Autism genes converge on asynchronous development of shared neuron classes

May combine two claims.

Under the network-burst criteria, 89.3 ± 14% of segmented cells in control organoids and 95.5 ± 6.8% in mutant organoids participated in network bursting.

“Under these criteria, 89.3 ± 14 % (range from 60.5 % to 100 %) and 95.5 % ± 6.8 % (range from 77.6 % to 100 %) of segmented cells participated in network bursting in control and mutant organoids, respectively.”

May combine two claims. The quote only partly states this claim.

The three mutations share little overlap in DEGs/DEPs, indicating distinct molecular targets.

“DEGs caused by different mutations rarely overlapped”

May combine two claims. The quote only partly states this claim.

SUV420H1+/− reduces spontaneous network burst frequency and duration.

“Mutants showed a relative reduction in both frequency”

May combine two claims.

Western blot analysis showed presence of SUV420H1, ARID1B and CHD8 protein expression in control lines, and its reduction in the mutant lines.

“Western blot analysis showing presence of SUV420H1 (d), ARID1B (e) and CHD8 (f) protein expression in control lines, and its reduction in the mutant lines.”

The quote only partly states this claim.

In HUES66 CHD8+/- organoids at 109 days in vitro, WGCNA modules from the partition of interest differed between mutant and control cells (adjusted p-values from linear mixed models).

“Adjusted p-values show differences between control and mutant based on linear mixed models (see Methods).”

May combine two claims.

In Mito210 ARID1B+/- organoids at 90 days in vitro, two of three mutants had more GABAergic interneurons than controls, while none of three controls did (adjusted p = 0.19, logistic mixed models).

“Two out of three mutant organoids show an increase in GABAergic interneurons, vs. zero out of three controls (adjusted p = 0.19, logistic mixed models).”

May combine two claims.

Most cell types had very few significantly differentially accessible regions (range 6-34), except apical radial glia cells, which had 515 differentially accessible regions.

“We noticed that most cell types had very few significantly differentially accessible regions (range 6-34, except for apical radial glia cells, the most prevalent, and thus most powered, cell type at this time point, which had 515 differentially accessible regions)”

May combine two claims. The quote only partly states this claim.

SUV420H1+/− mutants show consistent GABAergic neurons at one month; controls have rare or absent GABAergic neurons until ~3.5 months.

“mutants showed a consistent presence of GABAergic neurons in all backgrounds”

May combine two claims. The quote only partly states this claim.

GABAergic phenotype severity in SUV420H1 varies across lines: Mito294 most severe, PGP1 intermediate, Mito210 mildest.

“the Mito294 SUV420H1 line showed the most dramatic increase in GABAergic neurons”

Probably not a substantial claim: may not pass S3 (load-bearing).

As a control, GO term enrichment for 100 random gene sets of the same size sampled from genes expressed in each dataset found no significant enrichment of these terms.

“As control, we calculated GO term enrichment for 100 random gene sets of the same size sampled from genes expressed in each dataset, and found no significant enrichment of these terms (see Methods).”

Probably not a substantial claim: may not pass S3 (load-bearing).

At one month, GABAergic populations included neurons expressing broad GABAergic markers, GABAergic neuron progenitors, and cycling GABAergic neuron progenitors.

“For the GABAergic populations, one month organoids included neurons expressing broad markers of GABAergic identity (labelled as “GABAergic neurons”), progenitor cells expressing markers of GABAergic lineage identity (“GABAergic Neuron Progenitors”), and progenitor cells with high expression of cell cycle markers in addition to the progenitor identity markers (“Cycling GABAergic Neuron Progenitors””

May combine two claims. The quote only partly states this claim.

Mito210 SUV420H1+/− shows increased immature deep-layer projection neurons at one month, rescued by three months.

“the milder GABAergic phenotype allowed us to detect an increase in immature deep-layer projection neurons”

May combine two claims. The quote only partly states this claim.

Mito210 ARID1B+/− shows increased GABAergic neurons/progenitors and reduced newborn deep-layer projection neurons at one month.

