day 18 control organoids were markedly depleted by the PD173074 treatment.To determine if FGFR1 inhibition affected the ongoing cortical neuronogenesis, we pulse labeled dividing cells with BrdU in 14-day organoids (6th day of PD173074 treatment). After an additional 4 days, BrdU-labeled control or PD173074-treated organoids were sectioned and co-immunostained with rat anti-BrdU and with mouse anti-Pan-Neu.

In control organoids, the BrdU- labeled cells were found in the VZ, as well as migratedCells were counted in identical seven ROIs in sections of control and PD173074- treated organoids. Numbers represent mean + SEM; % of cells in rosettes and cortical ROI are shown.Pairwise comparisons were done using t-test with unequal variancesFGFR1 subcellular distribution in the CZ of hESC H9 cerebral organoids. In several independent experiments, the expression of FGFR1 in control hESC H9 organoids showed similar patterns (Fig. 8a) as in the control iPSC organoids (Fig. 5a). FGFR1 was highly expressed in the VZ cells (Fig. 8a). The FGFR1 staining was less intense in the IZ and stronger again in the CZ. In many CZ cells, FGFR1 localized within the cell nuclei (Fig. 8a) and in prominent nuclear speckles (Fig. 8, b1). To quantify the changes in FGFR1 subcellular distribution, we imaged DAPI and FGFR1 co-stained sections using confocal microscopy (Fig. 8b). In the CZ of control organoids, 52% of cells had nFGFR1 colocalized with DAPI. In PD173074-treated organoids there was over a three-fold reduction in the number of cells with nFGFR1 in the CZ (Fig. 8c), likely reﬂecting loss of the positive fgfr1 gene activation by its protein.

Discussion
In this study, we have expanded cerebral organoids as an experimental platform to analyze the early human cortical development in healthy and disease states and to investigate disease mechanisms. Using the protocol of Lancaster et al.,40 in which the ﬁrst step, an efﬁcient generation of EBs, was modiﬁed as in Brennand et al.,33 we successfully generated cerebral organoids from hESCs (H9 and HUES8 lines) and from seven human iPSC lines. We observed time-dependent generation and develop- ment of organoids, recapitulating the inside-out pattern of human cortical development. The VZ in the center of the rosettes contained proliferating GFAP+ radial glia, the IZ contained migrating doublecortin+ neuroblasts andimmature βIII-tubulin+ neurons, and the CZ incorpo- rated pioneer neurons (TBR1+), and built cortical-like layers of the βIII-tubulin+ and Pan-Neu+ neurons, and calretinin+ interneurons. We employed quantitative cellcounting, image intensity measurements, and computa- tional tools to quantify and analyze normal and patholo- gical tissue samples.An important observation made repeatedly during this study was that the cellular structures and zones of the organoid rosettes and their development were con- sistently reproducible among hESC H9, HUES8, and the four control iPSC line organoids, but were noticeably disturbed in all schizophrenia iPSC organoids and afterpharmacological inhibition of FGFR1. In the organoids developed from either hESCs or control iPSCs, the pro- liferating cells were restricted to 2–3 layers surrounding the lumen of the VZ rosettes. Relatively few cells that migrated to the IZ remained in a proliferative state, but essentially, no such cells were found in the CZ. One of the most striking features of the schizophrenia iPSC orga- noids was the disruption of the developmental strata. Speciﬁcally, we observed signiﬁcantly increased pro- liferation and movement of Ki67+ NPCs into the IZ, and development of atypically placed deep subcortical neu- rons. These changes were accompanied by signiﬁcant loss of the TBR1 pioneer neurons from the top cortical layers, while they remained abundant deep in the IZ. Together, these ﬁndings illuminated an abnormal subcortical neu- ronogenesis in schizophrenia tissue and were consistent with the increased, premature neuronal generation, pre- dicted from the transcriptome studies of the NPCs derived from schizophrenia iPSCs29.

The expanded subcortical neurogenesis was accom- panied by opposite changes in the CZ, where fewer mature neurons developed in schizophrenia organoids. This was shown by a signiﬁcant reduction in cortical Pan- Neu intensity and was corroborated by visibly diminished Pan-Neu ﬁbers in the schizophrenia organoids. The spe- ciﬁc depletion of the neurons in the cortex, but not in the IZ, may reﬂect the loss of cortical signals that guide the cortico-petal migration, i.e. reduced cortical deposition of reelin and pioneer TBR1 neurons. The altered intracor- tical connectivity in schizophrenia organoids was further denoted by the changes in the orientation of the calretinin+ interneurons. Unlike the horizontal organization of calretinin+ neurites in the control cortex, the schizo- phrenia interneurons displayed a random directionality of neurites, indicating perturbed intercortical connectivity. The relationship between glutamatergic pyramidal neu- rons and GABAergic interneurons (of which calretinin- positive neurons are a subtype of GABAergic inter- neurons), has been suggested in relation to thedevelopment of schizophrenia in humans58. So far, attempts to count calretinin neurons in the schizophrenia brain showed lack of signiﬁcant numerical changes59. Our study suggests that changes in interneuron directionality may disrupt cortical connections and communication in schizophrenia.In summary (Fig. 8d), the impaired development of cortical neurons, interneurons, and cortical connections in schizophrenia may be instigated by reduced cortico- petal migration of the neuroblasts and immature neurons. In addition, the disruption of cortical neuronal pro- graming in schizophrenia organoids coincided with the presence of the Ki67+ proliferating cells in the CZ, sug- gesting that these cells have failed to exit the cell cycle and differentiate.The impaired cortical development of schizophrenia organoids correlated with a striking loss of nFGFR1 from the CZ cells observed already at 2 weeks and was veriﬁed by a statistically signiﬁcant loss in 5-week organoids of all investigated patients.

