Lawrence Berkeley National Laboratory Recent Work

Title Germline Chd8 haploinsufficiency alters brain development in mouse.

Permalink https://escholarship.org/uc/item/0ck9n78k

Journal Nature neuroscience, 20(8)

ISSN 1097-6256

Authors Gompers, Andrea L Su-Feher, Linda Ellegood, Jacob et al.

Publication Date 2017-08-01

DOI 10.1038/nn.4592

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. work. Correspondence work. should Correspondence be addressed to A.S.N. ( California, Merced, California, USA. Department, Berkeley, California, USA. for Pediatric Regenerative Medicine, University of California, Davis, Sacramento, California, USA. of Medicine, University of California, Davis, Davis, California, USA. University of California, Davis, Davis, California, USA. 1 stages early embryonic problems tinal gastrointes and (ID) disability intellectual mild-to-severe phology, CHD8 DNA- helicase binding protein (chromodomain 8) CHD8 remodeler chromatin the novo de NDDs. driving mechanisms and cellular developmental may reveal eling genes affect transcriptional regulation during brain development disorder (ASD) and rare for strong ticularly roles in neurodevelopmental disorders (NDDs) haveefforts linked mutations of chromatin genes causal with specific, case-sequencing systems, organ across function factors remodeling cancer and development in critical are at the chromatin level, and proteins that control chromatin packaging proliferation and differentiation are dependent on genomic regulation Pluripotency, or repression. activation transcriptional either yielding state, chromatin local the control complexes remodeling Chromatin types cell metazoan of function and development the to central is of potential a cell and DNAthe transcriptional determines packaging offers an initial picture of the consequences of validated increased neuronal and proliferation splicing developmental perturbation in of dysregulation RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we and neuroimmune signaling. We identified a module co-expression with peak expression in early brain development featuring in We applied network analysis to gene characterize neurodevelopmental expression, revealing widespread changes transcriptional regional brain volume, validating that phenotypes of displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased We examined the impact of germline heterozygous frameshift The chromatin remodeling gene Konstantinos S Zarbalis Axel Visel Iva Zdilar Tyler W Stradleigh Andrea L Gompers development in mouse Germline nature nature Received 7 September 2016; accepted 23 May 2017; published online 26 June 2017; Department Department of Psychiatry and Behavioral Sciences, University of California, Davis, Davis, California, USA. A key gene that has emerged from studies profiling rare and and rare profiling studies from emerged has that gene key A Chd8 mutations exhibit macrocephaly, distinct craniofacial mor craniofacial distinct macrocephaly, exhibit mutations coding variation in ASD is the gene gene the is ASD in variation coding + NEUR / del5 1 6 , – mice across pathways disrupted in disorders, including neurodevelopmental synaptic neurogenesis, processes 2 OSCI 8 7 9 , , Shreya Louis , . Homozygous deletion of of deletion Homozygous .

8 . Understanding how mutations to chromatin remod 9

. . In addition to ASD, human individuals harboring , Len , A Len Pennacchio EN C 1 1 , 1 E 0 2 . .

, Chd8 , 2 Chd8 10 , , Michael C Pride advance online publication online advance , Linda Su-Feher de novo de 5 9 , , Jill L Silverman knockdown in recapitulates knockdown zebrafish Department Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 1 7 , 2 Department Department of Energy Joint Genome Institute, Walnut Creek, California, USA. CHD8

mutations in autism spectrum spectrum autism in mutations

, , Gaurav Kaushik haploinsufficiency alters brain 2 . Although many chromatin chromatin many Although . represents a central node in gene neurodevelopmental networks implicated in autism. 6 Chd8 , 7 , , Diane E Dickel [email protected] CHD8 3 3– Mouse Mouse Imaging Centre, The Hospital for Sick Children, Toronto, Ontario, Canada. 1 1 , 6 2 in mice is lethal at lethal is mice in . This finding is par , 4 , 10 , , Melanie D Schaffler Chd8 1

, which encodes encodes which , , 4 & Alex S Nord

, Jacob Ellegood Chd8 5 haploinsufficiency for haploinsufficiency brain development. Department Department of Pathology and Laboratory Medicine, Shriners Hospitals for Children, Institute

5 , , Brandon J Mannion + ). / del5 6 Chd8 , , Jason P Lerch 1 - - - - mice overlap pathology reported in humans with .

mice harboring 5-bp or 14-bp deletions in in deletions or 14-bp 5-bp harboring mice generate to oocytes C57BL/6N of targeting CRISPR/Cas9 Weused megalencephaly Mice harboring heterozygous germline RESULTS brain. mouse developing scriptional phenotypes associated with genome engineering and assayed neuroanatomic, behavioral and tran ated a germline 5-base-pair (bp) deletion in ing chromatin biology to NDD pathology. Toward this goal, we gener development could reveal specific and generalizable mechanisms link impact functional of germline heterozygous the Characterizing regulation. transcriptional and indirect direct via and behavior structure brain neurodevelopment, in regulating Chd8 phenotypes required in neurogenesis and that mutations to enhancers tion in brain by development binding to relevant gene promoters and func ment. It regulatory this that CHD8 achieves has proposed been develop in function CHD8 of conservation evolutionary of degree phenotypes and gastrointestinal macrocephaly mutation on in neurodevelopment mice. 1 3 , doi:10.1038/nn.459 2 , 10

1 , Nycole A Copping ,

4 , , A Ayanna Wade 11– 6 Lawrence Lawrence Berkeley National Laboratory, Functional Genomics 13 1 3 , 4 1 , . Although recent mouse studies indicate that that indicate studies mouse recent . Although 5 9 6 , important questions remain regarding the role of of role the regarding remain questions important , , , Jacqueline N Crawley , , Ingrid Plajzer-Frick 2 Department Department of Neurobiology, Physiology and Behavior, 2 Chd8 + / 8 1 del5 School School of Natural Sciences, University of 1 , 4 , 10 , 2 10

These These authors contributed equally to this mice. This integrative analysis , M Asrafuzzaman Riyadh

, Rinaldo , Catta-PretaRinaldo Chd8 Chd8 6 Chd8 Chd8 4 , , Veena Afzal CHD8 haploinsufficiency in the MIND MIND Institute, School 1 Chd8 , 4 Chd8 9 mutation exhibit , , exon 5, upstream of upstream 5, exon 1 using CRISPR/Cas9

1 CHD8 t r a mutation on brain , suggesting a high , suggesting +/ cause behavioral del5 mutations. mice C I 6 , Chd8 1 e l

, 2 5

, 10 is s

, ,  - - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. fertile irrespective of sex. of irrespective fertile heterozygous but ( mutants, homozygous in lethality embryonic in with an study earlier all experiments to eliminate potential off-target mutations. Consistent erations before experiments, and further multiple litters were used for gen four at for least bred were mice male Heterozygous mice. (WT) tion lines were carrier via C57BL/6N breeding expanded to wild-type mutations human identified of majority the P samples; ( hypothalamus. HTh, thalamus; Th, hippocampus; HC, callosum; corpus CC, striatum; St, neocortex; NCx, area; neocortical measured shading, Pink n (**** cerebellum. Cb, midbrain; MB, cortex; Cx, bulb; olfactory OB, megalencephaly. of indicative n of ( 5. exon in mouse of targeting 1 Figure t r a  and western blot on analysis brain lysates, using an Chd8 N-terminus h Chd8 = 10 male, 10 female). ( female). 10 male, = 10 = 9 WT, 9 b

values derived using Student’s Student’s using derived values (280 kD e c a ) Plots of cortical area, thickness at 30% and 70% distance from the dorsal midline and cortical hemispheric circumference (dots represent individual individual represent (dots circumference hemispheric cortical and midline dorsal the from distance 70% and 30% at thickness area, cortical of ) Plots (37 kD We performed quantitative reverse-transcription PCR (qRT-PCR) PCR reverse-transcription We quantitative performed Chd8 Gapdh Chd8 + WT Del a) a) + /

C I del5 / del5 Chd8 n = 4 mice each for both genotypes; area, * area, genotypes; both for each = 4 mice d WT ) mice were viable, reached a normal lifespan and were were and lifespan normal a reached viable, were mice ) Chd8 ) qRT-PCR showing reduction of RNA in in RNA of reduction ) qRT-PCR showing mice, showing reduction of Chd8 protein in in protein Chd8 of reduction showing mice, e l E1 + / HT del5 fe 4.5 Chd8pr RNA injectionof Cas9 + r s tiliz / Chd8 del5 sgRNA mouse model. ( model. mouse 390 5-bp deletionin ed oocyt mRNA + 1 390 ). ( ). 0 Chd8/Gapdh ratio , , the presumed 0.5 2.0 (bottom). ( (bottom). 1. 1. f ot 0 0 5 ) Whole-mount brain of of brain ) Whole-mount g ein es ) Representative coronal sections of WT and and WT of sections coronal ) Representative WT 400 * t Chd8 -test for for -test HT 400 a b ) Location of case mutations in human human in mutations case of ) Location ) Schematic of mouse line generation. Het, heterozygous; del, deletion. ( deletion. del, heterozygous; Het, generation. line mouse of ) Schematic

ex Surr (280 kD on Chd8 (37 kD mother Gapdh 5 d og Chd8 , , at e 41 a) a) and and frameshift alleles resulted alleles frameshift e 0 41 9 CHD8 C Ex Chd8 0 WT ( Chd8 ase mutations on 5gRNA h Fig. 1a Fig. P0 Chd8pr and using one-way ANOVA for for ANOVA one-way using and Chd8 P mouse human HT = 0.0328; thickness (30%) (30%) thickness = 0.0328; + d / del5

Relative expression Het Chd8 5-bp del + 0.5 2.0 of / – 1. 1. del5 mouse at P0 reveals increased cortical length (denoted by orange arrow and line), line), and arrow orange by (denoted length cortical increased reveals P0 at mouse Chd8 fspr 0 0 5 c Chd8/Gapdh ratio ot WT F0 0.5 ). F0 muta F0 ). 1. 1. ein + (HT) forebrain at P0 (** P0 at forebrain (HT) 0 5 0 ing Chd8 / del5 WT WT forebrain at E14.5 (* E14.5 at forebrain ** mRNA ** HT HT Chd8 - - CHD8 + Fig. 1a Fig. in expression lower of trends ent isoform Chd8 short a as reported been We a also observed smaller ~110 kDa band, similar in size to what has in significantly reduced consistently was which kDa), (~280 Chd8 full-length and tein at embryonic day (E)14.5, postnatal day (P)0 (ChIP-seq) and in Heterozygousadults sequencing mutation( resulted in deep decreased by chromatin followed Chd8 for immunoprecipitation used previously used been has that antibody h g f /

del5 2 mm +/del5

3.5 Chd8 WT 7. 9 brains at P7 visualized with Nissl stain. Scale bar, 1 mm. Scale stain. Nissl with visualized P7 at brains Supplementary Fig. 1a Fig. Supplementary 0 0 (top) and corresponding guideRNA sequence homology for Cas9- for homology sequence guideRNA corresponding and (top) P f OB 70% . Error bars represent mean mean represent bars . Error = 0.224, thickness (70%) (70%) thickness = 0.224, – c WT P ). We performed RNA sequencing on E12.5 WT mice and and mice WT E12.5 on sequencing RNA We performed ). Ar advance online publication online advance = 0.0076; = 0.0076; P ea Chd8 = 0.02; = 0.02; HT St * NCx + WT P0 wholemountbr / P del5 m µ Cx 1,200 HC < 0.0001; WT WT < 0.0001; n 600 n = 6 WT, 6 mice at all stages ( stages all at mice 0 = 9 WT, 8 30% P7 braincor Thic WT (30% Th kness – HTh c NS HT c MB ) Sequence trace showing 5-bp deletion deletion 5-bp showing trace ) Sequence ) ain ). We identified a band representing representing band a identified We ). CC ± Chd8 P onal sectionNissl Chd8 s.e.m. Cb = 0.268; length, ** length, = 0.268; n Chd8 = 4 male, 6 female; 6 female; = 4 male,

+ µm 1,200 + / del5 600 / del5 1 3 0 + , which displayed inconsist displayed which , Supplementary Fig. 1a Fig. Supplementary ) and P0 (** P0 ) and / ). ( ). del5

P0 Thic WT nature nature Chd8 e (70% mice ( mice ) Western blot blot ) Western kness Chd8 200 400 600 NS HT ) 0 transcript and pro +/ P F WT Co del P Supplementary Supplementary emales NEUR = 0.0026). = 0.0026). = 0.0089; = 0.0089; 5 r

mm tical length Chd8 **** 15 10 HT 0 5 OSCI

Hemispher

WT WT + Fig. 1d length / Males del5 ( µ **** EN HT ** HT

m)

–

c C 1 e , 2 ). ). e E - - .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. by an increase in length ( length in increase an by in larger ~8% both measurements ( of brains though ( respectively), midline dorsal the from distance 70% and 30% at thickness cortical in observed were differences sig No nificant observed. were anomalies neuropathological overt no and ( P7 at examined were sections brain coronal Nissl-stained ( sexes between differences no substantial brains was ~7% longer than matched WT littermates ( tions (P0), as macrocephaly is a hallmark trait in patients with allele. deletion 5-bp the harboring mice analyzed we ies, scripts underwent degradation. For the majority of the following stud frequencies relative to WT allele reads, suggesting the frameshift tran libraries. For both mutant lines, the overlapping lines reads sequence deletion observing below), 14-bp described (RNA-seq, and 5-bp the both from littermates matched Unpaired ( traveled P ( ( in ( or F ( in littermates WT to relative differences exhibit not do (WT: object familiarized ( assays cued ( both in conditioning tone–shock after freezing reduced memory, including and learning in deficits 2 Figure nature nature ( at P7 cephaly t i a e 1,18 = 0.3978) or ( or = 0.3978) 1,17

We tested for differences in brain size in in size brain in differences for Wetested Ultrasonic vocalizations Time sniffing Percent of time freezing f g 12 10 ) chamber entries ( entries ) chamber Chd8 ) time sniffing ( sniffing ) time 20 40 60 80

= 9.744, * = 9.744, (s per 10 min) = 0.1634, = 0.1634, (number per 5 min) 9 0 0 0 200 15 10 . The maximal cortical anteroposterior length of of length anteroposterior cortical maximal The . 1,000 1,250 50 250 500 750

0 0 0 NEUR t Chd8 t 1,38 deletion in both 5-bp and 14-bp libraries but not in WT WT in not but libraries 14-bp and 5-bp both in deletion 0 -tests used for for used -tests Chd8 WT Tr t = 1.1795, = 1.1795, aining 1,37

+ Mouse snif Object snif n n OSCI Chd8 / = WT Fig. 1 Fig. =20 +/ del5 P k P n del ) number of marbles buried ( buried marbles of ) number = 2.7064, * = 2.7064, = 0.0059), ( = 0.0059), 19 = WT = 0.8722). ( = 0.8722). 5 * mice exhibit cognitive deficits but no ASD-relevant social or repetitive phenotypes. ( phenotypes. repetitive or social ASD-relevant no but deficits cognitive exhibit mice

t 10 n 1,17 EN = + h Fig. 1 / F f 19 f del5 ). C F 1,37 = 0.9409, = 0.9409, a P E 1,19 Chd8 , ,

brains ( brains = 0.2455). All data shown from first cohort. Male mice were used in in used were mice Male cohort. first from shown data All = 0.2455). Chd8 C P n b = 0.11, = 0.11, ont h = advance online publication online advance n = 0.0026), confirming cerebral megalen cerebral confirming 0.0026), = and and = 11.503, * = 11.503, = ). ). The overall neocortical section area was P +/ ex 18 e +/ j del = 0.0104). ( = 0.0104). 9 * ) time sniffing a novel mouse (WT: mouse a novel sniffing ) time , * del t k 5 Chd8 Chd8 ) Repetitive behavior. behavior. ) Repetitive g 5 – P P l P ; repeated-measures ANOVA used for for used ANOVA ; repeated-measures = 0.0328), driven predominantly predominantly driven 0.0328), = = 0.3599), ( = 0.3599), b = 0.73). ( = 0.73). f j + deletion reads occurred at lower Percent of time freezing / Time spent grooming Entries del5 10 P (s per 10 min) (number per 10 min) 20 40 60 80 0 0 = 0.0031; = 0.0031; 15 12 20 30 40 50 10 10 Fig. Fig. 1 mice trended larger across across larger trended mice c P 50 0 t ) Novel object. object. ) Novel 0 0 0 0 = 0.224 and and 0.224 = 1,38 Chd8 g Bef – = 1.0151, = 1.0151, h i f or No No ) Male–female social interactions. interactions. social ) Male–female ). ). Whole-mount and ) time following ( following ) time n e cue n =20 WT v v + =20 el mouse el object WT d / Chd8 del5 Chd8 ) time spent in chamber with a novel mouse (WT: mouse a novel with chamber in spent ) time P < 0.0001) with mice at birth birth at mice CHD8 + + Chd8 / del5 / P P del5 Chd8

= 0.3165). ( = 0.3165). = 0.268, 0.268, = Chd8 Af Fig. 1 Fig. Chd8 : : F n t mice do not exhibit any differences in ( in differences any exhibit not do mice er cue =20 F + n muta = / 1,18 1,18 del5 +/ + t +/ 1,17 del * 19 / del del5 g 5 = 3.2825, = 3.2825, mice fail to show significant difference in exploration between a novel and and a novel between exploration in difference significant show to fail mice = 7.00369, * = 7.00369, ), ), 5 - - - - - = 0.5785, = 0.5785,

c – c k e l independent cohorts of adult adult behaviors of cohorts repetitive independent and standard social two cognitive, using of each battery assays behavioral tailored a performed We adult of phenotyping Behavioral contextual and cued conditioning and novel object recognition recognition object novel and conditioning cued and contextual and WT of cohort ent independ a second, with Replication recognition. in deficits cating 11.5030, = ( investigation object novel more exhibit whereas to failed object, familiar the than object ( P cue auditory the to scores freezing lower t in to compared controls ( the context chamber littermate WT after h training. 48 at cue auditory an to and h 24 at context environmental an to recognition. associations object learned for memory novel evaluated and conditioning Fear conditioning fear using evaluated Fig. female; 9 male, 11 cohort: second female; 10 male, 11 cohort: (first littermates WT and female) 11 male, 11 cohort: second female; 10 ) Open field. field. ) Open

; one-way ANOVA used for for used ANOVA ; one-way Time sniffing (s per 5 min)

Number of g 1,37 = 0.0104). WT littermates spent more time exploring the novel novel the exploring time more spent littermates WT 0.0104). = 20 30 40 10

marbles buried Total sniff time 0 12 15 18 = 3.3492, 3.3492, = 6 9 0 3 (s per 10 min) 2 P 14 10 and and = 0.0867). ( = 0.0867). 35 70 P P No F Chd8 0 0 5 = 0.5705) or ( or = 0.5705) amiliar object n = 0.0164; = 0.0164; Chd8 v =20 WT el object P n upeetr Fg 2 Fig. Supplementary Chd8 + = 0.0031; 0.0031; = * =20 WT / del5 + P n / = WT del5 = 0.0019). 0.0019). = + g mice exhibit no differences between WT littermates littermates WT between differences no exhibit mice / 10 – del5 d i a mutant mice exhibited less freezing when placed placed when freezing less exhibited mutant mice Chd8 ; males and females were used in all other panels. panels. other all in used were females and ; males – ) context ( ) context Chd8 f mice do not exhibit any differences in distance distance in differences any exhibit not do mice ) Three-chamber social approach. approach. social ) Three-chamber Chd8 i n ) ultrasonic vocalizations with an estrus female female estrus an with vocalizations ) ultrasonic n = Chd8 Chd8 =20 a + f Chd8 +/ 19 , . Error bars represent mean mean represent bars . Error / +/ F j b del5 del5 ) time spent self-grooming ( self-grooming spent ) time n del5 1,19 ) Fear conditioning. conditioning. ) Fear = Chd8 + +/ : : + 9 t / del5 1,37 = 16.31, * = 16.31, F Chd8 del5 / del5 1,17 d l : : = 3.3492, * = 3.3492, Time in chamber + mice similarly found deficits in in deficits found similarly mice Total distance travelled F = 12.8051, * = 12.8051, / h Chd8

del5 (s per 10 min) ). Learning and memory were were memory and Learning ). + 4,000 6,000 8,000 2,000 1,18 /

Total follow time 300 600 900 del5 0 mutant mice also exhibited exhibited also mice mutant (s per 10 min) 0 = 3.2825, 3.2825, = 24 32 16 mice (first cohort: 9 male, male, 9 cohort: (first mice P 0 8 +/ = 0.0007; = 0.0007; del5 Chd8 No Middle chamber No Fig. 2 Fig. P n Chd8 =20 v v WT = 0.0019) and ( and = 0.0019) mice n el mouse el object P =20 WT n + = 0.0023) = 0.0023) = WT t r a / P Fig. 2 Fig. + del5 b * ± 10 / = 0.0867), indi 0.0867), = t del5 s.e.m. ; ; 1,38 Chd8 Chd8 t mutant mice mice mutant 1,37 mice exhibit exhibit mice = 0.8552, = 0.8552, c Chd8 ; WT: WT: ; C I Chd8 = 2.7064, 2.7064, = + + n Chd8 1 / / n =20 del5 del5 6 = n

in two two in Fig. Fig. 2 +/ = +/ del5 19 e l +/ : b mice mice del5 9 del5 )

F * 1,19 s a  - - ;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. = 0.9409, Chd8 ( interaction social reciprocal male male–female ing in parameters social in observed were deficits No assay. the during genotypes both in activity exploratory or in the habituation phase ( affected by genotype in the phase social ( P F WT both in object and novel the sniffing spent time than greater was detected ( 0.0007; and WT assay,both for this in sociability of definition the meeting with object, than novel greater the was mouse novel the with chamber the behaviors in spent repetitive and social ( genotypes both in detected was sociability Normal of assays corroborative using memory. and learning in deficits ( Student’s in impairment cognitive and size region in present is thickness (* cortex WT, 18 (**** cortex cerebral of volume regional 3 Figure t r a  interaction interaction showed no genotype difference in number of emitted calls male–female during vocalizations Ultrasonic female. WT estrous the c b a Supplementary Fig. 2 Fig. Supplementary

