Cerebral organoids recapitulate cellular processes of cortical development

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 immature βIII-tubulin positive neurons in the outer layers (Fig. 1, a3, a4).

Fig. 1: Representative hESC (H9) cerebral organoids at (a) 8 and (b) 18 days of development (a1, b1) tile scanning of DAPI; yellow arrows point to cortical rosettes, enlarged in a2 and b2; at day 18, zones are outlined—ventricular zone (VZ), intermediate zone (IZ), and cortical zone (CZ). Immunostaining: (A4, B5) Ki67+ proliferating cells; (a3, b3, b4), doublecortin (DCX+) neuroblasts, βIII-tubulin+ immature neurons Full size image

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 outermost CZ contained horizontally aligned cortical layers (Fig. 1, b2).

Structures of the female hESC H9 line-derived organoids, 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 by young, 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, Supplementary 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 the βIII-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 to human iPSCs. To analyze whether early brain development may be altered in schizophrenia, we used iPSC lines reprogrammed from three schizophrenia and four control individuals, in which common dysregulated transcriptomes have been recently identified29. Our qualitative observations were verified by computational and statistical 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 residual lumens (Supplementary Fig. 2). No gross size differences were observed between control and schizophrenia iPSC organoids. However, a detailed cellular 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).

Fig. 2: Disorganized migration of proliferating cells and depletion of cortical neurons in schizophrenia iPSC cerebral organoids Organoids were coimmunostained for Ki67 (red) and Pan-Neu (green). Nuclei were stained with DAPI (blue). a 2-week organoids—images show representative sections of organoids, control (iPSC line BJ1) and schizophrenia (iPSC line 1835). In schizophrenia organoids, note the dispersion of proliferating (Ki67+) cells outside the VZ into IZ and CZ, fewer mature Pan-Neu+ neurons in CZ, and the appearance of Pan Neu+ neurons in the IZ. b 5-week iPSC organoids: control (line 2937) and schizophrenia (line 2038)—representative images of control and schizophrenia organoids. In schizophrenia organoids, note dispersion of Ki67+ cells into CZ, reduced density of Pan Neu+ neurites in basal CZ and the presence of Pan Neu+ cells with neurites in the IZ. 3D rotational confocal images of control (line 3651) and schizophrenia (line 1835) organoids are shown in Video 1a and b. Pan-Neu immunofluorescence intensity was measured in multiple randomly selected ROI (1 × 102 μm2 in basal cortex (*) and in IZ (**)). c 5-week organoids—Pan-Neu immunofluorescence intensity was measured in several ROIs (# shown on y-axis) of multiple organoids from three control and three schizophrenia patients. Note, significantly reduced Pan-Neu fluorescence intensity in basal cortex of the schizophrenia organoids and the lack of significant changes in the IZ. d Distribution of Pan-Neu intensity numbers in analyzed ROIs. Note the significant separation of the basal cortex plots in control and schizophrenia organoids and the lack of separation of the IZ plots Full size image

Staining with a monoclonal Pan-Neu antibody, which reacts with key somatic, nuclear, dendritic, and axonal proteins of the pan-neuronal architecture, revealed differentiated 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, Supplementary 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 processes formed in the cortex of the control organoids (Fig. 2b). Overall, density of the Pan-Neu fibers in schizophrenia 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 significant 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 significant 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).