“Mito210 ARID1B+/− organoids showed a consistent increase in the proportions of GABAergic neurons and their progenitors”

May combine two claims. The quote only partly states this claim.

HUES66 CHD8+/− shows increased GABAergic interneurons/progenitors at 3.5 months, persisting at six months.

“Mutants had an increased number of GABAergic interneurons and their progenitors”

Classified as probably not a substantial claim. May combine two claims.

To determine if PPI-weighted protein set distance was smaller than expected by chance, size-matched random sets were chosen from detected proteins and distance calculated 1000 times per pair, with P-values assigned by counting fractions.

“To determine if this distance was smaller than would be expected by chance, size-matched sets were randomly chosen from the proteins detected in each experiment, and distance between these random sets was calculated 1000 times per pair. P-values were assigned by counting the fractions of times this random distance was less than the actual distance value between differential sets.”

Classified as probably not a substantial claim. The quote only partly states this claim.

All datasets satisfied the assumptions required by the statistical tests used.

“All datasets met the assumptions of the applied statistical tests.”

Probably not a substantial claim: may not pass S3 (load-bearing).

After exclusions, 112 single organoids passed quality control and were considered in downstream analysis, with a total of 749,370 cells.

“This left a total of 112 single organoids that passed quality control and were considered in downstream analysis, with a total of 749,370 cells.”

Classified as probably not a substantial claim. May combine two claims.

For proteomic analysis, GO terms and KEGG pathways were calculated using GSEA, and FDR q-values < 0.05 were considered statistically significant.

“GO terms and KEGG pathways were calculated using the GSEA software (see Methods; Extended Data Fig. 12d–f) and FDR q-values < 0.05 were considered statistically significant.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For proteomic analysis, four mutant and four control organoids were used for SUV420H1; three mutant and three control and five mutant and four control organoids were used for CHD8 and ARID1B, respectively.

“four mutant and four control organoids were used for SUV420H1. Three mutant and three control and five mutant and four control organoids were used for CHD8 and ARID1B, respectively.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A 1% false discovery rate at the protein level for MS2 spectra assignments was achieved using a target-decoy database search.

“A MS2 spectra assignment false discovery rate (FDR) of 1% on protein level was achieved by applying the target-decoy database search.”

Classified as probably not a substantial claim.

Differentially accessible peaks per cluster were called using FindMarkers with a logistic regression framework and the number of fragments in peak regions as a latent variable.

“Concurrently, differentially accessible (DA) peaks per cluster were called using FindMarkers with the logistic regression framework with the number of fragments in peak regions as a latent variable.”

Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

Because most cell types had few significantly differentially accessible regions and the regions were almost entirely overlapping, differentially accessible regions were calculated using all cells together to improve power.

“Therefore, differentially accessible regions were calculated using all cells together to improve power.”

Classified as probably not a substantial claim.

For differential expression of modules, Seurat objects were downsampled to have an equal number of cells per organoid.

“To calculate differential expression of modules, Seurat objects were downsampled to have an equal number of cells per organoid, and then the AddModuleScore function was used, using gene lists from WGCNA results.”

Classified as probably not a substantial claim.

Adjusted p-values for differences in cell type proportions between control and mutant were based on logistic mixed models.

“Adjusted p-values for difference in cell type proportions between control and mutant (Fig. 1a–c; Fig. 2a–b; Fig. 3a–b; Extended Data Fig. 4c–f; Extended Data Fig. 5a–c; Extended Data Fig. 8b–c,e,g; Extended Data Fig. 9a–b,e; Extended Data Fig. 10b–d) were based on logistic mixed models (see scRNA-seq data analysis section).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Genes with less than 10 total reads were excluded from differential expression analysis.

“Genes with less than 10 total reads were excluded”

Classified as probably not a substantial claim.

For proteomic analysis, a moderated t-test was performed to detect statistically significant differential protein abundance between conditions, with an FDR threshold of 0.1.

“To detect statistically significant differential protein abundance between conditions a moderated t-test was performed (see Methods, FDR threshold of 0.1; Extended Data Fig. 12a–c).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Brain organoids were randomly selected for calcium imaging.