1,17 = 0.7555). The number of entries into the side chambers was not not was chambers side the into entries of number The 0.7555). =

Behaviors relevant to diagnostic symptoms of ASD were assessed assessed were ASD of symptoms diagnostic to relevant Behaviors False discovery rate 120 130 140 150 160 1% 5% 1% 30 31 32 33 34 35 Chd8 = 12.8051, 12.8051, = + / Chd8 del5 Chd8

Chd8 C I P WT WT

Chd8 Decreased Increased = 0.00147), amygdala (*** amygdala = 0.00147), Cortex Cortex t P F + -test used for for used -test males spent similar amounts of time sniffing ( amounts sniffing of time similar spent males = 0.3599) and following ( / 1,37 +/ del5 **** + del e l Chd8 HT HT / + * del5 haploinsufficiency drives megalencephaly and cognitive impairment in mouse. ( mouse. in impairment cognitive and megalencephaly drives haploinsufficiency 5 / = 2.16, ( mouserelativevolume(%totalbrainvolume) del5 Chd8 Axial above Fig. 2 Fig. P +/ ; see ; see s del = 0.0023), with no sex differences ( differences sex no with 0.0023), = : : Chd8 F 5 3.0 2.0 2.5 +/ mouseabsolutevolume(m 10 11 12 1,18 del 8 9 Chd8 Supplementary Table 1 Supplementary P e 5 a ; WT: WT: ; = 0.149). Time spent sniffing the novel mouse mousecorticalthickness = 9.744, = 9.744, and and Amygdala Amygdala + ), indicating that that indicating ), WT WT / del5 + / del5 F **** *** HT HT b mice ( mice 1,37 F ; error bars in in bars ; error mice. ( mice. 1,18 = 0.30, P = 0.0059). No sex differences were Nodifferences sex = 0.0059). = 7.00369, 7.00369, = 4.0 4.5 5.0 P 16 18 20 22 24 26 Fig. 2 Fig. 2 Fig. = 0.0010) and hippocampus (*** hippocampus and = 0.0010) P Off-axis behind Chd8 d < 0.0001), amygdala (**** amygdala < 0.0001), Hippocampus Hippocampus ) Voxel-wise differences in volume between between volume in differences ) Voxel-wise P WT WT Fig. Fig. 2 m = 0.584), indicating normal h 3 + Chd8 ) d a / ; for full regional statistical analysis and FDR values). ( values). FDR and analysis statistical regional full for del5 **** *** and and ; WT: WT: ; t HT HT 1,17 P f mice (context conditioning score; score; conditioning (context mice ; ; Chd8 = 0.0164; 0.0164; = = 0.5785, + F Fig. 2g Fig. b / 1,37 represent mean mean represent del5 F d 1,19 Fig. 2d Fig. = 0.11, + mice exhibited exhibited mice / F del5 = 16.31, 16.31, = 1,35 –

Fig. 2 Fig. False discovery rate i ie dur mice P ). WT and and WT ). Chd8 = 0.0985, 0.0985, = = 0.5705) – 5% 1% 1% P i = 0.73) ). Time ). Smaller Larger g + ; ; P / t P ± del5 < 0.0001) and hippocampus **** hippocampus and < 0.0001) 1,17 s.e.m. 1 = 7 - : . P

Mouse: Brain v = 0.0004) after correction for absolute brain volume. ( volume. brain absolute for correction after = 0.0004) Relative pus (+10.3%, FDR pus < (+10.3%, FDR 1%) < and (+11.0%, amygdala 1%; robust increases in absolute volume across cortical regions, hippocam in reported results with divisions across the cortex, subcortical areas and cerebellum (full (FDR = 2%), respectively. We 159 brain assessed independent regions in larger were matter gray cerebral and matter white cerebral Similarly, 1%). = (FDR) rate a 7.5% increase in absolute volume in with affected, was most cortex analysis, In and regional connectivity. identify changes in absolute (mm to performed was MRI Structural phenotyping. of behavioral cohort Intact brains were collected from the same mice that comprised the first of Analysis other relevant measures of general health in adult health of relevant measures general other or weight body in observed were differences substantial No abilities. t Open-field locomotor activity did not differ between genotypes ( t 1.0151, t repetitive behaviors were in observed the self-grooming assay ( ( 1,38 1,38 1,36 Fig. 2 Fig. olume dif Chd8 = 0.8552, = 1.1795, 1.1795, = = −0.504, −0.504, = R i +/ Chd8 ; ; P 2 del : cortex = 0.1855, hippocampus = 0.148, amygdala = 0.1352). = 0.1352). amygdala = 0.148, hippocampus = 0.1855, : cortex t = 0.3165). No sex differences were detected (self-grooming: = No 0.3165). were (self-grooming: sex differences detected fe 1,17 5 advance online publication online advance r

vs WT ences +/ Chd8 = 0.1634, 0.1634, = Absolute P del P P = 0.3978) or numbers of marbles buried ( and WT littermates. ( littermates. WT and = 0.2455), indicating normal exploratory and motor and exploratory normal indicating 0.2455), = a = 0.619; marble burying: burying: marble 0.619; = ) MRI revealed significant increases in absolute absolute in increases significant revealed ) MRI +/ Supplementary Table 1 Supplementary del5 b Chd8 ) Increases in volume are still significant for the the for significant still are volume in ) Increases P adult brain structure via MRI via structure brain adult = 0.8722). No spontaneous stereotypies or stereotypies spontaneous No 0.8722). = P < 0.0001) in in < 0.0001) + / del5 mice at 5.4% (FDR = 3%) and 6.1% 6.1% and 3%) = (FDR 5.4% at mice 3 ) ) and relative regional brain volume e Percent Percent Percent e ) Concordance of specific brain brain specific of ) Concordance

Chd8 context freeze context freeze context freeze 100 100 100 20 40 60 80 80 20 40 60 20 40 60 80 Chd8 0 0 0 120 Chd8

t + 1 9 1,38 ). ). / nature nature to del5 + 18 Chd8 brainv / +/ = 1.4883, 1.4883, = del5 del mice (false discovery 1 0 5 Hippocampus Volume (mm c 140 R R (HT) mice ( mice (HT)

mice: Chd8 R ) Increased cortical cortical ) Increased Amygdala + 20 2 2 olume corr 2 =0.1352 =0.1855 Cortex / 1 1 =0.148 del5 NEUR cont Fig. 2 + mice showed mice 22 160 1 2 / P del5 e = 0.1449). 0.1449). = xt lear 3 OSCI elation ) mice. Fig. Fig. 3 1 3 n k 24 = 19 Fig. Fig. 2 Fig. 2 ; ning t 180 EN 1,38 4 HT WT HT WT HT WT a C = ). ). j l E - ; ;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. tensor imaging revealed no significant differences in fractional voxel-wise fractional or regional in the either in differences diffusivity mean or significant anisotropy no revealed imaging tensor ( trends similar showed differences Voxel-wise 0.3%). < FDR −3%, to (−1 volume relative decreased showed nuclei along ness, the particularly cingulate cortex ( ( off still larger,significantly were though cortex failed to surpass the FDR < volumes 5% cut relative volume, brain total for correction After nature nature and organization matter white that indicating measurements, zero: downregulated DE right DE genes; of zero: downregulated genes); zero: upregulated on Directionality genes. here with and genes autism- identified Chd8-associated upregulated P GO terms for DE genes and for are genes whose bound promoters by Chd8. ( * of of DE stages. three FDR DE shown for genes developmental across in expression plot that showing most fold subtle DE (FC). ( changes genes relatively (red) exhibit 4 Figure P = 2.8 × 10 = ** protein, 0.0248; Hnrnpa2b1 h Chd8 ChIP-seq Chd8 ChIP-seq f d a Fig. 3 Fig. Called peaks Called peaks Input control Input control Frequency Relative expression 10,00 15,00 20,00

4.0 4.5 5.0 5.5 6.0 5,00 NEUR Differential gene expression in gene expression Differential Nfe2l3 b 0 0 0 0 0 Adult (>P56) Mouse DPC(P0=21DPC) 21 DPC(P0) ). ). −10 12.5 DPC 14.5 DPC 17.5 DPC Chd8 12 RNA (left; Chd8 1 0 WT Kdm5b OSCI (FDR =1.4 among ) ) in our DE gene set, based on gene sampled sets vs. randomly (bars) (red number ( line). observed Enrichment ofautism-associatedgene Adnp Hnrnpa2b1 14 + / + del5

Chd8 EN 16 expression / Chd8 del5 2 0 10 kb and WT littermates across brain development. DPC, ( days post and conception. brain across development. WT littermates 10 kb C × P 18 +/del5 n E mice also displayed increased cortical thick cortical increased displayed also mice 10 = mean Error bars 0.0055). represent = 6 WT, 4 +/del5

Differential expression mRNA sequencingof modeling (WGCNA) Expression network –2 Count 20 Dissect down-regulatedgenes advance online publication online advance and WTforebrain analysis (edgeR) ) 3 0 44 samples 22 Chd8 5.0 5.5 6.0 6.5 Chd8 4 0 Mouse DPC(P0=21DPC) +/del5 Chd8 Dpm Cbx3 12 WT + (FDR =2.5 Bcl11a / del5 s 1 14 0 + / del5 ) ) and (right; protein Fig. Fig. 3 16 expression Parikshak.ASD.DE.Dow Chd8 i neurodevelopment. ( neurodevelopment. Chd8.Mouse.Forebrai Parikshak.ASD.DE.U 18 b × Sanders.TADA.Q0. Fig. 3 Fig. g 10 Reg. ofcellcycleprocess(GO:0010564) Chromatin modification(GO:0016568 mRNA processing(GO:0006397)

+/del5 –log (P value)

c 2 0 2 4 6 8 20 ). ). Deep cerebellar –2 Hormozdiari.M1 GO biologicalprocessannotation –1.0 Darnell.FMR ) 22 d Willsey.Al –0.5 Gene setenrichment ). Diffusion Diffusion ). 8.0 8.5 9.0 9.5

log ± Mouse DPC(P0=21DPC) s.e.m. ( s.e.m. 0.0 c 2 P n p n 3 (FC) and l Hnrnpa2b1 n Term 12 z = 5 WT, 5 2 4 6 8 -scores generated via permutation test. Vertical via permutation generated -scores lines are at (FDR =1.9 0.5 WT a 14 d ) Schematic of our experimental pipeline. DPC, days postconception. ( DPC, ) days of pipeline. postconception. Schematic our experimental . Solid lines, means; dashed lines, . ±1 Solid lines, dashed means; lines, ( s.e.m. - - f

1.0 16 ) Chd8 ChIP-seq ) at Chd8 ChIP-seq h Autism genesets ) Enrichment of autism-associated genes ( ) of autism-associated Enrichment expression Chd8 ( and in memory, learning by task as the context assessed conditioning and amygdala ( ( behavioral performance. Increased absolute volume of cerebral cortex and volume brain between correlations test to us allowed of mice set ( littermates WT in that from in connectivity long-range × 18 R f 10 Chd8 -stat = 33.6, FDR < 0.1%), hippocampus ( -stat = FDR < hippocampus 33.6, 0.1%), z 2 Performing behavioral and structural MRI analyses on the same same the on analyses MRI structural and behavioral Performing c -score c : cortex = 0.1855, hippocampus = 0.148, amygdala = 0.1352; +/del5 –2 20 ) ) Relative expression ) ) Chd8 3.0 3.5 4.0 4.5 5.0 + 22 / del5 2 0 110/475 105/397 overlap 79/481 is the top differentially expressed gene; panel shows is relative expressed the top differentially ) ) in 10 Set Chd8 WT e x

-axis represents test gene -axis set represents used in (left of analysis mRNA relative Chd8 mRNA DEin Hnrnpa2b1 f forebrain (FDR<20% -stat = 38.6, FDR < 0.1%) were correlated with deficits

4 expression +/del5 0.0 0.5 1.0 1.5 3.17 5.69 1.47 20 P d + value ) Example expression patterns in patterns ) expression Example / del5 Hnrnpa2b1 × × × Katayama.DE.Dow WT Sugathan.DE.Down j Chd8 10 10 10 Katayama.DE.U Cotney.DE.Dow Sugathan.DE.Up (HT) forebrain at (HT) forebrain P0 (Student’s Durak.DE.Dow –4 –14 –17 30 Chd8 Cotney.DE.U Mouse DPC(P0=21DPC) and expression(FDR=3.2 Durak.DE.U HT * Chd8 8.56 6.62 +/del5 P 0.0317 Supplementary Fig. 3 Fig. Supplementary Adnp Hnrnpa2b1 value Adj. 40 × × ) (37 kDa) (35 kDa) 10 10 i qRT-PCR validation + , Gapdh j p n n p p n / 15 ) Comparison ) of DE Comparison down- and –11 –14 loci. ( del5 Q e 50 mice was not grossly different different grossly not was mice < 0.3 from Sanders overlap ) ) Validation of DE expression 35/481 56/475 51/397 Chd8 10 WT Set g ) Representative significant significant ) Representative Bound byChd8inforebrain genesets f (ChIP-seq × HT -stat -stat = FDR < 29.0, 0.1%) 10 0 5 60 z 1.62 9.47 4.06 = –23 z P -score ) ± value

× Hnrnpa2b1/Gapdh × × 2. t r a Chd8 0.0 0.5 1.0 1.5 10 10 10 P 70 value<10 ). –16 –15 –5 t -test; RNA, -test; 5 Western blot + WT / 1.57 1.37 del5 et et al. 80 P C I 0.0021 10 b value Adj. forebrain forebrain ) ) Volcano HT ** × × –4 Fig. 3 10 10 1 )

9 e l 15 –12 –12 ,

e s ). 

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. We applied RNA-seq in forebrain dissected from four developmentalfourWefromforebrain RNA-seqapplied dissectedin Chd8 in neurodevelopment across expression gene Differential relationship. region-specific than rather general was and a volume brain performance conditioning fear between tion correla the that suggesting responses, context and cued and regions brain other between present were correlations Similar by volume. brain driven was relationship This genes); DE upregulated zero: In modules. within genes relevant ( ( genes. individual of expression represents area shaded mean, represents Line module. each in genes all for expression shows plot Each module. each for log bar, mean Scale modules. expression gene developmental five the for ( 5 Figure t r a  stages (E12.5, E14.5, E17.5 and P0) and adult mice (age > P56; e a

) Top: number of DE genes in each module. Bottom: enrichment of down- or upregulated DE genes per module. ( module. per genes DE upregulated or down- of enrichment Bottom: module. each in genes DE ) Top:of number bar: Scale stages. and samples all across genes DE of expression representing ) Heatmap b e c a

+/ log2 CPM –5 10 15 M.gray M.gray del5

C I E12.5 0 5 DE genes with correlated expression patterns across brain development reveal perturbations to early and later neurodevelopmental pathways. pathways. neurodevelopmental later and early to perturbations reveal development brain across patterns expression correlated with genes DE M. M. M. M. M. M. M. M. 1,000 4 3 2 1 4 3 2 1 mice 15 e l

E14.5 E12.

10 E12.5 5 50 s

0 E17.5 5 E14.5 M. E14.5 z 1 -score 0 0 E17.5 P0 E17. 5 50 P0 0

Adult P 5 z 10 -scores generated via permutation test. permutation via generated -scores 1,000 d

15 Adult ) Functional enrichment of representative GO biological process annotations for DE genes. Scale bar, fold enrichment. enrichment. bar, fold Scale genes. DE for annotations process biological GO representative of enrichment ) Functional e 0 M. M.gray M. M. M. – log2 CPM –5 10 15 g 4 1 3 2 0 5 , directionality on on , directionality E12.5 Adul Chd8 Chd8.Mouse.Forebrain f Sanders.TADA.Q0. t

E14.5 4 4. 5 5. 6 5 5 + Hormozdiari.M –2 –1 0 1 2 / Darnell.FMRP Katayama.D del5 E17.5 Willsey.All Durak.DE M. d mice with increased increased with mice 2 E 1 3 P0 10 x All.Down -axis represents test gene set used in analysis (left of zero: downregulated DE genes; right of right genes; DE downregulated zero: of (left analysis in used set gene test represents -axis Adult M.1.Down

5 M.2.Down log2 CPM –5 10 15 Fig. 4 E12.5 0 5 M.3.Down

0 M.4.Down

z M.gray.Down a E14.5 -score 2 ). - (CPM) of module genes; CPM, counts per million. ( million. per counts CPM, genes; module of (CPM)

5 E17.5 M.All.Up and 18 WT littermates (sample details in details (sample littermates WT 18 and tively, that were differentially expressed (DE; FDR < 0.20 ( corresponding( 0.05 < FDRto genes that were robustly expressed in our data andsets. sequencingAt batch,significance we testedcutoffs for differential expressiondevelopmentalstage sex, for accounted thatacross model statistical Usinga 11,936 nataloradult brains. Afterquality filtering, analyzedwe 26 rodevelopment but has decreased sensitivity for changes limited to Thisstrategypost wasdesigned tocapture changes during embryonic neu M .3 M.1.Up

10 P0 M.2.Up

Adult M.3.Up advance online publication online advance 15 M.4.Up z P -score. ( -score.

< 0.0369), we found 510, 1,040 and 2,195 genes, respec log CPM g 2 M.gray.Up –5 10 15 E12.5 0 5 Parikshak.ASD.M19 Parikshak.DEV.M17 Parikshak.DEV.M16 Parikshak.DEV.M13 Parikshak.ASD.M9 Parikshak.DEV.M3 Parikshak.DEV.M2 GO:0016358_dendrite development GO:0007411_axon guidance GO:0007420_brain development GO:0045664_regulation ofneurondifferentiation GO:0042391_regulation ofmembranepotential GO:0007268_synaptic transmissio GO:0034220_ion transmembranetranspor GO:0051726_regulation ofcellcycle GO:0006260_DNA replication GO:0006281_DNA repair GO:0008380_RNA splicing GO:0006397_mRNA processin GO:0006338_chromatin remodeling GO:0016568_chromatin modification GO:0022904_respiratory electrontransportchai GO:0008652_cellular aminoacidbiosyntheticprocess GO:0055114_oxidation-reduction process GO:0002682_regulation ofimmunesystemprocess GO:0002253_activation ofimmuneresponse GO:0030168_platelet activatio

b E14.5 ) Mean expression across developmental stages stages developmental across expression ) Mean

E17.5 M. 4 P 1

< 0.0021), FDR < 0.1 ( 0.1 < 0.0021),FDR < P0 0 f ,

g Adult ) Enrichment of autism- and Chd8- and autism- of ) Enrichment 5

log CPM n ± 2 g –5 10 15 1 s.d., and dots depict depict dots and 1 s.d., 0 5

E12.5 n Supplementary Table 2 TableSupplementary c Supplementary Table 3 nature nature ) Gene expression plots plots expression ) Gene z -scor

0 E14.5 t e n E17.5 M.gray NEUR 5 P < 0.0088) or0.0088) <

P0 OSCI Chd8 2. 1 1. 1. 1. 1. 2 2. M.gray M. M. M. M. 4 2 4 6 8 2 1 4 3 2 1

0 Adult EN + / del5 C ). ). E - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 0.10 and 0.20. and 0.10 Similar enrichment forwas observed DE genes at FDR cutoffs of 0.05, additional numerous with annotations enriched cycle, at lower levels ( cell and remodeling chromatin processing, RNA with associated annotations for genes DE among Reactome and terms (GO) forebrains ( P0 in levels protein differential validated additionally we in listed ( qRT-PCR via genes of set a ( stage each at in reductions substantial with development, across P widespread yet subtle. The top DE gene gene were expression that was changes in indicating neurodevelopmental atively small (99.5% < 1.5 absolute fold change across phases; thalamus. Th, striatum; St, (mean and WT ( terms. GO selected with associated M.1 in ( represents area shaded and ( cycle. cell and processing 6 Figure nature nature forebrain. in mouse adult map interactions of genomic regions Chd8 c = 2.20 × 10 × 2.20 = ) STRING protein–protein interaction network of M.1. DE genes are colored by annotation to GO biological process terms. ( terms. process biological GO to annotation by colored are genes DE M.1. of network interaction protein–protein ) STRING The majority of significant expression changes in To identify direct regulatory targets of Chd8, we used ChIP-seq to ChIP-seq we used of Chd8, targets regulatory Todirect identify ± s.e.m. with dots representing individual samples) of EdU positive cells per area. Student’s Student’s area. per cells positive EdU of samples) individual representing dots with s.e.m. Chd8

NEUR An early neurodevelopmental expression network (M.1) regulated by by regulated (M.1) network expression neurodevelopmental early An d b a Supplementary Table 4 Table Supplementary

–100 log CPM

Fig. 4d 2 100 RNA processing Katnal2, Mlxip,Naa15,Nxf1,Otud4,Phf21b,Rnf38, 2700049A03Rik, Adnp,Baz2b,Cblb,Creb1,G2e3, –5 15 10 + (GO:0006396) 0 5 0 / del5 OSCI −27 E12.5 E12.5 Senp1, Slc35b2,St18,Supt16,Traf2,Trip12 Fig. 4 Fig. Papolg M.1: differentialexpressioneffectsizebystage Ncapg Sf3b1 Af2b1 Dhx9 Ints9 Ints6 Sfpq Down Up mice ( mice , FDR = 3.18 × 10 × 3.18 = FDR , , e ASD-relevant DEgenesinM. EN ). ). Gene set enrichment analysis of Gene Ontology c M.1: C ). We validated expression changes at P0 for for P0 at changes expression validated We ). E14.5 E14.5 n E = 4 mice each for both genotypes). Scale bar, 200 Scale genotypes). both for each = 4 mice

a genee (GO:0016568) neur ± ) M.1 gene expression plotted across brain development. Dots represent individual genes, line represents mean expression expression mean represents line genes, individual represent Dots development. brain across plotted expression gene ) M.1 modification advance online publication online advance Chromatin 1 1 s.d. ( 1 s.d. 8 Kdm5b Ep300 Chd1 Other Cntln pathways identified strong enrichment enrichment strong identified pathways Top1 Sirt1 ode Ctcf Rb1 E17.5 E17.5 xpr upeetr Fg 4 Fig. Supplementary v elopment ession acr ), including including ), b ) Relative mean differential expression of up- and downregulated genes in M.1 across brain development. development. brain across M.1 in genes downregulated and up- of expression differential mean ) Relative Supplementary Supplementary Tables 5 −23 (GO:0007049) P0 P0 ). ). Cell cycle Tmem67 e oss Chd8 1 Smc3 ) Left: representative coronal sections of E13.5 brain stained for EdU (magenta) and DAPI (blue) in (blue) DAPI and (magenta) EdU for stained brain E13.5 of sections coronal representative ) Left: Rb1 Ninl Chd8 (log Hnrnpa2b1 Adult Adult expression declined declined expression Chd8 2 fold change = 0.59, Chd8 e c ; primers are are primers ; + DAPI EdU WT E13.5 / del5 , for which which for , + Chd8