The loss of nFGFR1 was limited to cortical cells, as FGFR1 remained highly expressed in the VZ and IZ of schizophrenia organoids throughout the 5 weeks of development. Given the well-established role of nFGFR1 in cell differentiation and neuronal develop- ment18,20 (-reviews), the loss of nFGFR1 from the nuclei of differentiating cortical neuronal cells in schizophrenia organoids likely underlies the impaired neuronal differ- entiation and cortical maldevelopment observed in schi- zophrenia. Furthermore, blocking of nFGFR1 in NPCs and NCCs impacted a number of the key ontogenic and neuro-ontogenic gene categories involved in neuronal development, forebrain development, cell adhesion and migration, axon formation, and guidance and in the established neuro-ontogenic pathways. Our genomic analyses also revealed that the loss of nFGFR1 function in NPCs affected several of the reelin signaling genes (Sup- plementary Fig. 6c), which together with the loss of reelin,denotes the malfunctioning reelin mechanism in the schizophrenia cortex.The importance of these genomic ﬁndings transcends the current investigation centered on schizophrenia. Demonstration that the developmental gene programs known to be targeted directly by nFGFR119,29 are indeed regulated by the nuclear receptor, and further sub- stantiates the panontogenic-INFS theory20.In addition to investigating the loss of nFGFR1 function, our experiments showed that overactive nFGFR1 also affects the neuro-ontogenic programs. Excessive nFGFR1 gene targeting was shown in schizophrenia NPCs during their differentiation29. Thus, an over-activation by nFGFR1 of neurodevelopment gene programs in sub- cortical NPCs could lead to their premature migration and differentiation observed in the subcortical regions of the schizophrenia organoids.Within the list of the nFGFR1-regulated genes (Sup- plementary Table 7), ∼75% were previously shown to bind to nFGFR1 in control and/or schizophrenia iPSC-deriveddifferentiating NCCs29. Thus, the effects of transfected dominant negative and constitutively active nuclear forms of FGFR1 predominantly reﬂect direct gene control by the nuclear receptor.As nFGFR1 has been designated as a central neuro- ontogenic factor19,20, we propose that the depletion of cortical nFGFR1 in schizophrenia organoids leads to dysregulation of the key neuro-ontogenic programs and, consequently, arrests cortical neuronal development.

Indeed, the inhibition of FGFR1 signaling (the pre- dominant FGFR expressed) with PD173074, depleted nFGFR1 in the CZ of control hESC organoids, and inhibited cortical development, similar to as observed in the schizophrenia organoids. The BrdU pulse-chase experiment showed that blocking FGFR1 reduced cor- tical migration of new born cells and their differentiation to neurons. Furthermore, PD173074 exposes the role of FGFR1 in the formation of doublecortin expressing neu- roblasts and TBR1 expressing neuroblasts/pioneer neu- rons. It also demonstrated the importance of FGFR1 signaling in the development of calretinin interneurons.The early cortical malformations revealed by this organoid study could underlie the disruption of cortical layers, cell paucity, hypoplasia, abnormal connections, and white matter tracks observed in adult schizophrenia brains by pathological examination and/or in vivo imaging17,60.Why do schizophrenia symptoms appear in adolescence or young adulthood if they are a consequence of early prenatal brain maldevelopment? In schizophrenia patients, the malformations are often found in the pre- frontal cortex60,61, in which myelination and maturation occur at the time when the ﬁrst severe symptoms emerge.As the prefrontal cortex becomes increasingly incorpo- rated into the human brain circuitry, its developmental malformations may reveal itself in a complex clinical syndrome. Perhaps, the most severe forms of childhood schizophrenia, with abundant hallucinations and motor dysfunctions62, arise also in the cortical regions, which mature earlier and are home to the sensory and motor functions63. These and other compelling questions await further investigation.

In the present study, we observed similar anatomical changes in the organoids of all three patients that have distinct genetic backgrounds and DNA sequences, which could be linked to schizophrenia. However, the iPSC- derived NPCs all showed a common disruption of 1376 genes in the developmental transcriptome29. Thus, our studies reinforce the watershed model of schizophrenia, whereby different mutations lead to the common dysre-gulation of the neuroontogenic genome and to the common cortical maldevelopment. Our ﬁndings designate INFS as a common important mechanism in the disease. According to the model shown in Fig. 8d, nFGFR1 hyperactivity leads initially to the excessive migration of proliferating NPCs and then their premature differentiation within subcortical regions. Later, during carcinogenesis, turning off nFGFR1 in the schizophrenia cortical cells disrupts their further development. Future investigation, including restoration of INFS in the developing schizophrenia cortex, will test this hypothesis and potentially be used in preventive FIIN-2 therapies.