“Brain organoids were randomly selected and transferred to a recording chamber containing BrainPhys.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Automated motion correction and cell segmentation were performed with Suite2p, followed by manual curation of segmented cells.

“automated motion correction and cell segmentation were performed using Suite2p49, followed by manual curation of segmented cells”

Probably not a substantial claim: may not pass S3 (load-bearing).

In two instances, one organoid was excluded from analysis because it was an outlier.

“In two cases, one organoid was excluded from the analysis as outliers.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The total number of cells sequenced for scATAC-seq was 45,988.

“The total number of cells sequenced was 45,988.”

Classified as probably not a substantial claim.

For scATAC-seq, cells with 1500-20,000 fragments in peak regions, at least 35% of reads in peaks, a nucleosome signal of less than 4, and a TSS Enrichment score of greater than 2 were retained for further analysis.

“After consideration of QC metrics recommended in that package, cells with 1500-20,000 fragments in peak regions, at least 35% of reads in peaks, a nucleosome signal of less than 4, and a TSS Enrichment score of greater than 2 were retained for further analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For scATAC-seq analysis, three SUV420H1 mutant and three control organoids were used for each of the one month and three month timepoints, totaling twelve individually sequenced organoids.

“In summary, three SUV420H1 mutant and three control organoids were used for each of the one month and three month timepoints, being a total of twelve individually sequenced organoids.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For organoid size analysis, SUV420H1+/− organoids: n = 132 control, n = 132 mutant, from 6 batches; ARID1B+/−: n = 109 control, n = 122 mutant, from 4 batches; CHD8+/−: n = 472 control, n = 482 mutant, from 7 batches.

“In summary, SUV420H1+/− organoids: n = 132 for total control organoids, n = 132 for total mutant organoids, from 6 experimental batches. For ARID1B+/− organoids: n = 109 for total control organoids, n = 122 for total mutant organoids, from 4 experimental batches. For CHD8+/− organoids: n = 472 for total control organoids, n = 482 for total mutant organoids, from 7 experimental batches.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Heterozygous edited clones were expanded, and their genotypes were re-confirmed after expansion.

“Heterozygous clones were expanded and the genotypes were re-confirmed post expansion.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Edited clones were screened by PCR and Sanger sequencing to confirm insertions and deletions.

“Insertions/deletions in individual clones were screened via PCR amplification using primers flanking the guide.”

• Also stated in: § CRISPR-mediated gene editing · plain

  “Clones were screened by PCR and Sanger sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In HUES66 CHD8 organoids at 3.5 months batch II, a mutant organoid was excluded because it mostly contained interneuron lineage cells, with very few projection neuron cells.

“in the HUES66 CHD8 organoids at 3.5 months batch II, a mutant organoid was excluded because it mostly contained interneuron lineage cells, with very few projection neuron cells.”

Classified as probably not a substantial claim.

For module differential expression, linear mixed-effect models included the organoid as a random intercept.

“For each module, linear mixed-effect models were fit to the data, with the modules scores as the output, the organoid the cell belongs to as a random intercept, and with or without the control-versus-mutant state as a predictor.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For calcium imaging analysis, five organoids were analyzed per genotype.

“five organoids were analyzed per genotype.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The top LSI component was discarded because it correlated strongly with sequencing depth, and components 2-30 were used for downstream analysis.

“The top LSI component was discarded as it correlated strongly with sequencing depth, and components 2-30 were used for downstream analysis.”

May combine two claims.

Cortical organoids show proper neurodevelopment and express known ASD risk genes.

“We first verified that these organoids initiate appropriate neurodevelopment and express known ASD risk genes”

Classified as probably not a substantial claim.

For pseudotime analysis, cells were first subset to contain an equal amount from control and mutant.

“The cells were first subset to contain an equal amount from control and mutant.”

Probably not a substantial claim: may not pass S3 (load-bearing).

P-values for module differential expression were adjusted across modules using the Benjamini-Hochberg correction.