Fig. 3: Quantification of disorganized migration of proliferating NPCs in schizophrenia compared to control cerebral organoids a Exemplary images showing Ki67+ (red) proliferating NPCs in the center of the rosette of a control organoid (line 2937) and their dispersion in a schizophrenia organoid (line 2038) (nuclei were stained with DAPI). b Increased density of proliferating cells in schizophrenia organoids. ROIs were outlined on organoid images from three control and three schizophrenia patients, as shown in (c1). Bar graph shows significantly higher average numbers of the KI67+ proliferating cells in schizophrenia ROIs than in control ROIs (17 control and 20 schizophrenia ROIs quantified). c Global Minimum Spanning Tree (MST) analysis of Ki67+ NPC dispersion within ROIs (c1—examples) was carried out using 17 control and 20 schizophrenia ROIs from three control cases and three schizophrenia patients (total of 649 and 1070 cells analyzed, respectively). The shortest connecting edges between cells were identified in pixels (c2) using MST calculating program and were grouped into bins (c3). Bin 1 contains edges of 0–5 pixels, bin 2 of 5–10 pixels, etc. Frequency indicates average numbers of cells per bin in all ROIs measured. Schizophrenia organoids displayed a shift towards longer MST distances. Two-Way ANOVA showed a significant interaction between organoid phenotype (control vs. disease) and the MST distances Full size image

To quantitatively assess changes involving the Ki67+ NPCs, we analyzed images of the organoids of three controls and three patients. Computer-based cell identification 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 development of neurons in the subcortical region, and impaired neuronal development in the forming cortex of the schizophrenia organoids.

Changes in NPCs in schizophrenia iPSC 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 cortex47. TBR1 is necessary for neuronal differentiation of NPCs and is a potential master regulator in autism spectrum disorders48. At 5 weeks of control iPSC organoid 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, Supplementary 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 superficial pioneer TBR1+ neuroblasts.

Fig. 4: (a) Decreased nuclear TBR1 (red) expression in the upper cortical zone of 5-week schizophrenia organoids Nuclei were stained with DAPI. Images show representative sections of control (iPSC line BJ1) and schizophrenia (iPSC line 2038) organoids. Total number of DAPI-stained nuclei and the number of nuclei expressing TBR1 were counted in multiple randomly selected ROI (5 × 103 μm2, ∼50 cells/ ROI) within the upper cortical layers (*6 cells deep) of three control individuals and three patients. Percent of (TBR1 + DAPI)/DAPI-stained nuclei was determined for each ROI. Graph shows distribution of the % of TBR1 expressing cells in the individual ROIs (26 control and 33 schizophrenia ROIs). The difference between control and schizophrenia mean values was significant (t-test). Individual value plots are shown in Supplementary Fig. 5b. b Decreased reelin expression in schizophrenia organoid cortex. Images show control (BJ1) and schizophrenia (1835) organoids. Note the lack of reelin staining in 2-week organoids. In 5-week organoids, reelin immunofluorescence intensity was determined in randomly selected ROIs (3 × 103 μm2) in the upper CZ (*) and in the IZ (**) regions of three control individuals and three patients using Zen 2.0 Blue Imaging software (22 control and 17 schizophrenia upper CZ ROIs and the same number of IZ ROIs). ANOVA of four groups followed by Tukey posthoc test showed a significant decrease in the reelin expression in the schizophrenia upper CZ and a lack of significant differences between control and schizophrenia in the IZ. Individual value plots are shown in Supplementary Fig. 5. c Morphology and orientation of cortical calretinin interneurons. c1—images of control and schizophrenia organoids. A total of 770 control and 547 schizophrenia calretinin interneurons were measured in 20 and 16 ROIs, respectively, in the organoids from three control and three schizophrenia patients. The average cell density (d = number of cells/ROI) was not significantly different between control and schizophrenia (Supplementary Fig. 3a, b). c2—graph shows cell distribution (cumulative frequency) relative to their total length, including the cell body and neurites. An average cell body had a length of ∼50 pixels, 18 μm. A two-sample Kolmogorov–Smirnov test of cumulative density function (CDF shown in the inset) of control and schizophrenia groups found no significant difference between the length of control and schizophrenia interneurons. c3—angles between the long axis of each cell and the cortical surface organoids were computed as described in the Supplementary Methods. Graph shows distribution of cells (cumulative frequency) in bins corresponding to the deviation angles from the cortical surface. A two-sample Kolmogorov–Smirnov CDF test (CDFs shown in the inset) of control and schizophrenia groups yielded a highly significant difference (p-value of <13.9 × 10−7) between the orientation of control and schizophrenia interneurons, relative to the cortical surface Full size image

We next analyzed expression of reelin, a secreted glycosylated protein deposited by the neurons of the marginal 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, Supplementary Fig. 5b).