/ Chr RNA pr C were rel del5 Fig. Fig. 4 ell cy and St omatin modif mice mice + cle Cx / ocessing 6 del5 b ), ), ). ). HC -

E13.5 cellproliferation(EdU) ication Chd8 tested for overlap between DE downregulated and upregulated genes and upregulated DE downregulated for tested overlap between as such of reduction with ciated 5a Fig. Supplementary ( YY1 as such motifs of secondary enrichment significant, weaker, yet ChIP-seq Chd8 in but we recruitment, genomic found indirect suggesting regions, peak motif sequence primary a of evidence find not did We binding. promoter Chd8 for genes DE upregulated among ( genes downregulated among binding for enrichment strong observed we here, generated ChIP-seq Chd8 forebrain adult the in binding Chd8 exhibit not did DE genes ( and Chd8-bound strong concordance in annotation enriched functional terms between We set. data at in forebrain sively our promoters adult gene observed and ( criteria enrichment stringent using peaks 708 identified we After merging two independent samples with similar binding patterns, M.1 int µ e m. Below white dotted line is the measured neocortical area. Right: plot plot Right: area. neocortical measured the is line dotted white Below m. = 4.2 × 10 × 4.2 = In agreement with other studies of transcriptional changes asso changes transcriptional of studies other with agreement In Th Supplementary Table 3 Supplementary haploinsufficiency involved in chromatin modification, RNA RNA modification, chromatin in involved haploinsufficiency eraction net Kdm5b Chd8 −31 +/ wo del5 and and ), NRF1 ( NRF1 ), rk t Bcl11a -test, * -test, Fig. 4 Fig. ). e = 8.5 × 10 × 8.5 = were DE in in DE were Chd8 P = 0.0388. Cx, cortex; HC, hippocampus; hippocampus; HC, cortex; Cx, = 0.0388. Fig. Fig. 4 i ). Chd8 binding occurred nearly exclu nearly occurred binding Chd8 ). ). In contrast, we found no enrichment enrichment no found we contrast, In ). expression g ). ). While the majority of DE genes

−24 Number of EdU cells 1,000 Chd8 ) and NFYB ( NFYB and ) 500 0 d ) ASD-relevant DE genes genes DE ) ASD-relevant 11 + WT / EdU per cortex , del5 1 2 , autism risk genes genes risk autism , + mice ( mice cells t r a HT * e = 1.3 × 10 × 1.3 = Fig. 4 Fig. C I

Fig. 4 Fig. d e l ). We). −17 s  - - f ;

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ostatic processes, consistent with developmental timing ( timing developmental with consistent processes, ostatic and Parikshak.DEV.M17) linked to synaptic developmental and home modules ASD-relevant Gria1 ( ( rons gradually FMRP that for enriched targets expression were M.2 low in genes early Downregulated by increased. characterized was M.2 networks down regulated DE genes M.1 overlapped genes in early binding. expressed ASD-relevant Chd8 of targets for enriched module only largest the the ( genes included risk autism it downregulated DE and of number regulation, cell-cycle and RNA organization, processing chromatin with associated strongly was ment, neurodevelop across expression decreasing by characterized M.1, enriched for downregulated genes, while the other three modules modules three (M.2, M.4 and were M.grey) other for enriched upregulated genes ( the while genes, downregulated for enriched ( terms annotation and stage-specific for enriched were ules and expression trajectory modules across forebrain (WGCNA development co- ( analysis per gene network weighted expression used We identifying processes. toward stage-specific to development turbation brain during trajectories mutation. germline versus knockdown of impact differential suggesting further knockdown), after upregulated but here (downregulated genes differentiation nal neuro for including data, our versus data knockdown the in effects DE specific some of direction in reversal a Wenoted backgrounds. or and genetic alleles across different after knockdown consequences ( model germline independent the in smaller data knockdown the in larger generally were sensitivity sizes DE effect studies, and two other the and data our from genes ( DE studies two other the in and here iden tified genes between overlap significant observed we Nonetheless, model Table 3 E14.5 brain ventricle Chd8 heterozygous germline of studies independent from data RNA-seq neurodevelopmental reanalyze to here used methods same the used ( studies previous in identified genes DE regulated in genes DE downregulated down of studies independent in expression tial cortex. ASD mortem post in downregulated genes with enrichment identify not Wedid brains ASD postmortem from cortex in upregulated were that response immune with associated genes and genes upregulated DE gets studies other by identified ( genes risk autism with genes DE 4 Fig. test, (permutation enrichment strong representing in 37 our were DE data, at expressed FDR < and downregulated, 0.20 Chd8 and to relevant (FDR < gene sets autism and genetics 0.20) published t r a  Supplementary Fig. 5b Fig. Supplementary Fig. 4 Fig. Supplementary Fig. 5d Fig. Supplementary

We next explored how DE genes are organized into expression expression into organized are genes DE how explored next We differen with consistent was DE data our whether we asked Next, 2 Supplementary Table 7 Table Supplementary Supplementary Table 5 Table Supplementary 2 mutation regulation. Of 141 ASD risk genes based on case mutations case on based genes risk ASD 141 Of regulation. ( 11– . DE genes from M.2 were enriched for later developmental developmental later for enriched were M.2 from genes DE . h Supplementary Tables 5 5 Tables Supplementary 1 2 i P 5 ). We similarly observed enrichment among downregulated downregulated among enrichment observed Wesimilarly ). ). ). ) and with FMRP (fragile X mental retardation protein) tar protein) retardation mental X (fragile FMRP with and ) 2 C I and upregulated in the independent independent the in upregulated and = 0.04). Finally, we observed enrichment ( 13 , GO and Reactome terms that are hallmarks of mature neu that are hallmarks terms , GO and Reactome 2 Chd8 6 , 1 (Parikshak.DEV.M2 and Parikshak.DEV.M3; Parikshak.DEV.M3; and (Parikshak.DEV.M2 5 e l . We observed consistent enrichment among up- and and up- among enrichment consistent observed We . 1 was more strongly downregulated in the knockdown knockdown the in downregulated strongly more was 3 and s 1 5 in utero ( 2 Supplementary Fig. 5b 6 , (Parikshak.DEV.M13, Parikshak.DEV.M16 (Parikshak.DEV.M13, c ) and autism risk genes such as as such genes risk autism and ) ), suggesting differences in transcriptional in transcriptional differences ), suggesting Chd8 knockdown via ). DE genes assigned to specific mod specific to assigned genes DE ). ). Two modules (M.1 and M.3) were were M.3) and (M.1 modules Two ). and + / del5 6 forebrain and up- and down and up- and forebrain ) and include synaptic genes genes synaptic include and ) 2 5 ) to identify five discrete discrete five identify to ) Chd8 Fig. 4 Fig. 1 Chd8 3 compared to our data data our to compared Chd8 , c mutation or knock or mutation and j ). For overlapping overlapping For ). P shRNA delivery to Fig. 5 Fig. = 0.012) between mouse model mouse Fig. 4 Fig. P Supplementary = 2.8 × 10 × 2.8 = f j ). M.1 was was M.1 ). Cers4 ). We also We also ). Fig. 5a i. 5 Fig. Fig. 5 Fig. Fig. Fig. 5 i. 5 Fig. 1 5 23 and and and and 20 −10 , g g e 2 1 – , 2 1 4 3 ). ). ). ). ). ). d c 1 9 ------. . ;

an increase in the overall population as measured over the entire cortical cortical entire the over measured as population overall the in increase an ( E14.5. 7 Figure edges = 1,479, expected edges = 512, enrichment = 2.89, STRING STRING 2.89, = P enrichment 512, = edges expected 1,479, = edges genes in M.1 had more interactions than expected by chance (observed (STRING interactions tein–protein downregulation peaked from E14.5 to E17.5 ( E14.5; at peaked genes M.1 of genes Upregulation study. our DE in all identified of ~39% for accounting upregulated), 224 regulated, down (641 0.20 < FDR at DE were that M.1 in genes We865 found genes ( for downregulated enriched was significantly ( expression ing gets in mouse brain ( for autism risk early developmental modules processes. biological and stages across M.1; our in These results genes show that the impact of upregulated for enriched ASD.M19, enriched (Parikshak. microglia or M.4) and M.2 our in (Parikshak.ASD.M9, genes upregulated for astrocytes of identity func cell immune to and linked tion genes of expression increased by terized cortex ASD example, (for maturation neuronal in involved genes risk autism include and guidance) axon example, (for associated processes development terms transient GO with for enriched were brain, adult in expression lower with P0, to E12.5 from expression rising exhibited genes M.3 zone. subventricular LV,SVZ, Str, striatum; ventricle, lateral Chd8 Student’s via statistics in genotypes both for of of Chd8 EdU all over line) dashed EdU in ( ( segments ( hemisphere c f e < 0.0001). Interacting genes in M.1 were enriched for GO terms terms GO for enriched were M.1 in genes Interacting 0.0001). < ) ) Tbr2 2 We further focused on M.1, which showed the strongest enrichment c a g mm Chd8+/del5 WT (** (* . . ( + 0.08 0.09 0.10 0.11 0.12 0.13 0.14 b a + + Ki67 E14.5 d P / / del5 del5 , P = 0.0165) and and = 0.0165) h VZ 50 +

a = 0.0086). Analyses performed at E14.5; E14.5; at performed Analyses = 0.0086). 50 intermediate progenitors are significantly reduced, quantified quantified reduced, significantly are progenitors intermediate ) The rate of neurogenesis (Q fraction), measured as a ratio of a as ratio measured fraction), (Q neurogenesis of rate ) The Chd8 , % Bcl11a mice; CP, cortical plate; Cx, cortex; IZ; intermediate zone; zone; intermediate IZ; cortex; CP,Cx, mice; plate; cortical ( mice b WT − b % advance online publication online advance cells positioned basal to the proliferative VZ and SVZ ( SVZ and VZ proliferative the to basal positioned cells St Area ) Immunofluorescent analysis of Pax6 of analysis ) Immunofluorescent ), quantified in in quantified ), 2 a 4 Cx ), quantified in in quantified ), HT + , we identified enrichment with two modules charac modules two with enrichment identified we , * / del5 Pax6 h ). ). Compared to genes identified as DE in postmortem Fig. 6 Fig. ; 20-h EdU pulse; ** pulse; EdU ; 20-h Th mutants exhibit altered cortical neurogenesis at neurogenesis cortical altered exhibit mutants

e No. of cells per 200 µm

Fig. 5f DAPI – VZ t 150 200 250 300 b g -test. Error bars represent mean mean represent bars Error -test. a ; ; + (** ), had the greatest number of DE genes and and genes DE of number greatest the had ), cells within the same segment, is increased in increased is segment, same the within cells n Pax f = 3 mice each for both genotypes in in genotypes both for each = 3 mice . Scale bars, 500 500 bars, . Scale WT P , e g = 0.0032), respectively. ( respectively. = 0.0032), 6 , and within midcortical 200- midcortical within , and ). M.1 showed a general trend of decreas + cells HT ** h g 2 P 7 d c Chd8 No. of cells per 200 µm SVZ = 0.0057). ( = 0.0057). ) showed that DE (FDR < 0.10) < 0.10) (FDR DE that ) showed 150 200 250 Tbr2 haploinsufficiency reaches

2 Tbr2 µ

6 WT m in m in and for Chd8 binding tar nature nature

Fig. 6 DAPI + + radial glial cells reveals reveals cells glial radial cells HT ** n a = 4 mice each each = 4 mice e and 100 100 and b , ). Analysis of pro f ± g ) Quantifications ) Quantifications P NEUR s.e.m. HT, s.e.m. VZ SVZ IZ CP ) Quantification ) Quantification

Q fraction Ed = 8.8 × 10 0.10 0.15 0.20 0.35 µ U

m-wide m-wide Ed Total numberof

Ki67 OSCI Number of Ed U µ + i. 5 Fig. h m in m in Ki67 U WT d . All . All +

cells ;

DAPI – cells EN

HT −26 ** b – g C d ). ). ). ). E ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. To examine whether alterations in developmental genes play a func a play genes developmental in alterations To whether examine Chd8 in progenitors neural of proliferation prenatal Increased ( terms GO highlighted the to annotated many including genes, autism-relevant 38 DE include M.1 genes expression. gene and regulation Chd8 of level the at esses proc three these among interconnectedness highlight results these cycle ( cell and modification chromatin processing, RNA including adjusted points, time developmental across regression Linear Chd8 ( (chr3:126,644,528–126,650,725). ( change. splicing of direction and events developmental and genotype E17.5 between ( gray). (light WT E17.5 vs. WT E14.5 between spliced differentially ( right). (top C complex spliceosomal active the and left) (top pre-mRNA nascent of schematics simplified depict Images once. represented ( ( cleavage ( pre-mRNPs of formation the to annotated 8 Figure nature nature tional role in neuronal development that could lead to megalencephaly, c b b a Fig. 6 Fig. Mutually exclusiveexon c Alternative Alternative ) We detected 591 differential isoforms (dark gray) by MISO between E17.5 E17.5 between MISO by gray) (dark isoforms differential 591 ) We detected ) Schematic of the Reactome RNA-processing pathways. DE genes in the pathways are denoted as squares colored by module. Each gene is gene Each module. by colored squares as denoted are pathways the in genes DE pathways. RNA-processing Reactome the of ) Schematic Relative expression Alternative firstexon Alternative lastexon (RPKM) + 5.5 6.0 6.5 7.0 7.5 / +/ del5 c Tandem Retained intron del5

Srsf7 ). While protein–protein interaction databases have biases, biases, have databases interaction protein–protein While ). NEUR Skipped exon RNA processing pathways are enriched for differentially expressed genes in in genes expressed differentially for enriched are pathways processing RNA Upf3b , 7 E17.5 WT,Welch’s , 7 E17.5 mice; P0 11 3 5 5 ′ ′ Mouse DPC(P0=21DPC) splicesite splicesite 12 mice ′ WT expression(FDR=5 Snrnp70 Prpf40a Hnrnpa2b1 3 Ccar1 Srrm Rbm Ddx5 Dhx9 Hnrnph2 Hnrnph1 Hnrnpa3 Hnrnpa1 OSCI Hnrnpm Hnrnpd ′ Fus Hnrnpf Chd8 UTR 14 Gtf2f1 ). DE FDR shown; RPKM, reads per kilobase of transcript per million mapped reads. Solid lines, means; dashed lines, ±1 s.e.m. s.e.m. ±1 lines, dashed means; lines, Solid reads. mapped million per transcript of kilobase per reads RPKM, shown; FDR DE ). Chd8 1 5 16 Alternative transcriptevents identified byMISObetween EN +/ + 5 0 del / del5 5 C 18 E17.5 HTandWT Hnrnpu E Hnrnpr Srsf10 Polr2g Polr2e Polr2c Polr2a Srrm Ptbp1 Rbm (HT) vs. E17.5 WT (dark gray) and E14.5 WT vs. E17.5 WT (light gray) comparisons, showing strong correlation between between correlation strong showing comparisons, gray) (light WT E17.5 vs. WT E14.5 and gray) (dark WT E17.5 vs. (HT) Polr2f Srsf7 Srsf5 Srsf3 Srsf1

Srrt 0 E14.5 HTvsE17.5WT E17.5 HTvsWT 20 advance online publication online advance Count 2 x × 10 100 Fig. 6 Fig. 22 –2 3 ) ′ 150 f ) Isoform-specific qRT-PCR the of ) Isoform-specific d

). Relative expression

Hnrnpa1 (RPKM) Hnrnpa1 3.0 3.5 4.0 4.5 5.0 Formation ofpre-RNPs t d -test, E14.5 WT vs. E17.5 E17.5 vs. WT E14.5 -test, PSI (E14.5 WT vs E17.5 WT)

∆ spliceosomal Mouse DPC(P0=21DPC) 12 E14.5 E17.5 Formation of expression(FDR=3.6 WT mRNA transcription –0.5 ), formation of the spliceosomal E complex ( E complex spliceosomal the of formation ), complex 0.5 1.0 –1.0 (E17.5 14 0 & 5 Chd8 spliceosomal A ′ capping HT B complexes Formation of 16 –0.5 Chd8 +/ E del R 5 18 2

+/ = 0.77, = 0.77, ∆ del PSI 20 0 5 vs.E17.5WT) d & ) Scatterplot of change in percentage spliced in ( in spliced percentage in change of ) Scatterplot × |∆ |∆ e 10 PS PS 22 ) Genome Browser representation of the alternatively spliced spliced alternatively the of representation Browser ) Genome 0.5 I I | | Ank2 –3 P >0.10 <0.10 - - Chd8 Not DE M.gray M. M. M. M.

= 4.53 × 10 = 4.53 ) WT 4 3 2 1 1.0 for focal cortical lesions. To lesions. changes in for proliferative cortical delineate focal further evidence no found and lamination to alterations gross no observed Brn2 by immunostaining at P0 and P7 ( P7 and P0 at immunostaining by Brn2 cortical the cytoarchitecture via analysis examined of layer-specific markers we Tbr1, Ctip2 and DE, were structure cortical and opment devel the brain with associated in genes of number a since Additionally, neurogenesis perturbed indicating ( animals mutant of zones respectively) SVZ, and (VZ subventricular and ventricular cortical germinal the in cells EdU in increase 15.9% a observed we pulse, 1.5-h a After E13.5. at 5-ethynyl-2 performed we exon in WT and E17.5 E17.5 and WT in exon Chd8 Relative expression + /

del5 (RPKM) e 5.5 6.0 6.5 7.0 7.5 8.0 E17.5 WT E12.5 WT E17.5 HT + , , *** / Dhx9 Chd8 del5 Ank2 Ank2 Formation ofactive Docking &transport −14 Mouse DPC(P0=21DPC) spliceosomal 12 and E17.5 WT brains. Of these, 393 isoforms were also also were isoforms 393 these, Of brains. WT E17.5 and expression(FDR=7 P 0 7 0 7 0 7 WT through NPC Lariat formation, + = 0.001; E17.5 E17.5 = 0.001; . Error bars represent mean mean represent bars . Error complex / del5 and polyadenylation splice sitecleavage, 14 Cleavage of and exonligation Chd8 mice. ( mice. Srsf7 5 kb 16 Skipped exon +/ C del and and 18 5 Chd8 3 a 5 ′ ) Developmental expression of DE genes genes DE of expression ) Developmental end ′ and 20 Dhx9 ′ × -deoxyuridine (EdU) proliferation assays assays proliferation (EdU) -deoxyuridine + 10 Chd8 / del5 3 22 –2 ′ ), and lariat formation and 5 and formation lariat and ), ) mice ( mice + / del5 Relative expression f vs. E17.5 WT, E17.5 vs. * ∆

± (RPKM) n Fold change PSI) of isoforms detected detected isoforms of PSI) s.e.m. 4.0 4.5 5.0 5.5 = 7 E12.5, 9 E14.5, 7 E17.5 7 E17.5 9 E14.5, = 7 E12.5, (retained/skipped) Upf3b Supplementary Fig. 6 Fig. Supplementary Exon 10 15 20 Cwc27 Cwc22 Dhx38 Ddx46 Fip1/1 Elavl2 Cpsf7 Cpsf4 0 5 Clp Inclusion of

E12.5 Mouse DPC(P0=21DPC) 1 12 expression(FDR=6.9 1 WT Chd8 P E14.5 14 = 0.0388; 0.0388; = t r a Ank2 Chd8 Ank2

E17.5 HT *** Pabpn U2sur Rbm1 Sympk Srsf11 16 P

Sf3b

Sf3b Lsm Sart Exon + = 0.03). = 0.03). / +/ del5 exon exon del exonovertime

1 7 3 1

E17.5 WT 18 p

7

5 1 2 ′ * splice site site splice C I mutants. mutants. 20 Fig. 6 Fig.