“The ANOVA function was used to compare the models, and p-values were then adjusted across modules using the Benjamini-Hochberg correction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In Fig. 4, significance of the number of overlapping genes between two gene lists is reported as the Bonferroni-adjusted p-value of a hypergeometric test.

“In Fig. 4 for each comparison of two gene lists, circles inside box are colored and sized according to the significance of the number of overlapping genes in those two lists, reported as the Bonferroni-adjusted p-value of a hypergeometric test.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In previous work, MI scores for endogenous brain datasets were reported to range from 0.008 to 0.0649.

“In previous work, MI scores for endogenous brain datasets were reported to range from 0.008 to 0.0649.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Adjusted p-values for differences in distribution of module scores between control and mutants were based on linear mixed models.

“Adjusted p-values for differences in distribution of module scores between control and mutants (Fig. 1f; Fig. 2e; Fig. 3e; Extended Data Fig. 5e; Extended Data Fig. 8i; Extended Data Fig. 9h) were based on linear mixed models (see Pseudotime, gene module, and differential expression analysis section).”

Classified as probably not a substantial claim.

P-values for each cell type were adjusted for multiple hypothesis testing with the Benjamini-Hochberg correction.

“P-values for each cell type were then adjusted for multiple hypothesis testing using the Benjamini-Hochberg correction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For immunohistochemistry, at least three organoids of each condition were used for verifying expression of the indicated markers.

“at least three organoids of each condition were used for verifying the expression of the indicated markers in Extended Data Fig. 1a–c; Extended Data Fig. 3g; Extended Data Fig. 4a–b; Extended Data Fig. 8a,d,f; Extended Data Fig. 9c,d,f.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For bulk RNA-seq analysis, three organoids were sequenced per genotype, totaling 30 individual organoids.

“three organoids were sequenced per genotype being a total of 30 individual organoids.”

Probably not a substantial claim: may not pass S3 (load-bearing).

After transfection, pools of cells were harvested to test knockout efficiency.

“Post transfection, the pools of cells were harvested to test knock-out efficiency.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For scRNA-seq, three individual organoids per genotype were profiled in each dataset.

“In summary, in each dataset, three individual organoids per genotype were profiled.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For organoid size analysis, P-values were calculated from a two-sided t-test and then submitted to Bonferroni adjustment.

“P-values were calculated from a two-sided t-test and then submitted to Bonferroni adjustment.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For calcium imaging, P-values were calculated from two-tailed t-tests, and the P-value for cumulative frequency distribution of ISI was based on a two-sided Kolmogorov-Smirnov test.

“P-values were calculated from two-tailed t-tests (Fig. 1h–i). P-value for cumulative frequency distribution (Extended Data Fig. 7j) of ISI for control and SUV420H1+/− organoids based on two-sided Kolmogorov-Smirnov test.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In PGP1 SUV420H1 organoids at one month, a mutant organoid was excluded due to very low average nUMI and nGene in that sequencing lane.

“in PGP1 SUV420H1 organoids at one month, a mutant organoid was excluded due to very low average nUMI and nGene in that sequencing lane”

Probably not a substantial claim: may not pass S3 (load-bearing).

Densitometry band quantification was performed with Fiji and normalized to housekeeping genes (GAPDH or β-actin).

“Densitometry band quantification was done using Fiji software48 v2.0 and normalized on housekeeping genes (GAPDH or β-actin).”

Probably not a substantial claim: may not pass S3 (load-bearing).

For Western blot, each control and mutant protein lysate was blotted at least twice.

“each control and mutant protein lysate was blotted at least twice in Extended Data Fig. 3d–f.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For calcium imaging, spontaneous activity was recorded in three different z-planes, with 120 ± 803 neurons/plane in control organoids and 107 ± 75 neurons/plane in SUV420H1+/− organoids.

“Spontaneous activity was recorded in three different z-planes (120 ± 803 neurons/plane [range from 25 to 294 neurons/plane] in control organoids, and 107 ± 75 neurons/plane [range from 32 to 255 neurons/plane] in SUV420H1+/− organoids).”