Loss of directionality of calretinin interneurons in schizophrenia organoids

Calretinin is a cytoplasmic protein abundantly expressed 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 schizophrenia organoids (Fig. 3, c1, c2). Calretinin interneurons are known to form horizontal connections between cortical neurons and cortical fields51. Consistent with this function, calretinin+ processes ran predominantly 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.

Expression of FGFR1 protein is reduced in the cortex of schizophrenia organoids

Given the role of cytoplasmic FGFR1 in cell proliferation and nFGFR1 in neuronal differentiation and development, 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 organoids displayed a less dense population of FGFR1 expressing cells in the IZ. The CZ cells of control organoids expressed FGFR1 (Fig. 5a), with many cells predominantly 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.

Fig. 5: High expression of nuclear (n)FGFR1 in subcortical cells and the loss of nFGFR1 in cortical cells of schizophrenia organoids. a 2-week organoids: control (iPSC line BJ1) and schizophrenia (iPSC line 1835). Schizophrenia organoids have high FGFR1 expressing cells in VZ and dispersed in IZ. Few nFGFR1+ cells are present in CZ of the schizophrenia organoids. Images of whole sections are shown in Supplementary Fig. 4, a4 and a5. b 5-week organoids—control (BJ1) organoids express nFGFR1 in CZ and IZ (inset shows negative control—omitted primary FGFR1 antibody), and schizophrenia (1835) organoids show depletion of FGFR1 immunostaining in CZ. Arrow points to nuclei with FGFR1 speckles. 3D rotational confocal images of control (line 3651) and schizophrenia (line 1835) organoids are shown in Video 2a and b. c Quantification of the % of DAPI-stained nuclei that were immunopositive for nFGFR1 in multiple randomly selected ROI (3 × 103 μm2, ∼40 cells/ ROI) in the upper CZ. The % of nFGFR1+ DAPI-stained nuclei was determined for multiple ROIs from the three control individuals and three patients. The difference between control and schizophrenia mean values was significant (t-test). Plots show distribution of the % of nFGFR1 positive nuclei in individual control (18 ROIs) and schizophrenia (12 ROIs). The individual value plots are shown in Supplementary Fig. 5d Full size image

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 controls, 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; Supplementary 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 schizophrenia iPSC organoids was accompanied by a depletion of FGFR1 in the cortical cells.

Loss of nFGFR1 signaling affects developmental genome programing

Specifically blocking nFGFR1 signaling with a nuclear dominant negative FGFR1(SP-/NLS)(TK-) in hESCs or in human or mouse NPCs blocked their neuronal differentiation and development. On the other hand, transfection 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 development. Similar observations, albeit limited by the efficiency 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 differentiating 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 controls 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 significant (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. A total of 4704 genes showed expression levels that differed significantly (FC ≥ ± 1.5 and q-value > 0.05) between nondifferentiated NPCs and their NCC progeny induced by differentiating factors (cAMP/BDNF/GDNF) (Supplementary 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 NCCs pre-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 developmental 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 differentiation, axonal guidance and growth, synapse formation, glial development, and neuronal apoptosis. The same categories, with the exception of glial development, 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.

Table 2 Gene ontology analysis shows genes regulated between NPCs vs. NCCs, and genes affected by dominant negative nuclear FGFR1(SP-/NLS)TK-) or constitutively active nuclear FGFR1(SP-/NLS) (RNAseq) Full size table

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) overrepresented 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 differentiation (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 developmental 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 signaling, neurotrophin/Trk receptors, NGF, Erk/MAPK signaling, 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.

Table 3 Ingenuity pathway analysis (IPA)—selected IPA identified pathways that were regulated during cAMP/BDNF/GDNF-induced neuronal programming (NPC vs. NCC) and genes affected by dominant negative nuclear FGFR1(SP-/NLS)TK-) or constitutively active nuclear FGFR1(SP-/NLS) (RNAseq) Full size table

Graphs of selected nFGFR1-dependent pathways and their affected genes are shown in Supplementary Fig. 6.