e l ×

). We ). 10 P0 22 –5 e s ) ), ), +  -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. the the ~6-kb a of exclusion or inclusion event, opmentally relevant developmental state between WT E14.5 and E17.5. We validated devel DS in E17.5 suggests ( comparison WT E17.5 versus the WT in E14.5 values PSI with positively correlated comparison WT E17.5 E17.5 the in identified events DS of values (PSI) in E17.5 and WT E17.5 correlation versus WT E14.5 both in examined present events DS we between this, investigate To splicing. in changes and WT between ing WT mice E17.5 ( del5 mice is linked to ASD-relevant DS ( networks risk autism in reported been has but neurodevelopment and has not been functionally characterized in brain Table6 Supplementary perturbed splicing in in splicing perturbed ( via MISO analysis in changes splicing developmental validated Wealso in from WT levels E17.5 the decrease in and WT development across increase the exon inclusion validated expectations with consistent mortem ASD cortex mouse brain were enriched for DS-associated genes identified in post expression ( possibility that splicing changes explain some proportion of differential regulated( ated with a DS event in neurodevelopment in WT mice ( for differential splicing (DS) between WT and Mixture of Isoforms (MISO brain development linked to DE of RNA-processing genes. We used the examinedwhether required during neuronal differentiation that is implicated inexpression ID increases across development and is a neuron-specific factor ( database Reactome the in splicing mRNA and ing significantly overrepresented among genes annotated to RNA process divergentwereexpressiontrajectorieswith genes DownregulatedDE Chd8 in perturbed is processing RNA Neurodevelopmental Chd8 in megalencephaly for substrate a cellular represent may neuron production projection cortical to alteration that indicate results These in ~25% in by increase fraction Q an to corresponding increased, significantly were cells EdU Ki67. marker proliferation the 20 h, using over a defined cycle cell the exiting cells cortical of to fraction (Q) quit the assays determine pulse-chase performed we Finally, types. progenitor two P observing a significant decrease by ~24% in P observed ( a ~26% increase in Pax6 ~18% by increased, significantly was in VZ The hemisphere. cortical entire the over ( Chd8 t r a 1 in phenotypes neurodevelopmental Fig. 0 = 0.0032). Subsequently, we labeled Tbr2 = 0.0086), indicating different proliferative trajectories for these these for trajectories proliferative different indicating 0.0086), = At E17.5, MISO identified 591 DS events between WT and WT between DS 591 events identified AtMISO E17.5,

mice, of which 393 (~66%) were also DS between E14.5 WT and and WT E14.5 between DS also were (~66%) 393 which of mice, Ank2 7 + + +/ Chd8 ). ). First, we examined Pax6 / / del5 del5 del5 C I exon increased between E12.5 and E17.5 in WT ( WT in E17.5 and E12.5 between increased exon mice. mice, we assessed neural progenitor populations at E14.5 E14.5 at populations progenitor neural assessed we mice, P mice Supplementary Fig. 7a + = 1.2 × 10 × 1.2 = e l / del5 s versus E17.5 WT comparisons. Percentage spliced spliced Percentage comparisons. WT E17.5 versus Fig. Fig. 8 Chd8 Supplementary Fig. 7b Supplementary 2 Chd8 4 Chd8 ( Chd8 P Chd8 c Chd8 −10 ). These results suggest that differential splic ). that results suggest differential These = 1.2 × 10 Chd8 ). For example,For ). + / + ) but not upregulated DE genes, raising the raisingupregulatedgenes,not butDE ) del5 + / haploinsufficiency may contribute to the to the may contribute haploinsufficiency 3 del5 / + + 0 del5 ) program to examine our RNA-seq data 3 mice exhibitedmice aberrant splicing during / / + del5 del5 1 DS using a known neurogenic splicing / . qRT-PCR analysis of the the of analysis qRT-PCR . Supplementary Table 8 del5 mice corresponds to an intermediate to an intermediate corresponds mice + mice significantly overlapped down mice was linked to developmental developmental to linked was mice + radial glial cells, measuring VZ area −17 cells in embryos ( embryos Supplementary Fig. 7a ). DS genes identified in ), suggesting that DS in Chd8 Dhx9 i. 7a Fig. Chd8 + + Ank2 Fig. 8 Fig. / intermediate progenitors, Chd8 ). Our results suggest ). that Our suggest results Chd8 del5 Chd8 (M.1) decreases across decreases (M.1) Fig. 7d Fig. mice. 2 + exon 0 , / + + d , while while , e del5 Chd8 + / / ; ; del5 ). This correlation correlation This ). / del5 del5 P Chd8 brains ( , 006) We 0.0165). = mice ( 3 mice and across h mice ( ). Genes associ 1 + Srsf7 i. 8a Fig. ; ; . Inclusion of of Inclusion . / del5 P Upf3b + = 0.0057). 0.0057). = ). Ank2 / del5

Chd8 Chd8 embryos embryos detected detected Fig. 7c Fig. 7b Fig. 8 Fig. Fig. Fig. 8 28 Chd8 + versus versus , (M.3) , b Ki67 2 exon exon 9 and + + . We / / del5 del5 e , f , g + ), ), ). ). f − ------/ ; ;

ing to generate indel mutations in in mutations indel generate to ing Platt onic while stem cell targeting, Katayama of knockdown restricted an reported of downregulation with associated tions There are recent publica three behaviors. or repetitive sociability cal atypi no but impairments, memory and learning specifically mice, phenotypes. neurodevelopmental produces factor matin chro general a of haploinsufficiency why into insights mechanistic and phenotypes NDD-relevant revealed study Our species. between CHD8 types in our pheno neuroanatomical and genomic of presence The velopment. of of consequences the picture integrative initial an here present we haploinsufficiency, in in resulted mutations deletion 14-bp and 5-bp germline that validation After mutations. loss-of-function single-copy with ated associ strongly are which ID, and ASD like disorders of robiology neu the understanding to critical is development brain mammalian impact mutations germline heterozygous how constitutive Modeling DISCUSSION other other brain Additionally,amygdala. regions, including our components of fear conditioning and novel object recognition require while since maze spatial tasks are hippocampal-dependent, primarily conducted, tasks the to attributed be could phenotypes cognitive in Discrepancies assays. T-maze and Barnes the using learning sition relevant but report phenotypes to deficits report social normal acqui Katayama background. genetic or allele to due be may that our model. We differences in observed strength of expression changes to changes expression gene neurodevelopmental compare to directly Katayama on the comparison our focus heterozygous in phenotypes social specific report Katayama in reported those with agree behaviors of data on repetitive absence the Our challenge. a comparisons cross-approach make and differences down between mechanisms and consequences biological tory discrimination. Another difference is that Katayama Katayama that is difference Another discrimination. tory olfac impacting factors other to due be could or interaction social models these in mouse novel a discriminate to failure but sociability assay three-chamber normal of combination the such, As systems. processing and sensory of ety vari a by driven is which discrimination, novelty olfactory on dependent is social for Preference mouse. familiar and novel a between of failure on models other other for reported of differences genotype detection and signal larger a for allows which freezing, fear tual contex during freezing basal higher show generally C57BL/6N and Background strain differences in behavioral phenotypes are common, Katayama while mutation was generated and maintained on a C57BL/6N background, in genes implicated in ASD have not identified deficits in social social in deficits identified not have ASD in implicated genes in mutations with models mouse other of studies several that note also We variable. confounding a as aging avoid to weeks, 6–16 between ages, younger at sexes both using conducted was battery our while age, of weeks 12–50 of range age broad a with mice male only tested We report behavioral outcomes in our heterozygous heterozygous our in outcomes behavioral report We Our finding of normal sociability is consistent with results results with consistent is sociability normal of finding Our 1 mutations, suggesting similar neurodevelopmental pathologies 5 and our germline heterozygous model likely explain many many explain likely model heterozygous germline our and advance online publication online advance et al. Chd8 n utero in et al. et 13 Chd8 1 3 , 1 generated two lines of et al. et 4 + , report abnormalities in social behavior based based behavior social in abnormalities report , / 1 del5 +/− 3 Chd8 and Platt Platt and 3 1 2 knockdown of of knockdown mice paralleled the clinical signature of human mice to exhibit preference for social novelty novelty social for preference exhibit to mice 3 . harbored a different mutation on C57BL/6J. C57BL/6J. on mutation different a harbored +/− Chd8 emie models germline Chd8 1 et al. et 3 in upper cortical layer neurons. neurons. layer cortical upper in , 1 4 Chd8 et et al. could represent mild deficits in in deficits mild represent could haploinsufficiency on neurode haploinsufficiency 1 4 ; however, these studies also also studies these however, ; Chd8 Chd8 1 et et al. . The obvious differences in in differences obvious The . Chd8 4 used CRISPR/Cas9 target CRISPR/Cas9 used nature nature mutant mice via embry 1 expression, including including expression, 3 in mice. Durak Durak in mice. study as we were able able we were as study 1 3 , 1 4 . However, both both However, . NEUR Chd8 in utero in Chd8 mice. We mice. OSCI Chd8 Chd8 knock exon 5 exon t al. et et al. et et et al. EN + + / / del5 del5 C 1 1 1 E 3 5 3 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. apoptosis will be needed to understand the comprehensive impact of impact comprehensive the to understand be needed will apoptosis and proliferation cycle, cell of analysis Time-point-specific timing. non-cell-autonomous effects or due to differences in proliferation assay initial expansion symmetric of the radial glia pool), by the presence of could be explained by the timing of an in reported proliferation with the contrasts decreased eration in our experiments genitor populations involved. overall in increase The prolif observed for pro the consequences different with neurogenesis, of embryonic brain in reported been have patterns interaction genomic Chd8 contrasting that given critical especially is This binding. Chd8 of roles and patterns cell-specific and stage- determine to and tures expression cal signa and with changes differential cellular associated neuropathology. Further studies are needed to capture neuroanatomi transcrip tional regulation here, appears to indirect be an important by player in driven likely processing, RNA to disruption other NDD genetic models such with as seen As expression. gene target activating in levels expression WT for requirement and role direct suggest results our genes, cell cycle, including numerous genes implicated in ASD. Among and these processing RNA state, chromatin of control to central network interactive highly a represents which M.1, in genes of dysregulation in changes of hierarchy a response immune as ways path synaptic and differentiation neuronal remodeling, chromatin gene networks identified in ASD case sequencing studies, particularly Our results indicate convergent principle connecting neuropathology processes. and stages developmental across perturbations capture to at across neurodevelopment a level of sufficient detail loinsufficiency using analysis, biological network Our interest. of modules with expression and pathways correlated strongly and networks ASD-associated to relevant highly genes perturbed for stages developmental across consistent were changes expression Differential splicing. differential in development brain across in behavior and changes structural between relationships causal for test to needed be will work Future mice and appear largely overlapping in neurogenesis developmental in and length present anteroposterior cortical in Increases changes neurodevelopment. transcriptional widespread the by the in deficits excitatory (for example, neuroanatomy beyond group this within models mouse other Fmr1 The included models of group the with line in is which lum, cerebel the in structures smaller and lobes, parietal and frontal the particularly structures, cortical larger included 1 Group of aspects to autism related models mouse different 26 examined mod study mouse recent A relevant els. other in found those parallel here observed ASD-relevant evaluate mice. in behavior to used methods of sensitivity or ferences, dif background genetic or allele neurobiology, human and mouse behaviors nature nature Chd8 We report overall increased proliferation during the peak window window peak the during proliferation increased We overall report transcription in changes subtle captured analysis RNA-seq Our adult of brain the in changes structural The Chd8 7 haploinsufficiency on haploinsufficiency cell populations in the brain. It is possible mutant mice. Further examination may reveal similarities with similarities may reveal examination mutant Further mice. in vivo , 8 3 , and those identified in other ASD-relevant pathways such such pathways ASD-relevant other in identified those and , 4 NEUR , clustering these models into three distinct groups. Key Key groups. distinct three into models these clustering , Wdfy3 3 + 3 / , potentially due to evolutionary divergence between between divergence evolutionary to due potentially , del5 data, enabled characterization of the impact of OSCI in in utero Chd8 mouse most resembled the differences found in the the in found differences the resembled most mouse mutants, another model of megalencephaly in ASD EN 2 C 4 and FMRP binding FMRP and E

Chd8 advance online publication online advance Nrxn1 knockdown model knockdown Chd8 Chd8 + / del5 α Fmr1 in utero , , + + Chd8 brain development anchored by anchored development brain Nrxn1 Shank3 / / del5 del5 (ref. mice, as well as evidence of of evidence as well as mice, mouse described here. This This here. described mouse + / knockdown at E14.5 (after α del5 , , 2 mouse 2 En2 3 . Our findings suggest suggest findings Our . mice. 7 ) ) and 1 and and 5 . These differences differences . These 3 Chd8 5 Rbfox1 ), as suggested suggested as ), Fmr1 + 11– Chd8 Chd8 Chd8 Chd8

/ mutants. mutants. del5 (ref. 1 4 . mice mice Chd8 hap + + + 3 / / / del5 del5 del5 8 3 6 ), ), ------.

a bellwether for mutations affecting chromatin remodeling in autism. associated with mutations to resultsnotypes. Our offer insight into neurodevelopmental pathology Chd8 studies will be necessary to replicate and compare findingsgenesis and for acrossprioritizing risk genes frommutant genetic studies. Additional represent a resource for dissecting the pathways involvedmutations carry who in in NDD patho neurodevelopment, recapitulating traits observed in human individuals regional brain volume and perturbations ofincreased biological pathways deficits, across cognitive link experiments Our neurobiology. of axes behavioral and anatomical genomic, across findings significant mutation. germline heterozygous with occur to appear not did down caused an early shift from proliferation to differentiation after upregulated were in our M.3 module downregulated genes development neuronal this, pathways. In cellular of support with developmental further interfere stages early very tion greater in resulted knockdown that in considering differences to due are outcomes contrasting that 1. claims jurisdictional in published maps and affiliations. institutional reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial All authors contributed to manuscript revisions. L.S.-F., J.E., K.S.Z., N.A.C., J.N.C.,M.A.R., J.L.S. and A.S.N. drafted the manuscript. B.J.M. and A.S.N.; neuroanatomy: T.W.S.,A.L.G., M.A.R., I.Z., G.K., K.S.Z. A.L.G., J.P.L.; genomics and molecular genetics: L.S.-F., A.L.G., I.Z., A.A.W., R.C.-P., S.L., mouse behavior: N.A.C., M.C.P., M.D.S., J.N.C. and J.L.S.; mouse MRI: J.E. and Generation of mouse model: A.S.N., D.E.D., A.V., L.A.P., B.J.M., I.P.-F. and V.A.; N.A.C., J.P.L., J.N.C., J.L.S., K.S.Z. and A.S.N. the designed experiments. led components of the experiments and analysis. A.L.G., L.S.-F., J.E., M.A.R., A.L.G., L.S.-F., J.E., N.A.C. and areM.A.R. listed as joint first authors, as each Canada and the Ontario Brain Institute. J.E. and J.P.L. were supported by the Canadian Institute for Health Research, Brain Department of Energy Contract DE-AC02-05CH11231, University of California. conducted at the E.O. Lawrence Berkeley National Laboratory was performed under U01DE024427, R01HG003988, U54HG006997 and UM1HL098166. Research D.E.D. were supported by National Institutes of Health grants R24HL123879, by a Science Without Borders Fellowship from CNPq (Brazil). A.V., L.A.P. and Training Grant number T32-GM007377 from NIH-NIGMS. R.C.-P. was supported by grant number T32-GM008799 from NIGMS-NIH. A.A.W. was supported by by the UC Davis Floyd and Mary Schwall Fellowship in Medical Research and Center (U54 HD079125) and by NIGMS R35 GM119831. L.S.-F. was supported the UC Davis MIND Institute Intellectual and Developmental Disabilities Research was supported by institutional funds from the UC Davis Center for Neuroscience, by Sequencing was performed at the UC Berkeley and UC Davis DNA cores. This work online version of the pape Note: Any Supplementary Information and Source Data files are available in the the the in available are references, and codes accession statements of including Methods, and data availability any associated M 2. COM AUTH Acknowledgmen

et Our initial survey of mice heterozygous for mutation to Ho, L. & Crabtree, G.R. Chromatin remodelling during development. during remodelling Chromatin G.R. Crabtree, & L. Ho, Chen, T. & Dent, S.Y.R. Chromatin modifiers and remodellers: regulators of cellular of regulators remodellers: S.Y.R. and T.Dent, modifiers Chen, & Chromatin differentiation. (2010). 474–484 pape 1 P h 5 alleles and genetic backgrounds and to clarify dosage-specific phe O . As complete complete As . E ods TI R R r CON . NG FI TRIBUTI N l . . Publisher’s with neutral Nature remains regard to note: Springer Nat. Rev. Genet. Rev. Nat. A 1 0 NC , downregulation beyond haploinsufficiency may may haploinsufficiency beyond downregulation , ts r IA Chd8 . ON L in utero in CHD8 I N S loss impedes embryonic development at development embryonic impedes loss T CHD8 E

15 R . The transcriptionThe . datagenerated here E knockdown, suggesting that knock that suggesting knockdown, , 93–106 (2014). 93–106 , STS , a genetic model that appears to be http://www.nature Chd8 t r a o nline version of version nline Chd8 downregula Chd8 C I Nature revealed dosage, dosage, 1 e l 5

.com/

that 463 11 s - - - - , © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. t r a 12

Hormozdiari, F., Penn, O., Borenstein, E. & Eichler, E.E. The discovery of integrated S.J. Sanders, D. Croft, Silverman, J.L.,Babineau,B.A.,Oliver, C.F., Karras,M.N.&Crawley, J.N.Influence assays phenotyping Behavioural Crawley,J.N. & C. Lord, Yang,M., J.L., Silverman, O. Durak, Platt, R.J. Y. Katayama, J. Cotney, A. Sugathan, Nishiyama, M. R. Bernier, Iossifov,I. intellectual S. in Rubeis, De studies Genetic J.A. Veltman, & C. Gilissen, L.E.L.M., Vissers, S.E. McCarthy, cognition: disorder.spectrum autism to of neurobiology the of glimpses First development S.J. Sanders, neural From G.R. Crabtree, & W. Wu, J.L., Ronan, ee ewrs o ats ad eae disorders. related and (2015). autism for networks gene loci. risk 71 from biology and (2014). D472–D477 model autism. mouse of BTBR the in approach social on hyperactivity stimulant-induced of autism. of models mouse for signaling. Wnt and cycle cell of circuits. mice. (2015). uim ik ee drn hmn neurodevelopment. human during genes risk autism (2014). E4468–E4477 progenitors. neural in disorder spectrum autism Duplin. protein development. disorder. autism. disorders. related and disability disability. intellectual Psychiatry Mol. and autism with overlap genetic a support and remodeling Dev. Genet. Opin. chromatin. for roles unexpected C I Nature Nature Cell Rep. Cell Nature t al. et et al. et et al. t al. et et al. et Neuropharmacology e l t al. et

et al. et 537 et al. et Cell t al. et et al.

h Ratm ptwy knowledgebase. pathway Reactome The t al. et Chd8 mediates cortical neurogenesis via transcriptional regulation transcriptional via neurogenesis cortical mediates Chd8 515 Mol. Cell. Biol. Cell. Mol. Chd8

The contribution of contribution The 19 The autism-associated chromatin modifier CHD8 regulates other regulates CHD8 modifier chromatin autism-associated The t al. et 515 s Disruptive

, 675–679 (2016). 675–679 ,

33 158 , 652–658 (2014). 652–658 , 19 CHD8 regulates neurodevelopmental pathways associated with associated pathways neurodevelopmental regulates CHD8

Early embryonic death in mice lacking the beta-catenin-binding nihs no uim pcrm iodr eoi architecture genomic disorder spectrum autism into Insights , 209–215 (2014). 209–215 , CHD8 yatc tasrpinl n crmtn ee dsutd in disrupted genes chromatin and transcriptional Synaptic, , 216–221 (2014). 216–221 , Mutation Leads to autistic-like behaviors and impaired striatal , 80–92 (2015). 80–92 , , 335–350 (2017). 335–350 ,

, 263–276 (2014). 263–276 , e novo De haploinsufficiency results in autistic-like phenotypes in phenotypes autistic-like in results haploinsufficiency CHD8 Nat. Rev. Neurosci. Rev. Nat.

Neuron

68 uain i shzprna mlct chromatin implicate schizophrenia in mutations 24 Nat. Rev. Genet. Rev. Nat. Nat. Rev. Genet. Rev. Nat. Nat. Neurosci. Nat. , 210–222 (2013). 210–222 , , 8386–8394 (2004). 8386–8394 , mutations define a subtype of autism early in early ofautism a subtype define mutations de novo de

87 , 1215–1233 (2015). 1215–1233 , coding mutations to autism spectrum autism to mutations coding

19 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc.

17 11 14 , 1477–1488 (2016). 1477–1488 , , 9–18 (2016). 9–18 , eoe Res. Genome , 490–502 (2010). 490–502 , , 347–359 (2013). 347–359 , a. Commun. Nat. uli Ais Res. Acids Nucleic

25 142–154 ,

6 6404 ,

111 Curr.

42 , , 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 38. 37. 36.

Etherton, M.R., Blaiss, C.A., Powell, C.M. & Südhof, T.C. Mouse neurexin-1alpha Mouse T.C. Südhof, & C.M. Powell, C.A., Blaiss, M.R., Etherton, Ellegood,J. fear trace Brunner, D. and Delay K.M. Lattal, & K.J. Buck, J.D., Raybuck, M.E., Tipps, X. Zhang, RNA of design and Analysis C.B. Burge, & E.M. Airoldi, E.T., Wang, Y., Katz, Laumonnier,F. T. Alrahbeni, L.J. Jensen, N.N. Parikshak, Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network Parikshak, N.N. I. Voineagu, Darnell, J.C. A.J. Willsey, Lee, J.-A. Hagerman, R., Au, J. & Hagerman, P. FMR1 premutation and full mutation molecular L.A. Orosco, models to gain insight into the heterogeneity.insightgainthemodelsintoto disorder. spectrum autism of models mouse Mem. Learn. conditioning in C57BL/6 and DBA/2 mice: issues of measurement and performance. cortex. cerebral developing regulation. isoform identifying (2010). 1009–1015 for experiments sequencing or with autism. retardation without mental nonspecific with associated are neurons, in expressed cells. stem neural organisms. 630 in interactions autism. in circuits and pathways molecular analysis. autism. in patterns expression pathology. molecular autism. and function autism. of pathogenesis the in neurons projection related genes. related autism. to related mechanisms pathology. autism the of aspects reflecting (2009). impairments. cognitive with consistent changes behavioral and electrophysiological correlated causes deletion advance online publication online advance BMC Bioinformatics BMC et al. t al. et et al. et al. et

et al. et al. et 21 t al. et t al. et t al. et t al. et Cytoplasmic Rbfox1 regulates the expression of synaptic and autism- Neuron et al. et et al. , 380–393 (2014). 380–393 , Mol. Psychiatry Mol. Comprehensive analysis of the 16p11.2 deletion and null Cntnap2 t al. et Clustering autism:usingneuroanatomical differencesmouse 26 in Cell-type-specific alternative splicing governs cell fate inthe fate cell governs splicing alternative Cell-type-specific FMRP stalls ribosomal translocation on mRNAs linked to synaptic Coexpression networks implicate human midfetal deep cortical deep midfetal human implicate networks Coexpression Mol. Brain Mol. ul P3 fnto i ciia fr ernl ifrnito of differentiation neuronal for critical is function UPF3B Full os f dy i mc atr crba cria neurogenesis cortical cerebral alters mice in Wdfy3 of Loss rncitmc nlss f uitc ri rvas convergent reveals brain autistic of analysis Transcriptomic TIG -a lbl iw n rtis n ter functional their and proteins on view global 8--a STRING Mutations of the of Mutations Genome-wide changes in lncRNA, splicing, and regional gene Nature Cell

nertv fntoa gnmc nlss mlct specific implicate analyses genomic functional Integrative 89

, 113–128 (2016). 113–128 , 146 Cell

474

8 9 rc Nt. cd Si USA Sci. Acad. Natl. Proc. , 247–261 (2011). 247–261 , Nature

, 33 (2015). 33 , , 559 (2008). 559 ,

Nucleic Acids Res. Acids Nucleic J. Neurodev. Disord. Neurodev. J. 166 15 , 380–384 (2011). 380–384 , , 767–776 (2010). 767–776 , , 1147–1162.e15 (2016). 1147–1162.e15 ,

UPF3B 540 , 423–427 (2016). 423–427 , Nat. Commun. Nat. Cell Mol. PsychiatryMol. PLoS One PLoS gene, which encodes a protein widely protein a encodes which gene,

155

Cell nature nature 37

, 1008–1021 (2013). 1008–1021 , 3 , 211–224 (2011). 211–224 , , D412–D416 (2009). D412–D416 ,

155 10

, e0134572 (2015). e0134572 , 5

, 997–1007 (2013). 997–1007 ,

, 4692 (2014). 4692 , 20 106 NEUR , 118–125(2015)., a. Methods Nat. 17998–18003 , OSCI EN

C 7 E ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Blocker solution (Millipore), visualized using a LI-COR Odyssey CLx system CLx Odyssey LI-COR a using visualized (Millipore),solution Blocker Fluorescent in overnight incubated were antibodies primary Sigma-Aldrich) (ref. (Invitrogen). Anti-Chd8 (ref. NovexblottingsystemTriswesternthe 3–8%using gelacetateaprotein onof plate reader to assess protein concentration using a standard curve. We andran 22the supernatant was used for a BCA Bradford assay usingprotease inhibitor thecocktail (Roche). Aftersonication, samples Spectramaxwere spun down 190 Triton0.25% andwith NP40 0.5%glycerol,EDTA, 10%mM 1 NaCl, nM 140 TrismM 8, 50 littermates),pH HCl,in neonatesadults werelysed P0P60 and W Welch’s stages and between to differentiallyaspecific spliced exon of isoform analysis of from reverse-transcription PCR on cDNA libraries prepared from forebrains dissecteddiscarded. To validate differential splicing, we used isoform-specific quantitativeweregroups both from valueslowest and ∆∆CT.highest Tothe noise, reduce on normalized relative gene expression between WT and failed. Cycle counts were normalized to replicates technical if excluded were Samples used. were samples female and Table 4 listed in the “Genomics” section, below. Primers are reportedprotocols same in the followingprepared librariescDNA using P0, qRT-PCRat qRT-P number of animals used. a 12-h light–dark cycle. Efforts were made to minimize pain and distress and the Subject mice were housed in a temperature-controlled vivarium maintained on University of California Davis and the Lawrence Berkeley National Laboratory. ies were approved by the Institutional Animal Care and Use Committees at the both males and females were used unless otherwise described. All mouse stud Chd8 andcomparedandatP0whole-mount E14.5 in length cortical brains P0 from We examined Chd8 protein and transcript in levels via western reported blot andprimers qRT-PCR the with analyses, in included animals all for sequence-verified and PCR allele-specific via identified were tions caused by Cas9 targeting would persist in our mutant wild-type animals vastly reduces the likelihood that any potentialfemales (Charlesoff-target River). Extensivemuta crossing of heterozygous mutation carriers to DavisUC andmaintained bycrossing carriersmalewithC57BL/6N wild-type genotyping was performed using allele-specific PCR with sequence verification. Chd8 protein levels. Deletion alleles were subcloned to verify DNA changes, and mediated decay based on reduced frequency of mutant transcripts and decreased to be 2,582 amino acids. Both deletion alleles are predicted to result in nonsense- tively,before stopa codon isreached. The full-length Chd8 protein ispredicted tion frameshifts are predicted to result in 16 and 14 altered amino acids, respec amino acid sequence through position 604, after which the 5-bp and 14-bp dele (14-bp deletion) of the target sequence. The deletions occur at position 1,814 (5-bp deletionsdeletion)(mm9:chr14:52,847,249–52,847,262) or in 1,813 identified F0 pups carrying 5-bp (mm9: chr14:52,847,259–52,847,263) and 14-bpmutations were genotyped and bred to expand lines that harboredBerkeley Nationala mutation. Laboratory. We F0s (induced on C57BL/6N background) carrying lihood of off-target mutations. Initial Cas9 targeting was performed at Lawrence This sequence maps uniquely to the target site via sites,BLAT identifying (mm9), one reducingin exon 5 thewith sequencelike GAGGAGGAGGTCGATGTAAC. mouseoocytes. We scanned the methodsaccordingdescribedto (5 bp and 14 bp) in mouse C56BL/6Ngeneratetooocytes mouse twolinesharboring frameshift deletions G ONLINE MET doi: (NationalFIJI Institutesquantifiedin andblots,western Health). For Chd8 of nrto of eneration estern blot analysis. The colony of animals carrying the 5-bp 10.1038/nn.4592 4 + Chd8 0 / C del5 ) (1:1,000, ab31645; Abcam) and anti-Gapdh (ref. . For qRT-PCR analysis, t R. -test and linear regression. mice and matched WT littermates. For all experiments, samples from ifrnil xrsin f eetd ee agt ws eiid by verified was targets gene selected of expression Differential + / del5 Chd8 and matched WT littermates. We used primers validated for validated primers We used littermates. WT matched and H Ank2 ODS Chd8 Chd8 uat mice. mutant Isolated forebrain from E14.5 embryos (male and female expression during development mRNA (uc007tot.1). Both alleles match wild-type Chd8 + Chd8 / del5 1 and matched WT littermates were analyzed using exon 5. Guide RNA was designed and synthesized 2 n ) (1:1,000, ab114126; Abcam), anti-Hnrnpa2b1 Chd8 3 = 9 WT and 7 9 , pooled with Cas9 mRNA and injectedintomRNAandCas9 with pooled , e sd a9mdae mtgnss of mutagenesis Cas9-mediated used We coding sequence for unique gRNA target ActB Srsf7 Chd8 . Unpaired . Comparisons. of splicing across Chd8 deletion allele was rederived at Chd8 + Supplementary Table 4 Table Supplementary / del5 exonthat 5 overlapped 3 t Chd8 1 -tests were performed and designed primers 4 forebrains from male Chd8 1 ) (1:10,000, G8795; + / Supplementary line. Genotypes del5 brains using µ g - - - - - .

“Morphological analysis,” above. Slides were incubated for 24 h at 4 °C in a in °C 4 at h 24 for incubated were Slides analysis,”above.“Morphological L imaging was carried out on a Nikon A1 laser scanning confocal microscope. centered at this position a 200- tricle from the corticostriatal boundary to the apex, defined the 50% positionposition ofand cortical segments for cell type counts, we measured the cortical ven lent anteroposterior positions between genotypes. To determine the equivaatoutdorsoventral carried were cells Waltham,quantificationslabeled of All MA). ies(488 and 594) were at used 1:200 concentrations (Thermo Fisher Scientific, antibod secondary Fluor Alexa MA). Danvers, Signaling, Cell 1:200, rabbit, rabbit,Abcam,1:400, Cambridge, United Kingdom) (ref.Ki67or www.abcam (ref. following standard protocols and using primary antibodies directed against Pax6 VZ. All immunolabeling was carried out on slide-mounted cryosections (18 Chd8 and WT from sampleeach One Q-fraction. countsrepresentedtwothe the of tioned basal to the SVZ counted, followed by the count of brief,all EdU midcortical 200- ured individual morphological parameters using the Keyence BZ analyzer analyzer BZ Keyence the using parameters morphological individual ured We microscope. meas BZ Keyence a on acquired were hemispheres entire of images and size), thalamic length, (hippocampal orientation for landmarks anatomical subcortical using genotypes across aligned were mm, −2 bregma to approximatelyadult PA).corresponding Hatfield, sections, Sciences, Select Microscopy (Electron DePeX with mounted and solution violet cresyl 0.1% a with Nissl-stained mounted on (SuperFrost Plus, slides glass Thermo-Fisher), were sections the Subsequently, Leica). 1000S, (VT vibratome a on sections from the in skull, embedded 2% LTE Agarose/PBS and cut coronally in 50- removed were brains fixation, After by solution. same the followed in fixation overnight (PBS), saline phosphate-buffered in (PFA) 4% with paraformaldehyde perfused transcardially were mice female Following and male litters. P7 separate anesthesia, two least at from embryos/pups on triplicate in M with FIJI software (National Institutes of Health). Axiovision 4.8 software. Analysis of cortical length measurements Zeisswas Lumar.V12performed stereoscope with a Ned Lumar 0.8× FwD 80-mm objective and mold to ensure positioning. specific Whole-mount images were taken with the agarosean in placed and dissected carefullywereBrains h. 24 PFAleast atfor P7, Analysis of cortical length at P0. Student’s compared via wereblot western via assayed Proteinlevels used. weresamples both males and females were used. For the Hnrnpa2b1 western blot, only male instructions. Q-fraction analysis followed previously established practices kitprotocol (Life Technologies, Carlsbad, CA) according to the manufacturer’s detectionEdU was performed with the Click-iT AlexaEdU Fluor 594 imaging pulse) or 18 Houston, TX), frozen in dry-ice-chilled methanol and sectioned at 16 15% and then 30% sucrose in PBS, placed in OCT compound (FisherAfter 2 HealthCare,h of further fixation in the same solution, embryofemales brainswere wereanesthetized immersed andin embryos transcardially perfusedbody weight EdU.with After 4%1.5 h (proliferationPFA/PBS. assay) or 20 h (Q-fraction analysis), the in used following were assays. samples female and male Both genotype. to blinded were Brains were perfused before isolation, embedding and sectioning. male breeding Experimentersby generated were analysis E nucleus. endopririform of dorsal endpoint the dorsal from the originating midline to the to perpendicular a line midline dorsal the the from dorsal to the ventral midline. circumferences Neocortical were measured measured from were circumferences Hemispheric area). (cortical ware soft FIJI or thickness) neocortical circumference, (hemispheric/neocortical amination assay.amination U aeig n imnfurset analysis. immunofluorescent and labeling dU orphological analysis. orphological Time-pregnant females were intraperitoneally injected at E13.5 with 50 mg/kg Chd8 4 + 2 / del5 ) (PRB-278P-100; rabbit, 1:100, Covance, Princeton, NJ), Tbr2 ( Tbr2 NJ), Princeton,Covance, (PRB-278P-100; 1:100,rabbit, ) male and female littermates were decapitated and heads placed in 4% t was removed from Q-fraction analysis due to difficulties in selecting the -test and one-way ANOVA for adult Chd8 levels. µ .com/TBR2– Eomes-a m (20-h pulse) on a cryostat (Leica Biosystems, Buffalo Grove, IL). P0 or P7 brains were fixed and sectioned as described for described as sectioned and fixed were brains P7 or P0 µ m-wide segments were imaged and EdU All histological experiments were at experiments performed least All histological µ m-wide box perpendicular to the ventricle. All For gross anatomical measurements at P0 and ntibody-EPR19012-ab183991.pd Chd8 + / nature nature del5 itr fr neuroanatomy for Litters mice with WT females. females. WT with mice NEUR + Ki67 + cells. The ratio 4 OSCI f 3 ; ab183991; − ) (#12202; ) µ cells posi m (1.5-h http:// EN 3 µ 6 µ . In m) C m E ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. described previously those to similar methods using cm), 23 × cm 60 × cm (40 arenas field open CA) Sacramento,TapWhite,Plastics, (P95 white matte opaque in level of vocalization during handling and responsebitingdowel a intensityof petting, responsivenesstomeasuring testswithassessed mousewasandauditoryreactivity eachofThelevel startle. righting reflex, corneal reflex, whisker twitch, pinnae response,forepawhanging, reaching,wire curl, eyeblink trunk includedtests Neurologicalreflex response tively. Righting reflex and any occurrences of physical abnormalities were noted. thermistor probe with lubricant and gently inserted 2 cm into the measured,rectum, using respeca hand held portable scale (Ohaus, Parsippany, NJ) and askin mousecolor, and body and limb tone. Body weight and basal temperature was wereassessed also on a ranking scale of 0–3 based on fur condition, whisker reflexescondition, were evaluated in adult mice as previously described approach tasks. The testing room was illuminated at ~40 lx. social and conditioning fear self-grooming, interactions, reciprocal the from tionor hyperactivity considered an essential control for effects on physical activity, for example, seda environmentpreviouslyassayedwasas described Disabilities Research Center Mouse Behavior Core. Developmental and Intellectual Institute MIND Davis UC the at performed interactions,social novel object recognition and fear conditioning. Testing was self-grooming, marble burying, three-chambered social approach, male–female WT littermates were tested in the following sequence: open field, general health, approach, in the middle of the behavioral battery. Adult social three-chambered the during happened This testing. behavioral during cohortdied first the in animalOnefemale). 9 male, cohort:11 secondfemale; second cohort: 11 male, 11 female) and WT littermates (first cohort: 11 male, 10 two independent cohorts of adult diagnosticandassociatedsymptoms ofautism core the torelevant neurobehavioralassays of battery standard a in evaluated the light cycle. blinded to genotype during testing and analysis. All tests were conducted during with 2–4 animals per cage and used experimenters and video scorer/processors cages home genotype mixed We used testing. behavioral for used animals on distress and the number of animals used. No previous analyses were performed Careand Use of Laboratory Animals. Efforts were madeto minimize pain and were conducted in accordance with the National InstitutesUniversity of Health of GuideCalifornia forDavis theInstitutional Animal Care and Use ium Committeemaintained andon a 12-h light–dark cycle. All procedures were approved by the Behavioral testing. study were validated and their use widely reported. were excluded. Male and female samples were used. All antibodies used for this done blind, by investigators who were unaware of groupprocessing withoutallocation. taking morphologicalNo criteriadata intopoints account. All histology was marks. Within each genotype, all brains were selected randomly for histological and similar sections from each brain were identified based on anatomical land (v. 4.20; Nikon Instruments). Images were imported into FIJI ImageJ (v. 1.50E lightcury source, appropriate fluorescent filter sets and NIS Elements software, mer 0.2), N.A. Apo; (Plan lens objective 4× a with microscope upright E600 NikonEclipse a on Fluoromount-Gimagedwere (SouthernBiotech). Sections in DAPI (D1306, Thermo-Fisher), rinsed in PBS-T and coverslip-mounted with Jackson ImmunoResearch). Slides were rinsed in PBS-T, counterstained for 2 h in fluorophore-conjugated secondary antibodies (711-545-152 and 712-165-153; brain 1:500 in PBS-T. These antibodies have been previously used for IHC analysis in anti-Brn2 (ref. (ref. Ctip2 anti-containingsolution antibody primary in °C 4 at h 24 for incubated and phosphate(1× Triton(v/v)bufferedand0.01% saline PBS-T in rinsed X-100), blockingsolution containing dilutedv/v)(5% normalPBS-T donkey in serum nature nature to the open field arena, a 10-min familiarization session and a 5-min recognition Novel object recognition. object Novel Adult general health and neurological reflexes. Open field locomotion. Chd8 46 , 4 + 7 / . The slides were then rinsed in PBS-T and incubated overnight at 4 °C 50 del5 NEUR 4 , 5 4 male and female mice and WT littermates, ages 2–4 months, were 6 ) (ab18465; Abcam), anti-Tbr1 (ref. anti-Tbr1 Abcam), (ab18465; ) . The experiment consisted of three sessions: a 30-min exposure 30-min a sessions:three consistedofexperiment The . 4 5 OSCI ) (sc-6029; Santa Cruz Biotechnology) antibodies, each diluted Subject mice were housed in a temperature-controlled vivar 17 EN , 51 , General exploratory locomotion in a novel open field 5 C 2 , which, could confound theinterpretation of results E The novel object recognition test was conducted was test recognition object novel The Chd8 5 5 + . / del5 mice (first cohort: 9 male, 10 female; 1 General health and neurological 6 . We. testingperformedtheon 49– 4 4 5 1 ) (ab31940; Abcam) and Abcam) (ab31940; ) . Open field activity wasactivity field Open . Chd8 53 + , 5 / 4 del5 . General health and matched 4 8 - - - - - )

for levels of general exploratory locomotion. by sex. Number of entries into the side chambers served as a within-task control relativelya inactivestrain, aged weeks,10–14and matched subjectmicetheto 10 min of habituation to the entire arena. Novel stimulus mice were 129Sv/ImJ, initial the during confirmed preferencewas side innate chamber.of side Lack bers containing a novel object in one side chamber and a bers novelduring mousea 10 in minthe habituationother session, then allowed to explorein the centerthe threechamber forcham 10 min, then allowed to explore all three empty cham provided uniform, low-level illumination. The subject mouse was first contained every pair of three-chambered units. Infrared lighting (Nightvisionexperts.com) above directly positioned York,was Photo,New NY) B&H ICD-49, (Ikegami camera infrared-sensitive top-mounted A time. sniff defined enclosure, cup products/Galaxy-P wire cup, Galaxy Cup, Kitchen Plus, lusextendingfrom cmnovel each2 objectornovel mouseenclosure (inverted annu the as assay. defined werethe ofZones phase each forchamber each in time EthoVisiondetected software,the XTusing defined zones, Threements. compart of division manual maximal providing while cm), 10 × cm (5 bers doors (12 cm × 33 cm) were designed to create optimal entrywaysfinished acrylic (P95 White, betweenTap cham Plastics, Sacramento, CA). Opaque retractable 60 cm × 23 cm) was a rectangular, three-chambered box made from matte white bers were employed to maximize throughput. The updated apparatus Information (40 cm × Noldus 9.0, (Version Technologies, Leesburg, software VA) and videotracking modified nonreflective materials XT for the cham Ethovision apparatus using methods similar to those previously described Testing was performed under dim light (~15 lx). scored as previouslyscoredas described cone with ribbed sides. plasticorangehardsoft plasticmagneticsafetyconeaand small plastictoys: a were used Objects bias. side innate an of lack the confirmed phase iarization general exploration. Time spent sniffing two identical objects during the famil familiarobject. Total time spent sniffingbothobjects wasas measureaused of the novelthan object thesubstantiallyspending assniffing moretimedefined object and the nose–object distance was 2 cm or less. Recognition memory was defined as time spent sniffing the object when the nose was oriented toward the 9.0, Noldus Information Technologies, Leesburg, VA). Object investigation was were videotaped and scored with Ethovision XT videotracking software (Versionobject and the novel object. The familiarization session and the recognition test recognition test, during which time it was allowed to freely explore the familiar the familiarization session, each subject was returned to its open field for a 5 min locatedbeen during in the familiarization phase. Sixty minutes after the end of clean novel object were placed in the arena, where the two identical objects had field was cleaned with 70% ethanol and let dry. One clean familiar object andwhich onewere transferred from the testing room to a nearby holding area. Afterthe familiarization The session, open subjects were returned to their holdingcages, habituated been hadit which freelyexploreinandallowed to field min. for10 identical objects were placed in the arena. Each subject was returned to the open field and placed in a clean temporary holding cage for approximately 2 min. Twofor 10 min for the habituation phase. The mouse was 30then min.removed Twenty-four from the hoursopen later, each subject was returnedtest. On today 1, the each opensubject was fieldhabituated arena to a clean, empty open field arena for the investigator. ‘Buried’ was defined as by greater tallied than 50% was covered buried by beddingmarbles of number the which after period, exploration with a filter top lid. Each mouse was placed in the center of the cage for a 30-min top of 2–3-cm deep bedding in a clean standard mouse cage (27 ×marbles (1516.5 mm in diameter)× were 12.5arranged in acm) symmetrical 4 × 5-cm grid on described previously as measured was marbles the all the body regions during the second 10 min of the test session. and was unscored. Each subject was scored for cumulative time spent grooming habituationwas period 10-min first The min. Sessionswereforvideotaped20 (Security Cameras Direct) was placed ~1 m from the cages to record the behavior.sessions. The room was illuminated at ~40 lx. competingA front-mountedpotentially a cameraCCTV is which bedding, the in digging eliminate to empty were Cages high). cm 20 × wide cm 23.5 × long cm (46 cage mousestandard Social approach. Repetitiveself-grooming. Repetitive marble burying. Social approach was tested in an automated three-chambered encil-Utility-Cu Spontaneous repetitive self-grooming behavior was Marble burying and digging in the bedding to cover 17 , 4 9 . Each mouse was placed individuallyplacedmousewas into Each a . p https://www.spect ). Directionfacinghead,toward).ofthe the 49 , rumdiversified.com/w 57– 5 9 . Twenty black glass glass Twentyblack . doi: 10.1038/nn.4592 49 , 6 0 . Automated hs/ 5 8 ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. used to create fractional anisotropy (FA), mean diffusivity (MD), axial diffusivitywere analyzed using the FSL software package (FMRIB, Oxford UK), which was Jones30scheme and 30 high an image with 0.078-mm isotropic voxels. We acquired five m(FOV) of 14 × 14 × 25 mm remainingformstheaverage. of10 1 TEsechoes, five We field-of-viewa used of 6. Parameters for the DTI sequence were: TR = 270 ms, first TE = 30 ms and 3D diffusion-weighted fast spin-echo sequence (FSE), with an echo train length D total imaging time was 14 h. 630 mm mm 25 × 20 × 20 to equal field-of-view acquisition of tomical MRI scans: T2-weighted, 3-D fast spin-echo sequence, with a cylindrical changes via anatomical imaging, we used the following parameters for the ana noid coils were used to image the brains in parallel CA) was used to image the brains within their skulls. Sixteen custom-built sole before MRI scanning. 0.1 M PBS containing 2 mM ProHance and 0.02% sodium azide for at least 7 d PFAincubated4% in ProHance mM 2 + overnighttransferredthen °C, atto4 laginous nose tip were removed. The brain and remaining skull structures were were perfused and then decapitated, and the skin, lower jaw, ears and the carti by 30 mL of 4% paraformaldehyde (PFA) containing 2 mM ProHance ProHancemM 2and (Sigma)Gadoliniumheparin (a contrast agent) followed effect) and intracardially perfused with 30 mL of 0.1 M PBS containing 10 U/mL undergonehadbehavioral theassays weretoisofluraneanesthetized with(4% from the first cohort ( M software (Med Associates). Cumulative time spent freezing in each condition was quantified by VideoFreeze followed by a 3-min presentation of the tone CS and a 90-s exploration period. acclimation3-minconsisted test aperiod of cued The off. turnedlighting was in a novel environment with distinct visual, tactile and olfactory cues. Overhead training day. Cued fear conditioning, conducted 48 h after training, was assessed overhead lighting, vanilla odor and chamber cues identical to those used on the training,absencethein of tonethe and footshock but presencethein of 100lx dilution). A 5-min test of contextual fear conditioning was performed 24 h after swabbed with vanilla odor cue (prepared from vanilla extract; McCormick; 1:100 sented. The environment was well lit (~100 lx), with a stainlessintershock steelinterval, grid 90 floors) andand a 2.5-min period during which no s; durationfootshock,1 stimuli0.5-mA s; tone,duration30 (80-dB pairings (CS–US)were pre Training consisted of a 2-mincubicle. acclimationsound-attenuating period followed a by three tone–shock in enclosed and Associates) Med 1.12.0.0, sion Med Associates) interfaced with a PC installed with VideoFreeze software (ver USA)aspreviously described using an automated fear-conditioning chamber (Med Associates, St Albans, VT, manually by a highly trained investigator blinded to genotype. tion spectrograms were displayed using Avisoft software and calls were softwareidentified (Noldus, Leesburg, VA) as previously described sniffing and following were scored using Noldus Observer 8.0XT event LEDrecording illumination (~10 lx). Duration of nose-to-nose sniffing, nose-to-anogenitalating environmental chamber (Lafayette Instruments, Lafayette, IN) under dim the resolution was 16 bits. The entire apparatus was contained in a 20 sound-attenucm above the cage. Sampling frequency for the microphone was 250 kHz, and microphone capsule CM15; Avisoft Bioacoustics, Berlin, condenser Germany)UltraSoundGate(Avisoftmicrophone ultrasonic quality. An was video mounted an angle from the Noldus PhenoTyper arena (Noldus, Leesburg, VA) for Aclosed-circuit optimaltelevision camera (Panasonic, Secaucus, NJ) was positioned at subject was paired for 5-min with a freely moving unfamiliar estrous WT female.test was conducted as previously described doi: (AD) and radial diffusivity (RD) maps for each of the brains used in this study. iffusion tensor imaging. tensor iffusion RI within brains of the subjects assessed in behavioral assays. After perfusion, a multichannel 7.0 Tesla MRI scanner (Agilent Inc., Palo Alto, Fear conditioning. Male–female social interaction. 10.1038/nn.4592 3 . Our parameters output an image with 0.040-mm isotropic voxels. The b -value ( k -space 6 7 . Total. imaging timewas ~12After h. acquisition, imagesthe Delay contextual and cued fear conditioning was conducted b 6 = 2,147 s/mm Chd8 6 , a TR of 350 ms and TEs of 12 ms per echo for 6 echoes, + Diffusion tensor imaging (DTI) was done using a using done was (DTI) imaging tensor Diffusion 3 / del5 and a matrix size of 180 × 180 × 324 mm 6 2 . Theconditioning. chamber cm 23 × 25 ×(32 : 9 male, 10 female; WT: 8 male, 10 female) that The male–female reciprocal social interaction 2 ) images in 30 different directions, using the 3 and matrix size equal to 504 × 504 × 504 × 504 to equal size matrixand 49 , 6 1 . Briefly, each freely moving male 6 5 . In order to detect volumetric 4 9 . Ultrasonic vocaliza b = 0 s/mm