Deconstruction of mRNA networks by loss of nFGFR1 signaling

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 differentiation 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 control β-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).

Fig. 6: (a) Histogram of pairwise mRNA correlations Correlation was performed using three controls and three patients and triplicate cell samples. NPCs were transfected with control DNA or FGFR1(SP-/NLS)(TK-) and 24 h later were stimulated for 48 h with neuronal differentiation inducing media with cAMP/BDNF/GDNF (NCCs). Genes (861), which were affected by dominant negative nuclear FGFR1(SP-/NLS)(TK-) were analyzed. Genes that showed the highest positive (+0.9 to +1.0) correlations (changing in the same direction) are represented by the gray bar. Genes that showed the highest negative (−0.9 to −1.0) correlations (changing in the opposite directions) are shown as a black bar. b, c Among the FGFR1(SP-/NLS)(TK-) regulated genes, top 200 of the positively correlated genes (b) and top 200, which were negatively correlated genes (c) were selected for the circular network analysis. Gray lines link pairs of genes whose correlation is greater than 0.9. In the control β-galactosidase set, three separate networks were formed. In the FGFR1(SP-/NLS)(TK-) transfected cells, two weakly correlated networks and few individual correlated genes are observed. GO categories overrepresented by 200 top connected genes are listed Full size image

Fig. 7: Treatment with PD173074 (days 8 and 18) affects cortical development in hESC H9 organoids a Double staining for DAPI and doublecortin (DBX, neuroblasts). b PD173074 reduces expression of calretinin in hESC organoids. c BrdU pulse-chase experiment. c—control hESC organoids, d—PD173074 treated (days 8 and 18) organoids. PD173074 inhibits cortical migration and neuronal differentiation of newborn cells in hESC organoids. Sections were coimmunostained for BrdU (red), Pan-Neu (green), and DAPI (blue). Merged and individual stains are shown. Inhibition of FGFR1 with PD173074 inhibits migration and formation of new BrdU+ cortical neurons in the CZ Full size image

FGFR inhibitor, PD173074, models loss of nFGFR1 and cortical maldevelopment in hESC cerebral organoids

Given 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; 1.0 FGFR1 > 0.16 FGFR2 > 0.26 FGFR3 > 0.008 FGFR4 ), the effects of PD173074 would likely reflect the FGFR1 blockade. After the initial 8 days of cortical development, structurally stable hESC cerebral organoids were incubated with 100 nM PD173074 or in drug-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 migrated into the IZ and CZ (Fig. 7c). Counting cells in multiple rosettes showed BrdU+ cells abundantly present in all zones of the control organoids in the following order: VZ > IZ > CZ. In contrast, in PD173074-treated organoids, BrdU+ cells were most abundant in the IZ with only few cells present in the cortex (IZ > VZ > CZ) (Fig. 7c and Table 4).

Table 4 Effect of PD173074 on distribution of BrdU+ cells and double labeled BrdU+ plus Pan-Neu+ cells in rosettes and in the cortical zone Full size table

In control organoids, BrdU and Pan-Neu colocalized predominantly in the same cells of the marginal CZ (Fig. 7c, Supplementary Fig. 9a), indicating that the dividing VZ cells had migrated out to cortical layers and differentiated into neurons. Treatment with PD173074 markedly altered this process (Fig. 7c, Supplementary Fig. 9b). The BrdU+ cells remained largely arrested in the IZ. Relatively few BrdU+ cells that were found in the CZ differentiated to Pan-Neu positive neurons (Table 4). Thus, blocking FGFR1-inhibited cortical migration of the newborn cells and formation of mature neurons.

PD173074 was shown previously to reduce nuclear nFGFR1 accumulation observed in pancreatic tumors57. Hence, we examined the PD173074 effect on FGFR1 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 protein19,57.