Perfusion. 63 3 , yielding 2 , 6 images 4 . Mice Mice 3 ------,

G false discovery rate across the brain. Multiple comparisons in this study were controlled forexamined usingon thea voxelwise basis to localize the differences found within regions or bulbsolfactoryand white matter structures (i.e., corpus callosum), ventricles, cerebellum, brain stem and RD) of 159 different segmented structures, encompassing corticalwhich allowedlobes, us largeto assess the volume or mean diffusion measures (FA, MD, AD calculated by warping a pre-existing classified MRI atlas ontogenotypes.Significantvolume changes intensityand be differencesthencould the population atlas, analyze the intensity differences of all measures (FA, MD, AD and RD) between ume at each voxel. For the diffusion measurements, the registration allowed determinantsus of to the deformation fields were then calculated as measures of vol being to model how the deformation fields relate to genotype needed to take each individual mouse’s anatomy into ion. Forthis the volumefinal measurements, atlas this allowed us tospace, analyze the deformationsthe goal fash unbiased an in other each with alignmentinto images all deform to was b anatomystudysample.40- Notethe ofthe that average the representing atlas population a create to averaged and transform tools)normalization were performed with a combination of mni_autoreg tools followedparameter)by12 andnonlinearly registered together in the mouse brains the images (or Structural BatchEffect function. Normalized log function followed by removing the sequencing batch effect viaperformed. theNormalized expressionlimma levels removewere generated using the edgeR rpkm as the variable for testing. Stage-specific differential expression testing was also opmental stage and sequencing run factor-based covariates and using genotype performed with edgeR was analysis expression differential and generated, were estimates dispersion with no obvious separation between wild-type and stage, developmental was samples across variance of driver strongest the that 11,936 genes for differential testing. Multidimensional scaling analysis indicated least two individual samples were included for analysis, resulting in a final set of expression analysis using edgeR differential for infomation sample with along input as used were samples all D to with primers reported in idate DE and DS analysis using cDNA libraries preparedtranscript using frequency.the same protocols,We performed quantitative reverseand 14-bptranscription deletion at PCR to val BLAST distribution.py were discarded. Unaligned reads were quality checked strongby FastQC.3 suite tool RSeQC full the controlinformationusing generatequality to used werereads aligned andannotation trackknownGene to genes were counted at the gene level using subreads featureCountsthe mouse genome (mm9) using STAR (version 2.4.2a) at the UC Davis DNA Technologies Core. Reads from RNA-seq were aligned to samplesotherweresequenced all Laboratory; SequencingGenomics Berkeley UC the at sequenced were samples E12.5 sequencing. for submission before orpaired-end 100-bpstrategy.(E12.5) quantifiedwaslibrary and pooled Each IlluminaHiSeqplatform using single-enda 50-bp (E14.5, E17.5, andP0 adult) Stranded mRNA kits; 6–12 samples per lane were pooled and sequenced on the instrument.Stranded mRNA sequencing libraries were prepared using TruSeq isolatedAmbionusingRNAqueous AgilentassayedandBioAnalyzeranusing each in reported arenumbers at exact stage; genotype each of females and males included Samples genotype. to blind performed were Dissections collected. was head developing the of tion por anterior the when E12.5, except ages all for skull and tissue surface ing forebrainincludedwholeDissection >P56). plotsas hemispheres removafter (representedadultsin from and P0 and E17.5 E14.5, littermatesE12.5, at WT = 0 s/mm enomics. ifferential expression analysis and permutation testing. ActB 8 and compared via standard methods as discussed in the methods. ′ 0 bias using geneBody_coverage.py or poor exon distribution using read_ was used to identify reads that mapped to either the reference or 5-bp M 2 Bulk forebrainsBulk weremicrodissectedfrom DTI images were registered separately. The result of the registration RI registration and analysis. 7 68 6 . We reported combined sex results in the main text. 70 , Chd8 74 8 , 7 1 , 1 7 using a generalized linear model including sex, devel Supplementary Table 4 . All scans were then resampled with the appropriatethe with resampled then were scans All . 5 , in all brains.Further, all in measurements, these be could exon 5 to verify genotype and test for deletion allele 8 1 . Genes with at least 10 reads per million in at b = 0 s/mm 2 Supplementary TableSupplementary 2 (RPKM) values were used for plotting of To visualize and compare any changes 2 images for DTI) were linearly (6 µ . Cycle counts were normalized m anatomicalthe m andimages nature nature Chd8 7 7 9 7 . Samples that exhibitedthat Samples . Chd8 . Aligned reads mapping 6 + 9 and ANTS (advanced / del5 Raw count data for + NEUR 72 samples. Tagwise / del5 . Total. wasRNA , 6 7 8 3 . Registrations. . The Jacobian and matchedand 7 OSCI 8 . The mm9 EN C E ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. the finding that most DE gene expression changes are far smaller than changes withlargely sameresultsthe samplefor consistentasisfull which the set, with only wild-type samples, only tion were placed into the module M.grey. We repeated module generation using orderby gene size set and named numerically. Genes with no network correla reorderedweredescendingand in method generatedwere this modulesusing eigengenes.Fourmodule expressionwith profilegene correlating the bylated were merged together. The module connectivity (kME) of each gene was calcu module eigengene, ME. Modules with highly correlated MEs (correlation > 0.80) and modules. The expression profile of a given module was summarized by the were used to calculate correlation between sample traits (for example, genotype) clustered together in these modules. In addition, Pearson’spositivelywithcorrelated Genes expressionoverlaptopological high wereand correlation coefficients WGCNAin function Hybrid cutree the using base, tree clusterhierarchical the ofbranches wereModules used as the input to calculate co-expression modules using washierarchical transformed toclustering. a topological overlap matrix (TOM). The matrix 1-TOM ( was approximatetopology exhibiting scale-free thresholdingsoft power estimatedwas toderiveand anused adjacencymatrix The samples.relevant all computedfor was genes all between midcorrelation biweight the using correlationmatrix A sample.one least at in higher or 0.25 co-expressionsigned networksusingany ofexpressedvaluegene RPKM atan WGCN available as UCSC TrackHubs for upload to the UCSC Genome Browser. For comparison, all datasets were run through the same analysis pipeline and are default parameters sets using ENRICHR promoters, functional annotation was performed on the promoter-bound gene gene at were peaks all nearly As scripts. custom using annotated and regions nomodel–shiftsize = 150 -p 0.0001 10,30–nolambda– = 300–mfold = 50–bw = mm–tsize = BAM–gsize = format and control data using MACS1.4. ChIP-seq experiment versus matched input control, as well as for merged ChIP identification and local significance testing. Peaks were called for each individual mapped then to the andmouse genome (mm9) sequences, using the BWAlow-quality and algorithm artifacts remove to filtered were reads strategy.Resulting 50-bp single-end a using instrument 4000 HiSeq Illumina V2, indexed for multiplexed runs of four libraries per lane and sequenced on an ChIP samples were prepared using the Nugen Ovation Ultralow Library System Chd8 ChIP-seq in a previous publication neuronalor brainfor used been has antibody This Abcam).(ab114126; Chd8 for antibodies using performed was immunoprecipitationChromatin (ChIP) tissues mouse for adapted protocols ChIP-seq standard using instrument using formaldehyde and lysed with SDS, and the DNA was C sonicated on a Covaris are available upon request. the observed overlap. All analysis was performed in R using custom scripts that distribution,this onempirical Based distribution. expected the ofderivationstandard and mean the estimateto us samplinggenes.RandomDEenablingrepeatedgeneswith times, 100,000was annotatedthe followedin geneset, bytesting for overlap randomlyof selected as genes of number same the ofsampling randomiterative via generated was overlappingthe ascalculated overlap expected genes,distributionwhereasthe was overlap Observed overlap. expected with observed comparing sets, gene ferentially down- or upregulated genes identified in the full model and annotated in overall findings. resulted in identification of DE genes with lower CPM but no major differences which inclusion, gene for criteria coverage reduced with obtained results DE heatmapssummary expressionandof dataforindividual genes. We examined nature nature for genes across developmental stages. hIP-seq analysis. hIP-seq After peak calling, enriched regions were filtered to remove ENCODE blacklist MACS call: macs14 -t chip.bam–control = input.bam –name = chip_output– WeMACS used BWA call: bwa aln -t 6 -l 25 mm9 sample.fastq.gz Permutationtestingperformed wasbytesting inclusionfor set dif between A. NEUR We used the WGCNA package WGCNA the Weused OSCI 8 8 6 Adult mouse forebrain was dissected on ice, cross-linked ice, on dissected was forebrain mouse Adult 4 . We additionally analyzed available Chd8 ChIP-seq data to identify significant peaks, disabling model-based peak peak disablingmodel-based significantpeaks, identify to 8 5 EN . We performed C E Chd8 8 7 , with minimum module size set to 500 genes. 500 to set size module minimum with , + / del5 z de novo samples and with/without adult samples, -scoresand 1 2 . DNA libraries of matched input and 2 5 in R (version 3.2.3) to construct to 3.2.3) (version R in R motif discovery using RSAT with 2 > 0.85). The adjacency matrix adjacency The 0.85). > P values were calculated forwerecalculatedvalues 8 3 . 11– 8 1 2 3 - - - - . .

M substantial differences observed for either methods comparison. DAPPLEprotein–proteinwithSTRINGcompared interaction and comparedTopGO enrichment results withgoseq M.1ule exhibited asignificant enrichment in protein–protein interactions. We considering only experimentally defined and database interactions. Only mod STRINGusing performed was sets gene DE generation module-specific for ted for GO terms of interest. Protein–protein interactionenriched. Heatmapsenrichment showing and positive network log above. Genes with expected/observed ratios of at least 1.5-fold were considered genesexpressed ourstudyin onminimum based read-count cutoffs described of set background comparedthe againstwas genes DE of set test the analysis, of (cutoff sets genes stage-specific and module genes from the full model (cutoff of FDR < 0.20) for all genes and by expression examined down- and upregulated genes separately, repeating the analysis on DE we analysis, comparisons.GO multipleFortestingforaccounts generally that analysis set gene for recommended strategy a test, Fisher the and framework testing ‘weight01’ internal the We used 20. of terms) GO to annotated genes of (number size node minimal a required annotationsand Process Biological GO to testing our restricted we presentedhere,analysis relationships. the For TopGO the used We GO.db). org.Hs.eg.db, (org.Hs.egGO2ALLEGS, Bioconductor fromdownloadedHumanwaspublished data Ontology(GO)sets. gene Gene and genes DE between overlapfor test to performed Permutationwas testing G ence and power analyses. Effects of genotype and sex were evaluated using using ANOVA,multifactor evaluated published as previously were sex and genotype of Effects analyses. power experi and past ence on based chosen were indicated sizes Groups two-tailed. were were levels atset significance All sexes. and/or between points time across two-way comparisons for or used one-way were ANOVA and repeated-measures genotypes, between comparisons parameter gle Student’s including methods, assay-specific established using considered were Sexes 7.0a. performed was testing factor. Statistical fixed the as separately,genotype with Prism GraphPad using generated were Plots analyzed using two-tailed Welch’s assays. For isoform-specific qPCR, comparisons across stage and genotype were of three biological replicates (individual animals) were used for all genotypes one-wayand ANOVA followed by all analyses comparing two groups. For comparisons of three or more data sets, plots were generated using GraphPad Prism 7.0a. Two-tailed M A tested.formally not and normal be to assumed was bution westernblots and histology assays. Aside from behavioral analyses, data distri RNA-seq,for litters across randomized was collection Data blind. performed ChIP-seq, western blots or qRT-PCR. All other data collection and analyses were and analysis were not performed blind to the conditions of the experiments for sizes are similar to those reported in previous publicationssample other our but studies, behavioral of exception the with sizes, sample Statistical analysis. = 10,–delta-psi = 0.1,–bayes-factor = 100. events) based on the following parameters: –num-total = 100,–num-sum-inc-excstagesResults were(forfilteredexample,versus (filter_WT). E17.5 WT E14.5 stage (for example, E17.5 Comparisons (compare_miso) were run between either genotype for a specific was run to compute PSI scores (miso-run) using standard, unfiltered Standardparameters. GFF alternate-event annotations for mm9 (version 1) were used. MISOstandard MISO software package fastmiso ( cantly impacting MISO results across genotype. Analysis wassample performedhad lower read coverageusing comparedthe to the other samples and was signifi (S159) from the E17.5 developmental stage (for example, E17.5 and WT andgenotype onE17.5 based merged and sorted were files bam aligned individual to identify alternative transcript events in our RNA-seq data. For MISO analysis, ene ethods IS Behavior. Neuroanatomy, biochemistry and immunostaining. O O analysis. 8 ntology enrichment and protein–protein interaction network analysis. 8 program to test for enrichment of GO terms indicating parent:childindicatingterms GO ofenrichmentfor test programto C hecklist Data were analyzed with Statistica software (Tulsa, OK, USA). USA). OK, (Tulsa, software Statistica with analyzed were Data The Mixture-of-Isoforms (MISO) statistical model is available.

General. Chd8 Chd8 + / No statistical methods were used to predetermine del5 post hoc + / group was discarded from analysis because the del5 t -test and linear regression. versus E17.5 WT) or pairwise between WT two-tailed 2 (expected/observed) values were plot https://github.com/yarden/MIS 16 , 49 P t 8 -tests were used. A minimum , 9 < 0.05). For all enrichment all For 0.05). < 50 , correcting, for gene length, , All statistical analyses and 59 , 6 Chd8 2 . Significant ANOVAs . Significant P < and 0.05 all 12 doi: t -tests were used for , + 1 / 3 del5 10.1038/nn.4592 . Data collection Supplementary t ). One sample -test for sin for -test 3 9 0 0 was used , with no with , t -tests -tests O 2 7 ). ------,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 44. 43. 42. 41. 40. 39. See above for description and parameters. ( performed using Enrichr ( annotated.Comparison of enriched gene betweenRNAsets and ChIP-seq was Peaksremovesion1.4.2).filteredwereto then ENCODE blacklistregions and aligned to mm9 via BWA (version 0.6.2) and peaks were called via MACS (ver wrapper using standard the TopGO (version 2.20.0) program. ChIP-seq data workflow was and functions. Gene Ontology analysis was performed with a custom formed with a custom pipeline following the standard WGCNA (version 3.2.3)per was analysis network Co-expression script. R custom a with performed was Permutationtesting 3.24.15). (version limma and 3.10.5) (version edgeR sion analysis was done with a custom pipeline in R Studio using functions from formed on individual samples using RSeQC (version 2.6.3). Differential expres featureCountsgenes.gtfqualityandchecksper mm9 (version UCSC to 1.5.0) mm9 using RNA-seq aligner STAR (version 2.4.2a), featuresprocessing pipeline assignedwas used to via align subreadsraw sequencing samples to mouse genome ( Repository andanalysis are available in C data for ChIP-seq are available on GEO ( gene sets used in enrichment analysis: from the corresponding author upon reasonable request. DOIs for all published D as calculated by MISO analysis, individual events were considered significant if the Bayes factor > 100, z empirical if significant considered was enrichment testing, permutation For peaks were considered significant if P and FDR if significant statistically considered were differences analysis, sion failed to pass quality scoring and coverage criteria. For differential gene expres and processed blind to genotype. Samples from RNA-seq were removed if they within points 2 s.d. of mean the were in analysis. included data all and groups between similar were variances analyses, behavioral all For assays. all in analysis parametric via analyzed was thus and variables continuous using collected was tests, normality distribution passed were followed by Tukey’s honest difference test. significant analysis Behavioral doi: h -scores and values calculated by edgeR were < 0.20 and < 0.05, respectively. For ChIP-seq, ode availability. ode ataavailability. ttps://github.com/yarden/MISO Raw, aligned and gene-count data for RNA-seq and raw, aligned and peak call Parikshak Durak Katayama Hormozdiari Darnell Sugathan Willsey Cotney Parikshak Sanders Genomic analyses.

aao MJ, mly JG & aki, .. otctaai poeto neuron projection Corticothalamic J.D. Macklis, & J.G. Emsley, M.J., Galazo, Glucose B.L.G. Nyomba, & S. Mishra, A.G., Erickson, X.-H., Yao, K.H., Nguyen, of role multivalent and novel A D. Goldowitz, & D.J. T.J.,Swanson, Ha, J., Yeung, adipogenic Nogo in callosum corpus the of The Agenesis R.L. Schnaar, & M.G. S.-W.,Yoo,Motari, E. Mueller, & L. Hugendubler, S., Meruvu, X., Ma, C., Du, P. Mali, intraclass neuronal diversity. neuronal intraclass development beyond subtype specification: Fog2 and intersectional controls regulate (2015). 462–474 ability.binding IGF of devoid IGFBP-3 mutant its and IGFBP-3 human of effects differential mice: transgenic IGFBP-3 male aging in intolerance development. cerebellar in Pax6 mice. deficient receptor splicing. influences alternative and regulators splicing with interacts ZNF638 cofactor transcriptional (2013). 823–826 10.1038/nn.4592 et al. et al. et al. et al. et al. t al. et et al. https://github.co P et al. et al. et al. 1 values were > 2 and < 0.05, respectively. For differential splicing 1 5 2 2 1 2 : 1 et al. 2 9 N-udd ua gnm egneig i Cas9. via engineering genome human RNA-guided : 1 : https://d : : Thedata that support findingsthe ofstudy this are available 2 2 1 1 https://dx.doi.org/ https://dx.doi.o All custom scripts and TrackHubsandscripts custom processing All datafor used https://dx.doi.or h 4 6 3 : : : : ttps://dx.doi.org/10 https Samples for RNA-seq were randomly collected across litters https://d https://dx.doi.org/ https://dx.do 2 0 J. Lipid Res. Lipid J. 3 : 0 https://dx.doi.org/ . ://dx.doi.org/10.107 http://amp.pharm x.doi.org/10.1038/nn J. Comp. Neurol. Comp. J. SupplementarySoftware m/NordNeurogenomicsL x.doi.org/10.1038/na Neuron i.org/10.1038/nature rg/10.1016/j.cell.20

J. Neurosci. J. / g/10.1016/j.cell.2011.06.01 55 P ) was performed using standard parameters. values determined by MACS were < 0.0001. 10.1038/ncomms740

, 1886–1896 (2014). 1886–1896 , 91 10.1016/j.cell.2013.10.03 .1016/j.neuron.2015.09.01 , 90–106 (2016). 90–106 , GSE9933

10.1101/gr.178855.11 525 .mssm.edu/Enrichr

3/pnas.140526611 36 , 291–301 (2017). 291–301 , .440 , 9057–9069 (2016). 9057–9069 , 1 ture2061 ). 0 ; and 13.10.02 1935 and at the Nord Git Lab ab / 4 ). A custom sample-custom A ). ; 2 7 . ; Endocrinology 0 3 / ; ; ). MISO analysis 1 1 4 ; ; ; 6 Science ;

156 339 - - - - - , ,

56. 75. 74. 73. 72. 71. 70. 69. 68. 67. 66. 65. 64. 63. 62. 61. 60. 59. 58. 57. 55. 54. 53. 52. 51. 50. 49. 48. 47. 46. 45.

Yang, M., Lewis, F.C., Sarvi, M.S., Foley, G.M. & Crawley, J.N. 16p11.2 Deletion 16p11.2 J.N. Crawley, & G.M. Foley, M.S., F.C.,Sarvi, Lewis, M., Yang, Ullmann, J.F.P., Janke, A.L., Reutens, D. & Watson, C. Development of MRI-based of Development C. Watson, & J.F.P.,D. Ullmann, Reutens, A.L., Janke, Steadman,P.E. J.G. Sled, & R.M. Henkelman, S.L., Adamson, A.M., Flenniken, B.J., Nieman, J.P. Lerch, diffeomorphic Symmetric J.C. Gee, & M. Grossman, C.L., Epstein, B.B., Avants, B.B. Avants, intersubject 3D Automatic A.C. Evans, & T.M. Peters, P., Neelin, D.L., Collins, resolution High R.M. Henkelman, & N. Kabani, S., J.P.,Spring, Lerch, A.E., Dorr, formeasuring strategies A.Optimal &Simmons, M.A. Horsfield, D.K., Jones, B.J. Nieman, Bock, N.A. L.S. Cahill, perfusion heart Isolated R. Schulz, & A. Szelag, B., Grotthus, M., Skrzypiec-Spring, K.L. Bales, in vocalizations of repertoire Unusual J.N. Crawley, & L. Ricceri, M.L., Scattoni, social three-chambered Automated J.N. Crawley, & J.L. Silverman, M., Yang, C. Henderson, A. Thomas, I Group R. Paylor, & L.A. Yuva-Paylor, J.R., Perkins, N., Bui, A.M., Thomas, Rogers, D.C. Chadman, K.K. autism of models rodent in used methods Behavioural M.L. Scattoni, & M. Wöhr, J.L. Silverman, Silverman, J.L. Flannery,B.M. J.L. Silverman, J. Schindelin, and expressed diversely a is (SC1) SPARC-like1 A.J. Roskams, & S. Lloyd-Burton, recognition neural of Loss Y. Liu, & K. Watanabe, Y., Shimoda, Y., Yu, Z., Huang, I. Espuny-Camacho, Learn Mem. Learn mice display cognitive deficits in touchscreen learning and novelty recognition tasks. (1997). 711–713 assessment. phenotype comprehensive for protocol proposed a mice. knockin 251 developments. new and standards current disorders: spectrum autism. of model mouse T+tf/J ofautism. models Transl.Med. mouse in deficits social rescues and behaviors repetitive tetramethylenedisulfotetramine. with atlases of non-human brains. non-human of atlases magneticautismusingresonancea imaging atlas. (2006). magnetic by tomography. mouse computed mutant and a imaging of resonance skull and brain the in phenotyping Anatomical disease. Huntington’s of model brain. neurodegenerative and elderly of labeling automated evaluating cross-correlation: with registration image registration. image brain in space. Talairach standardized Tomogr.Assist. in data volumetric MR of registration 40 of mice. C57Bl/6J image adult resonance magnetic average an using atlas brain three-dimensional imaging. resonance Med. magnetic by systems anisotropic in diffusion phenotyping. extendbrainthephenotype cdf/cdfformice. (2012). 933–939 Methods according to Langendorff---still viable in the new millennium. model. 10 adult BTBR T+tf/J mice during three types of social encounters. 8.26–8.26.16 Unit 8, Chapter S.) (Wiley,2011). Wray, & P. Skolnick, D., Sibley, A., Holmes, in mice. for task approach 4 model byselective activation ofGABAB receptors with arbaclofen. (2009). anxiety. novelty-induced than more syndrome. X fragile for metabotropic glutamate receptor antagonists alter select behaviors in a mouse model n rdcs eeiie eair n w mue oes f autism. of models mouse pharmacology two in behavior repetitive reduces and Methods Nat. CNS. the in SPARC of the absence for compensate not does that protein matricellular regulated developmentally corticospinalmouse.the in axonstract developing of branching terminal and projection normal the delays NB-3molecule brain. mouse into (2017). transplanted neurons human , 152ra128 (2012). 152ra128 , , 44–56 (2011). 44–56 , , 5–17 (2013). 5–17 ,

42 Transl.Psychiatry

, 515–525 (1999). 515–525 , 55 et al.

t al. et , 113–126 (2007). 113–126 ,

t al. et t al. et t al. et 22 et al. et al. et 4

Magn. Reson. Med. Reson. Magn. 9 40 , 131ra51 (2012). 131ra51 , Autism Res. Autism et al. et

t al. et In vivo magnetic resonance imaging and semiautomated image analysis et al. et et al. t al. et et al. et al.et , 622–632 (2015). 622–632 , et al. et , 676–682 (2012). 676–682 , 18 t al. et , 2228–2239 (2015). 2228–2239 , otcl hcns maue fo MI n h YC2 mouse YAC128 the in MRI from measured thickness Cortical Marble burying reflects a repetitive and perseverative behavior and perseverative a repetitive reflects burying Marble Behavioral and functional analysis of mouse phenotype: SHIRPA, ogtr epsr t itaaa oyoi i a os autism mouse a in oxytocin intranasal to exposure Long-term A reproducible evaluation of ANTs similarity metric performance ANTsmetric of similarity evaluation reproducible A Preparation of fixed mouse brains for MRI. brains mouse fixed of Preparation , 192–205 (1994). 192–205 , Reversal of disease-related pathologies in the fragile X mouse X fragile the in pathologies disease-related of Reversal Negative allosteric modulation of the mGluR5 receptor reduces Minimal aberrant behavioral phenotypes of neuroligin-3 R451C Behavioral assessment of NIH Swiss mice acutely intoxicated acutely mice Swiss NIH of assessment Behavioral Geneticeffects cerebellaron structure acrossmouse models of ii a oe-ore ltom o booia-mg analysis. biological-image for platform open-source an Fiji: Low stress reactivity and neuroendocrine factors in the BTBR the in factors neuroendocrine and reactivity stress Low t al. et at pneh fr utpe os mgei resonance magnetic mouse multiple for spin-echo Fast AA rcpo aoit -alfn eess oil deficits social reverses R-baclofen agonist receptor GABAB Neuroimage Psychopharmacology (Berl.) Psychopharmacology

4 alak o Azemrs ies i stem-cell-derived in disease Alzheimer’s of Hallmarks

, e480 (2014). e480 , 1 urn Pooos n Neuroscience in Protocols Current Neuroimage , 147–158 (2008). 147–158 , Med. Image Anal. Image Med. J. Comp. Neurol. Comp. J. Neuroscience Neuroimage J. Comp. Neurol. Comp. J.

54

42 , 532–537 (2005). 532–537 , Neurotoxicol. Teratol.Neurotoxicol. scohraooy (Berl.) Psychopharmacology , 60–69 (2008). 60–69 , J. Comp. Neurol.Comp.J.

54 , 2033–2044 (2011). 2033–2044 ,

J. Neurosci.J. 41

171

, 243–251 (2008). 243–251 ,

523 12 hso. Genomics Physiol.

AutismRes. , 1197–1208 (2010). 1197–1208 , 520

, 26–41 (2008). 26–41 , nature nature 219 , 391–405 (2015). 391–405 , Neuron , 2575–2590 (2012). 2575–2590 ,

520 , 47–58 (2012). 47–58 , 26

, 4455–4459, (2006). 47 , 1227–1245(2012).,

J. Pharmacol. Toxicol.

93 , 36–45 (2015). 36–45 , 7 NEUR es Gre, C.R., Gerfen, (eds. Mamm. Genome Mamm. Genes Brain Behav. , 124–137(2014)., ea. ri Res. Brain Behav. 1066–1081.e8 ,

Sci. Transl. Med. Neuroimage 204

an Reson. Magn. 24 Neuropsycho­ OSCI 361–373 , 154–162 , . Comput. J. EN

Sci. 60 C

8 E , ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 83. 82. 81. 80. 79. 78. 77. 76. nature nature

Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler with alignment read short accurate and Fast R. Durbin, & H. Li, A.S. Nord, for package Bioconductor a edgeR: G.K. Smyth, & McCarthy,D.J. M.D., Robinson, experiments. RNA-seq Altschul, S.F., of Gish, W., control Miller, W., quality Myers, E.W. & Lipman, RSeQC: D.J. Basic local alignment W. Li, & S. Wang, L., Wang, Liao,Y., Smyth, G.K. &Shi, W. featureCounts: an efficient general purpose program for A. Dobin, in maps statistical of Thresholding T. Nichols, & N.A. Lazar, C.R., Genovese, transform. development. mammalian during (2010). 139–140 data. expression gene digital of analysis expression differential tool. search Bioinformatics assigningsequence readsgenomicto features. (2013). 15–21 (2002). rate. discovery false the using neuroimaging functional NEUR Bioinformatics t al. et t al. et J. Mol. Biol. Mol. J. OSCI

28 TR utaat nvra RAsq aligner. RNA-seq universal ultrafast STAR: ai ad evsv cags n eoewd ehne usage enhancer genome-wide in changes pervasive and Rapid , 2184–2185 (2012). 2184–2185 , EN C

E 215 25 , 1754–1760 (2009). 1754–1760 , , 403–410 (1990). 403–410 , Cell

155 , 1521–1531 (2013). 1521–1531 , Bioinformatics Neuroimage

30 , 923–930, (2014). Bioinformatics Bioinformatics

15 , 870–878 ,

26 29 , , 90. 89. 88. 87. 86. 85. 84.

Young, M.D., Wakefield, M.J., Smyth, G.K. & Oshlack, A. Geneontology A. E.J. Rossin, &Oshlack, G.K. Smyth, M.J., Wakefield, M.D., Young, S. co-expression Laere, Van gene weighted for framework general A S. Horvath, & B. Zhang, A. Medina-Rivera, E.Y.Chen, data. ChIP-seq from peaks identify to MACS Using Y. Zhang, & T. Liu, J., Feng, 7 biology. underlying suggest and interact physically disease mediated bias. selection for (2010). accounting RNA-seq: for analysis analysis. gene-expression genome-wide Affymetrix-based using cancer breast non-inflammatory to compared analysis. network Res. Acids tool. analysis Orchard, S. & Pearson, W.R.) Chapter 2, Unit 2.14 (Wiley,2011). 2.14 Unit 2, W.R.)Chapter Pearson, & S. Orchard, in , e1001273 (2011). e1001273 , Current Protocols in Bioinformatics in Protocols Current

et al. et 43 et al. et BMC Bioinformatics BMC , W50-6 (2015). W50-6 , t al. et Enrichr: interactive and collaborative HTML5 gene list enrichment list gene HTML5 collaborative and interactive Enrichr: Stat. Appl. Genet. Mol. Biol. Mol. Genet. Appl. Stat. Proteins encoded in genomic regions associated with immune- with associated regions genomic in encoded Proteins t al. et Distinct molecular phenotype of inflammatory breast cancer breast inflammatory of phenotype molecular Distinct ST 05 rgltr sqec aayi tools. analysis sequence regulatory 2015: RSAT Br. J. Cancer J. Br.

14 (eds. Bateman, A., Draghici, S., Khurana, E, Khurana, S., Draghici, A., Bateman, (eds. , 128 (2013). 128 ,

97 , 1165–1174 (2007). 1165–1174 ,

4 , e17 (2005). e17 , eoe Biol. Genome doi: 10.1038/nn.4592 PLoS Genet. PLoS

11 Nucleic R14 ,

nature neuroscience | reporting checklist

Corresponding Author: Nord # Main Figures: 8

Manuscript Number: NN-A57672 # Supplementary Figures: 7

Manuscript Type: Article # Supplementary Tables: 8

# Supplementary Videos: 0

Reporting Checklist for Nature Neuroscience This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. For more information, please read Reporting Life Sciences Research.  

Please note that in the event of publication, it is mandatory that authors include all relevant methodological and statistical information in the manuscript.

} Statistics reporting, by figure

z Please specify the following information for each panel reporting quantitative data, and where each item is reported (section, e.g. Results, & paragraph number).   zEach figure legend should ideally contain an exact sample size (n) for each experimental group/condition, where n is an exact number and not a  range, a clear definition of how n is defined (for example x cells from x slices from x animals from x litters, collected over x days), a description of the statistical test used, the results of the tests, any descriptive statistics and clearly defined error bars if applicable.   z For any experiments using custom statistics, please indicate the test used and stats obtained for each experiment.  z Each figure legend should include a statement of how many times the experiment shown was replicated in the lab; the details of sample  collection should be sufficiently clear so that the replicability of the experiment is obvious to the reader.  z For experiments reported in the text but not in the figures, please use the paragraph number instead of the figure number.

Note: Mean and standard deviation are not appropriate on small samples, and plotting independent data points is usually more informative. When technical replicates are reported, error and significance measures reflect the experimental variability and not the variability of the biological process; it is misleading not to state this clearly.

DEGREES OF DESCRIPTIVE STATS TEST USED n P VALUE FREEDOM & (AVERAGE, VARIANCE) F/t/z/R/ETC VALUE

EXACT WHICH TEST? DEFINED? REPORTED? EXACT VALUE VALUE VALUE FIGURE NUMBER SECTION & SECTION & SECTION & SECTION & SECTION & PARAGRAPH # PARAGRAPH # PARAGRAPH # PARAGRAPH # PARAGRAPH # March 2016 one-way Fig. 9, 9, 10, mice from at least 3 Methods error bars are Fig. Fig. 1a p = 0.044 F(3, 36) = 2.97 Fig. legend ANOVA legend 15 litters/group para 8 mean +/- SEM legend legend example

results, unpaired t- Results Results error bars are Results Results Results 15 slices from 10 mice p = 0.0006 t(28) = 2.808 para 6 test para 6 para 6 mean +/- SEM para 6 para 6 para 6 example

+ 1A- NA - 1C

1 nature neuroscience | reporting checklist DEGREES OF DESCRIPTIVE STATS TEST USED n P VALUE FREEDOM & (AVERAGE, VARIANCE) F/t/z/R/ETC VALUE

EXACT WHICH TEST? DEFINED? REPORTED? EXACT VALUE VALUE VALUE FIGURE NUMBER SECTION & SECTION & SECTION & SECTION & SECTION & PARAGRAPH # PARAGRAPH # PARAGRAPH # PARAGRAPH # PARAGRAPH #

+ Unpaired t- Results Error bars are Fig Fig t=4.037 1D 17 WT=9, HT=8 Fig legend p=0.0076 - test para 2 mean +/- SEM legend legend df=15 e14.5: t=2.765, + Unpaired t- Results e14.5: WT=6, HT=6 Error bars are Fig e14.5: p=0.02 Fig df=10 1E 12, 18 Fig legend - test para 2 P0: WT=9, HT=9 mean +/- SEM legend P0: p=0.0089 legend P0: t=2.977, df=16 Male: t=6.317, df=14 One way ANOVA Female: t=6.126, + ANOVA, Results WT-M=4, WT-F=6, Error bars are Fig p<0.0001 Fig 1F 10, 20 Fig legend df=12 - post hoc t- para 3 HT-F=10, HT-M=10 mean +/- SEM legend M: p<0.0001 legend Anova: test F: p<0.0001 R2=0.7764, F(3,26)=1.946 + Results 1G NA - para 3 Area: p=0.0328 Area: t=2.76, 30% df=6 Thickness: 30% Thickness: + Unpaired t- Results WT=4 (2 M, 2 F), Error bars are Fig p=0.224 Fig t=1.221, df=6 1H 8 Fig legend - test para 3 HT=4 (2 M, 2 F) mean +/- SEM legend 70% legend 70% Thickness: Thickness: t=1.355, df=6 p=0.268 Length: t=4.944, Length: df=6 p=0.0026 + Unpaired t- Results WT=20, HT=19 Results Error bars are Fig Fig Fig 2A 39 p=0.0019 t(1, 37)=3.3492 - test para 4 littermates para 4 mean +/- SEM legend legend legend + Unpaired t- Results WT=20, HT=19 Results Error bars are Fig Fig Fig 2B 39 p=0.0104 t(1, 37)=2.7064 - test para 4 littermates para 4 mean +/- SEM legend legend legend WT F(1, Repeated + Results WT=20, HT=19 Results Error bars are Fig WT p=0.0031 Fig 19)=11.5030, Fig 2C measures 39 - para 4 littermates para 4 mean +/- SEM legend HT p=0.0867 legend HT F(1, legend ANOVA 18)=3.2825 Repeated WT F(1, Repeated measures: WT Fig 19)=16.31 Fig measures + Results WT=20, HT=19 Results Error bars are Fig p=0.0007 legend, HT F(1, legend, 2D ANOVA & 39 - para 5 littermates para 4 mean +/- SEM legend HT p=0.0059 Results 18)=9.744 Results Two factor Two factor: para 5 Two factor: para 5 ANOVA p=0.149 F(1, 37)=2.16 WT F(1, Repeated WT p=0.0164 Fig 18)=7.00369, Fig measures + Results WT=19, HT=18 Results Error bars are Fig HT p=0.0023 legend, HT F(1, legend, 2E ANOVA & 37 - para 5 littermates para 4 mean +/- SEM legend Two factor: Results 17)=12.8051, results Two factor p=0.7555 para 5 Two factor: para 5 ANOVA F(1, 35)=0.0985 Fig p=0.73 F(1, 37)=0.11 + One-way Results WT=20, HT=19 Results Error bars are Fig Fig legend,

2F 39 Habituation: Habituation: F(1, March 2016 - ANOVA para 5 littermates para 4 mean +/- SEM legend legend results p=0.584 37)=0.30 para 5 + Unpaired t- Results WT=10, HT=9 Results Error bars are Fig Fig Fig 2G 19 p=0.3599 t(1, 17)=0.9409 - test para 5 littermates para 4 mean +/- SEM legend legend legend + Unpaired t- Results WT=10, HT=9 Results Error bars are Fig Fig Fig 2H 19 p=0.5705 t(1, 17)=0.5785 - test para 5 littermates para 4 mean +/- SEM legend legend legend + Unpaired t- Results WT=10, HT=9 Results Error bars are Fig Fig Fig 2I 19 p=0.8722 t(1, 17)=0.1634 - test para 5 littermates para 4 mean +/- SEM legend legend legend

2 nature neuroscience | reporting checklist WT=20, HT=20 Fig Fig p=0.3978 t(1, 38)=0.8552 + Unpaired t- Results littermates Results Error bars are Fig legend, legend, 2J 40 Sex diff: p = Sex diff: t(1, 36) - test para 5 (Sex differences: para 4 mean +/- SEM legend results results 0.619 = -0.504 WT=19, HT=19) para 5 para 5 Fig t(1, 38)=1.0151 + Unpaired t- Results WT=20, HT=20 Results Error bars are Fig p=0.3165 Fig legend, 2K 40 Sex diff: t(1, - test para 5 littermates para 4 mean +/- SEM legend p=0.1449 legend results 38)=1.4883 para 5 + Unpaired t- Results WT=20, HT=20 Results Error bars are Fig Fig Fig 2L 40 p=0.2455 t(1, 38)=1.1795 - test para 5 littermates para 4 mean +/- SEM legend legend legend Cortex: Cortex: t=4.511, p<0.0001, df=35 + Unpaired t- Results 18 HT (8 m, 10 f), All relevant stats Table Amygdala: Fig Amygdala: 3A 37 Methods - test para 6 19 WT (9 m, 10 f) reported S1 p<0.0001, legend t=4.57, df=35 Hippocampus: Hippocampus: p<0.0001 t=4.646, df=35 Cortex: Cortex: t=2.566 p=0.0147, df=35 Fig + Unpaired t- Results 18 HT (8 m, 10 f), Error bars are Table Amygdala: Amygdala: 3B 37 Methods legend, - test para 6 19 WT (9 m, 10 f) mean +/- SEM S1 p=0.0010, t=3.599 df=35 Table S1 Hippocampus: Hippocampus: p=0.0004 t=3.896 df=35 + Voxel-wise Results 18 HT (8 m, 10 f), All relevant stats Table All relevant All relevant stats 3C 37 Methods Table S1 Table S1 - FDR para 6 19 WT (9 m, 10 f) reported S1 stats reported reported Cortex: F- stat=33.6, Cortex: FDR<0.1% Generalized R2=0.1855, Fig Hippocampus: F- Results + linear Results 18 HT (8 m, 10 f), % Time (exact Table Hippocampus: 3E 37 Methods legend, stat=29.0, para 7, - regression para 7 19 WT (9 m, 10 f) value) vs. Volume S1 R2=0.148, Table S1 FDR<0.1%, Table S1 model Amygdala Amygdala: F- R2=0.1352 stat=38.6, FDR<0.1% FDR- Full statistical + corrected Results 18 HT (8 m, 10 f), Full statistical Table Full statistical 3D 37 Methods results Table S1 Table S1 - Regional para 6 19 WT (9 m, 10 f) results reported S1 results reported reported Analysis + Results 4A NA - para 8 Generalized linear Full statistical + Results WT=18, HT=26 Full statistical Table Full statistical 4B regression 44 Tables S2 results Table S4 Table S3 - para 9 littermates results reported S3 results reported model reported (EdgeR) Generalized linear + Results WT=18, HT=26 Error bars are Table p=2.2E-27 Full statistical 4C regression 44 Table S2 Table S4 Table S3 - para 9 littermates mean +/- SEM S3 FDR=3.18E-23 results reported model (EdgeR) Kdm5b: Generalized FDR=1.4E-02, linear + Results WT=18, HT=26 Error bars are Table Bcl11a: Full statistical 4D regression 44 Table S2 Table S4 Table S3 - para 9 littermates mean +/- SEM S3 FDR=2.5E-02, results reported model Hnrnpa2b1: (EdgeR) FDR=1.9E-02 qPCR: t=2.691, Hnrnpa2b1: qPCR: + Unpaired t- Results Error bars are Fig Fig df=8 4E 10, 10 qPCR - WT=6, HT=4 Fig legend p=0.0248, - test para 9 mean +/- SEM legend legend WB: t=3.765, WB - WT=5, HT=5 WB: p=0.0055

df=8 March 2016 Results Methods + ChIP-seq WT=18, HT=26 Relevant stats Table MACS Relevant stats 4F para 44 Table S2 & Table Table S3 - peak calling littermates reported S3 p<0.0001 reported 10 S4

3 nature neuroscience | reporting checklist mRNA processing: p=6.62E-14, 1.37E-12, Enrichr GO Results Chromatin + gene set WT=18, HT=26 Relevant stats Relevant stats Fig, Table 4G para 44 Table S2 Fig mod: Fig - enrichment littermates reported in table reported in table S5 10 p=8.56E-11, analysis 1.57E-12; Reg of Cell Cycle: p=0.0317, 0.0021 Permutation test: gene set compared Results + for test and WT=18, HT=26 Relevant stats Table Permutation Results Relevant stats 4H para 44 Table S2 Table S7 - random littermates reported S4 p=2.8E-10 para reported 11 sample (100k) set against DE genes Z-scores shown top-to- bottom: Results 8.2, 6.3, 6.8, + Permutation WT=18, HT=26 Relevant stats Table 4I para 44 Table S2 4.8, 2.0, 0.3, Fig - test littermates reported S3 11 2.5 FMRP p=0.04 DE-up.ASD p=0.012 Z-scores shown top-to- Results + Permutation WT=18, HT=26 Relevant stats Table bottom: 4J para 44 Table S2 Fig - test littermates reported S3 10.2, 14.2, 10/12 4.8, 3.4, 15.0, 3.7, 3.2, 10.6 Generalized Results + linear WT=18, HT=26 Relevant stats Table 5A para 44 Table S2 - regression littermates reported S3 13 model Results + WT=18, HT=26 Relevant stats Table 5B WGCNA para 44 Table S2 - littermates reported S7 13 Generalized Results + linear WT=18, HT=26 Table All relevant All relevant stats 5C para 44 Table S2 Mean +/- 1 SD Table S4 Table S3 - regression littermates S3 stats reported reported 13 model GO Results + WT=18, HT=26 Relevant stats Table All relevant All relevant stats 5D Enrichment para 44 Table S2 Table S7 Table S7 - littermates reported S7 stats reported reported (TopGO) 13 Z-scores: M1: 10.2, NA Results M2: NA, 5.0 + Permutation WT=18, HT=26 Relevant stats Table Relevant stats 5E para 44 Table S2 M3: 11.3, NA Fig Table S7 - test littermates reported S7 reported 13 M4: NA, 9.7 Mgrey: NA, 10.9 March 2016

4 nature neuroscience | reporting checklist Z-scores top- to-bottom, by module: Katayama up: 12.60, 5.23, 4.85, 6.88, 3.55 Katayama down: 6.80, 7.22, 2.51, 2.92, 0.33 Durak up: 1.27, 3.39, 1.47, 4.24 Durak down: 10.1, 5.26, 3.58, 5.24 Results Chd8 up: 1.01, + Permutation WT=18, HT=26 Relevant stats Table Relevant stats Fig, Table 5F para 44 Table S2 0.96 Fig - test littermates reported S7 reported S7 13 Chd8 down: 10.2, 0.50, 0.77, 0.27 Sanders down: 5.81, 2.22, 1.06, 2.47 Willsey down: 5.13, 1.26, 3.95, 2.30 Hormozdiari down: 5.8, 1.68, 0.81 Darnell up: 0.88 Darnell down: 4.30, 0.22, 0.73, 0.31 Z-scores top- to-bottom, by module: M2 down: 11.51, 2.59, 7.35, 4.62 M3 up: 2.05, 0.79 M3 down: 9.33, 0.11, 1.71 Results + Permutation WT=18, HT=26 Relevant stats Table M13 up: 9.24, Relevant stats 5G para 44 Table S2 Fig Table S7 - test littermates reported S7 1.30 reported 13 M16 up: 3.07, 1.51 M16 down: 2.77 M17 up: 1.59 M17 down: 2.58 M9 up: 1.06, 7.55, 3.77 M19 up: 3.90, 0.52 Generalized linear Results + regression WT=18, HT=26 Table permutation p All relevant stats March 2016 6A para 44 Table S2 Mean +/- 1 SD Table S7 Table S7 - model & littermates S3 p=8.8e-26 reported 14 Permutation test Generalized Results + linear WT=18, HT=26 Mean Effect Size All relevant All relevant stats 6B para 44 Table S2 Fig Table S3 Table S3 - regression littermates in Pseudo-counts stats reported reported 14 model

5 nature neuroscience | reporting checklist STRING: protein- Reported as Results + protein WT=18, HT=26 calculated Results All relevant stats Results 6C para 44 Table S2 NA NA - interactions littermates using STRING: para 14 reported para 14 14 (observed vs p<0.0001 expected) Results + - 6D NA para 14 Results + Unpaired Error bars are Fig Fig t = 2.636 6E para 8 WT=4, HT=4 Fig legend p=0.0388 - type 2 t-test mean +/- SEM legend legend df = 6 15 Results + - 7A NA para 8 WT=4, HT=4 Fig legend NA NA 16 Results + - 7B NA para 8 WT=4, HT=4 Fig legend NA NA 16 Results + - 7C NA para 8 WT=4, HT=4 Fig legend NA NA 16 Results + - 7D NA para 8 WT=3, HT=3 Fig legend NA NA 16 Results + Unpaired t- Error bars are Fig Fig 7E para 8 WT=4, HT=4 Fig legend p=0.0165 t=3.294, df=6 - test mean +/- SEM legend legend 16 Results + Unpaired t- Error bars are Fig Fig 7F para 8 WT=4, HT=4 Fig legend p=0.0032 t=4.733, df=6 - test mean +/- SEM legend legend 16 Results + Unpaired t- Error bars are Fig Fig 7G para 8 WT=4, HT=4 Fig legend p=0.0086 t=3.84, df=6 - test mean +/- SEM legend legend 16 Results + Unpaired t- Error bars are Fig Fig 7H para 6 WT=3, HT=3 Fig legend p=0.0057 t=5.406, df=4 - test mean +/- SEM legend legend 16 FDR: Srsf7 = 5e-02 Generalized Results Hnrnpa1 + linear WT=18, HT=26 Fig Table S3, All relevant stats 8A para 44 Table S2 Mean +/- 1 SD =3.6-03 Table S3 - regression littermates legend Fig reported 17 Dhx9 = 7e-02 model Upf3b = 6.9e-05 1130 DE (FDR < Reactome Results + 0.20) genes All relevant stats Table All relevant All relevant stats 8B gene set para 1130 Table S2 Table S6 Table S6 - identified in reported S6 stats reported reported enrichment 17 reactome DB e17.5: WT=2, HT=7 MISO Results BF>100, All + (591 total events All relevant stats Table All relevant stats 8C statistical para 9 Table S8 relevant stats Table S8 Table S8 - on bar plot, 395 reported S8 reported model 18 reported dark grey) MISO Results BF>100, All + e17.5: WT=2, HT=7 All relevant stats Table All relevant stats 8D statistical para 9, 9 Table S8 relevant stats Table S8 Table S8 - e14.5: WT=3, HT=6 reported S8 reported model 18 reported + - 8E NA e14.5-e17.5: p=0.001 e14.5-e17.5: March 2016 Welch's t- e12.5=7 e17.5-e17.5: t=4.1708, test, Results e14.5=9 + Error bars are Fig p=0.03 Fig df=13.622 8F Generalized para 41 e17.5-WT=7, Fig legend - mean +/- SEM legend Dev time: legend e17.5-e17.5: linear 29 e17.5-HT=7 p=4.53E-14 t=-2.4352, regression P0=11 Dev time: df=11.632 R2=0.77

6 nature neuroscience | reporting checklist 280 kDa 280 kDa ANOVA: ANOVA: F=10.19, p=0.0118 R2=0.7726 110 kDa 110 kDa ANOVA: ANOVA: One way F=2.33, p=0.1676 ANOVA, WT=3 (280), WT=4 R2=0.3997 + Results Error bars are Fig 280 kDa: 5bp Fig S1A unpaired 10 (110), HT-5bp=3, Fig legend 280 5bp t=3.233, - para 2 mean +/- SEM legend p = 0.0319; legend post hoc t- HT-14bp=3 df=4 14bp test 280 14bp p=0.0328 t=3.203, df=4 110 kDa: 5bp 110 5bp t=1.951, p=0.1086, df=5; 14bp 14bp p = t=0.8174, df=5 0.4509 280 kDa: 280 kDa: + Unpaired t- Results Error bars are Fig p=0.0089 Fig t=2.977, df=16 S1B 18 WT=9, HT=9 Fig legend - test para 2 mean +/- SEM legend 110 kDa: legend 110 kDa: p=0.001 t=4.022, df=16 280 kDa: 280 kDa: + Unpaired t- Results Error bars are Fig p=0.9903 Fig t=2.765, df=10 S1C 12 WT=6, HT=6 Fig legend - test para 2 mean +/- SEM legend 110 kDa: legend 110 kDa: p=0.02 t=0.01248, df=10 + Unpaired t- Results Error bars are Fig Fig Fig S2A 42 WT=20, HT=22 Fig legend p=0.1046 t(1, 40)=1.6607 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2B 42 WT=20, HT=22 Fig legend p=0.0571 t(1, 40)=1.9593 - test para 4 mean +/- SEM legend legend legend WT F(1, Repeated + Results WT=21, HT=20 Error bars are Fig WT p=0.0453 Fig 20)=4.5583, Fig S2C measures 41 Fig legend - para 4 littermates mean +/- SEM legend HT p=0.3846 legend HT F(1, legend ANOVA 19)=0.7921

WT F(1, Repeated + Results Error bars are Fig WT p=0.0042 Fig 20)=10.438 Fig S2D measures 42 WT=21, HT=21 Fig legend - para 4 mean +/- SEM legend HT p=0.0008 legend HT F(1, legend ANOVA 20)=15.470 WT F(1, Repeated + Results Error bars are Fig WT p=0.0002 Fig 20)=20.3750 Fig S2E measures 42 WT=21, HT=21 Fig legend - para 4 mean +/- SEM legend HT p=0.00002 legend HT F(1, legend ANOVA 20)=30.3946 + One-way Results Error bars are Fig Fig Fig S2F 42 WT=21, HT=21 Fig legend p=0.583 F(1, 40)=0.307 - ANOVA para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2G 22 WT=11, HT=11 Fig legend p=0.8807 t(1, 20) = 0.1520 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2H 22 WT=11, HT=11 Fig legend p=0.8057 t(1, 20)=0.2492 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2I 22 WT=11, HT=11 Fig legend p=0.1167 t(1, 20)=1.6396 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2J 22 WT=11, HT=11 Fig legend p=0.4914 t(1, 20)=0.7010 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2K 44 WT=22, HT=22 Fig legend p=0.8047 t(1, 40)=0.2489 - test para 4 mean +/- SEM legend legend legend + Unpaired t- Results Error bars are Fig Fig Fig S2L 44 WT=22, HT=22 Fig legend p=0.3328 t(1, 40)=0.9799 - test para 4 mean +/- SEM legend legend legend All relevant + Voxel-wise Results 18 HT (8 m, 10 f), Table All relevant stats S3 37 Table S1 Effect size stats (FDR, p) Table S1 Table S1 - FDR para 6 19 WT (9 m, 10 f) S1 reported reported + Unpaired t- Results Error bars are Fig Fig

S4A 13 WT=6, HT=7 Fig legend p=0.5221 t=0.6612, df=11 March 2016 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4B 8 WT=4, HT=4 Fig legend p=0.5888 t=0.5663, df=7 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4C 11 WT=6, HT=5 Fig legend p=0.0851 t=1.935, df=9 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4D 11 WT=6, HT=5 Fig legend p=0.0383 t=2.425, df=9 - test para 9 mean +/- SEM legend legend

7 nature neuroscience | reporting checklist + Unpaired t- Results Error bars are Fig Fig S4E 10 WT=6, HT=4 Fig legend p=0.9069 t=0.1207, df=8 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4F 11 WT=6, HT=5 Fig legend p=0.0248 t=2.464, df=9 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4G 14 WT=7, HT=7 Fig legend p=0.0264 t=2.994, df=8 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4H 11 WT=6, HT=5 Fig legend p=0.5476 t=0.6248, df=9 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4I 11 WT=6, HT=5 Fig legend p=0.0581 t=2.17, df=9 - test para 9 mean +/- SEM legend legend + Unpaired t- Results Error bars are Fig Fig S4J 10 WT=6, HT=4 Fig legend p=0.1174 t=1.755, df=8 - test para 9 mean +/- SEM legend legend All relevant stats reported in table: RSAT de Results Predicted sites, YY1: + 708 ChIP-seq peaks All relevant stats S5A novo motif para 708 Methods logo motif bit Fig e=4.2E-31, Fig Fig - from WT=2 reported in table analysis 10 scores NRF1: e=8.5E-24, NFYB: e=1.3e-17 Scn2b: FDR=3.9E-02 Generalized Cacna1e: Results + linear WT=18, HT=26 Table FDR=1.2E-01 All relevant stats S5D para 44 Table S2 Mean +/- 1 SD Table S4 Table S4 - regression littermates S4 Cacna2d1: reported 13 model FDR=6.2E-02 Cacna1b: FDR=1.2E-01 Generalized Gompers: WT=18, Results + linear 44, 37, HT=26 littermates All relevant All relevant stats S5B para Table S2 NA NA Table S4 Table S4 - regression 77 Katayama: 37 stats reported reported 12 model Durak: 77 Generalized Gompers: WT=18, Results + linear 44, 37, HT=26 littermates All relevant All relevant stats S5C para Table S2 NA NA Table S4 Table S4 - regression 77 Katayama: 37 stats reported reported 12 model Durak: 77 P0: Student's t- Results P0: + P0: WT=8, HT=10 Fig df=4 S6A test (type 3, para 18, 17 Fig legend NA Tbr1: p=0.34 - P7: WT=7, HT=10 legend Tbr1: t=0.75 2-tailed) 15 Ctip2: p=0.81 Ctip2: t=0.46 Student's t- Results + P0: WT=8, HT=10 S6B test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15 Student's t- Results + P0: WT=8, HT=10 S6C test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15 Student's t- Results + P0: WT=8, HT=10 S6D test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15 Student's t- Results + P0: WT=8, HT=10 S6E test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15 Student's t- Results + P0: WT=8, HT=10 S6F test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15

Student's t- Results March 2016 + P0: WT=8, HT=10 S6G test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15 Student's t- Results + P0: WT=8, HT=10 S6H test (type 3, para 18, 17 Fig legend NA - P7: WT=7, HT=10 2-tailed) 15

8 nature neuroscience | reporting checklist P7: Tbr1: P7: Results df=4 + P0: WT=8, HT=10 p=0.88 Fig S6I NA para 18, 17 Fig legend NA Tbr1: t=0.75 - P7: WT=7, HT=10 Ctip2: p=0.69 legend 15 Brn2: p=0.32 Ctip2: t=0.46 Brn2: 0.75 Results + P0: WT=8, HT=10 S6J NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6K NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6L NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6M NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6N NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6O NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6P NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6Q NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Results + P0: WT=8, HT=10 S6R NA para 18, 17 Fig legend NA - P7: WT=7, HT=10 15 Permutation test: gene set Left: compared Results p=1.1e-03, + for test and WT=18, HT=26 Table Relevant stats S7A para 44 Table S2 Count (red line) Middle: Fig Table S3 - random littermates S3 reported 17 1.2e-10, sample Right: 1.2e-17 (100k) set against DE genes e14.5-e17.5- Dev time: Welch's t- e12.5=7 HT: p=0.30 e14.5-e17.5-HT: test, Results e14.5=9 e17.5-HT-WT: t=1.0847, + Error bars are Fig Fig S7B generalized para 41 e17.5-WT=7 Fig legend Fig p=0.62 df=13.9 - mean +/- SEM legend legend linear 18 e17.5-HT=7 Dev time: e17.5-HT-WT: regression P0=11 p=1.32E-05 t=0.51739, R2=0.37 df=10.666 + - S1D NA

} Representative figures

1. Are any representative images shown (including Western blots and Western blot data is presented in Figures 1E, 4E, and S1. immunohistochemistry/staining) in the paper? IHC data is presented in Figures 6E, 7A-D, S6. March 2016

If so, what figure(s)?

2. For each representative image, is there a clear statement of Yes to all, and is reported in the above statistics reporting table as how many times this experiment was successfully repeated and a well as the following: discussion of any limitations in repeatability? - Full western blot data and is displayed in Figure S1 - IHC data is described in figure legends (6E, 7A-D, S6) If so, where is this reported (section, paragraph #)? 9 nature neuroscience | reporting checklist } Statistics and general methods

1. Is there a justification of the sample size? Behavioral analysis sample size is justified in Paragraph 3, Statistics.

If so, how was it justified? No justification of sample size is reported as power analysis is not Where (section, paragraph #)? standardized for RNA-seq. However, sample size per group and condition is equal to or greater than generally accepted standard of Even if no sample size calculation was performed, authors should three biological replications per group. Our sample size is report why the sample size is adequate to measure their effect size. additionally sufficient for network analysis. This is reported in Para 2, Statistics.

No justification of sample size for other experiments, though biological replicate numbers are standard in the field and sufficient to capture significance and trends. This is reported in Para 1, Statistics.

2. Are statistical tests justified as appropriate for every figure? Behavioral analysis sample size is justified in Paragraph 3, Statistics.

Where (section, paragraph #)? We use standard statistical approaches with appropriate corrections for each experiment.

a. If there is a section summarizing the statistical methods in Behavioral analysis statistical tests are delineated within the text in the methods, is the statistical test for each experiment Paragraph 3, Statistics. clearly defined? For RNA-seq, yes, for each method described, in the relevant Methods sections.

All statistical methods reported in full in the text or supplement.

b. Do the data meet the assumptions of the specific statistical Behavioral analysis data meets the assumptions of the statistical test you chose (e.g. normality for a parametric test)? tests chosen. Data passed distribution normality tests, was collected using continuous variables and thus analyzed via Where is this described (section, paragraph #)? parametric analysis, in all assays. This is described in Para 3, Statistics.

Yes - QC performed to examine specific parameters and we used tests specifically designed for RNA-seq differential expression analysis. Additionally, we use non-parametric tests where necessary. This is described within the text in paragraph 1, subsection "RNA sequencing," of section "Methods."

c. Is there any estimate of variance within each group of data? Behavioral analysis variances are similar within each group and between groups that are being statistically compared. This is Is the variance similar between groups that are being described within the text in Paragraph 1 of "Behavioral Testing" in statistically compared? the "Methods". Where is this described (section, paragraph #)? For genomics, analysis methods are designed to be robust to sample variance. Further, we perform QC on individual samples to March 2016 ensure robust comparisons. This is described within the text in paragraph 1 of Genomics in Methods.

For other analysis (e.g. qPCR and western blot), we used standard approaches (e.g. Student's t-test and ANOVA) in the field.

d. Are tests specified as one- or two-sided? Always two-sided. 10 nature neuroscience | reporting checklist e. Are there adjustments for multiple comparisons? Yes for behavior. Yes for RNA-seq. Yes for MRI.

3. To promote transparency, Nature Neuroscience has stopped allowing All figures converted to show individual data points and summary/ bar graphs to report statistics in the papers it publishes. If you have variance measurements. bar graphs in your paper, please make sure to switch them to dot- plots (with central and dispersion statistics displayed) or to box-and- whisker plots to show data distributions.

4. Are criteria for excluding data points reported? We followed established laboratory protocol in which all data points that lie within 2 standard deviations of the mean are Was this criterion established prior to data collection? included in analysis. This is described within the text in Para 3 of Where is this described (section, paragraph #)? Statistics.

No data points excluded in RNA-seq after original quality filtering to remove samples with 3' coverage bias. This is described in the results and methods. For MISO analysis, one sample (e17.5 S159) was removed before pooling samples; this is reported in Para 1 of Miso Analysis in Methods.

For qPCR, the highest and lowest values for both WT and Chd8+/ del5 groups were removed to reduce variation. This was described in qRT-PCR in Methods; actual n's are listed in the figure legends.

One of each sample from WT and HT in the Q-fraction analysis was removed; this is mention in Paragraph 1 of section "EdU labeling and immunofluorescent analysis" in "Methods".

5. Define the method of randomization used to assign subjects (or Behavioral analysis was performed on mixed genotype home cages samples) to the experimental groups and to collect and process data. and blinded to experimenter and video scorer/processor. This is described within the text in paragraph 1 of "Nissl staining, EdU If no randomization was used, state so. labeling, and immunofluorescent analysis" in Methods. Where does this appear (section, paragraph #)? For RNA-seq: samples were randomly collected across litters from male het x female wt and processed without knowing genotype. This is described in paragraph 4 of Statistics.

Samples for western blot analysis were randomly collected from multiple litters. For all histology, samples were randomly selected for analysis, as described in Statistics, and additionally in methods.

6. Is a statement of the extent to which investigator knew the group The investigator was blinded to behavioral experiments and analysis allocation during the experiment and in assessing outcome included? and to histology. This is described within the text in the first paragraph of subsections "Behavioral testing" and "Lamination If no blinding was done, state so. Assay" in Methods. Where (section, paragraph #)?

All experiments were performed blind to genotype ("Results" March 2016 section, paragraph 1), unless denoted otherwise in Methods.

7. For experiments in live vertebrates, is a statement of compliance with A statement of compliance with ethical guidelines and regulations is ethical guidelines/regulations included? included in both the methods and subsection "Behavioral testing" of Methods. Where (section, paragraph #)?

11 nature neuroscience | reporting checklist 8. Is the species of the animals used reported? The species of animal used in is given and described within the text in Paragraph 1 of section "Mice harboring heterozygous germline Where (section, paragraph #)? Chd8 mutation exhibit megalencephaly" in "Results".

9. Is the strain of the animals (including background strains of KO/ Animal strain information is included in Paragraph 1 of section transgenic animals used) reported? "Mice harboring heterozygous germline Chd8 mutation exhibit megalencephaly" in Results, and in Methods. Where (section, paragraph #)?

10. Is the sex of the animals/subjects used reported? The sex of animals used in the behavioral analysis is described within the text in paragraph 1, section "Behavioral phenotyping of Where (section, paragraph #)? adult Chd8+/del5 mice " in Results, and additionally in Methods and Figure Legends.

Full RNA-seq sample info given in Table S2. Sex of animals used in western blots and histology was not described.

11. Is the age of the animals/subjects reported? The age of animals used in the behavioral analysis is described within the text in paragraph 1 of "Behavioral Testing" in Methods. Where (section, paragraph #)? The age of animals used in RNA-seq is described within the text in paragraph 1 of section "Genomics" in "Experimental Procedures".

The age of animals used in western blot is described within the figure legends and in text in the first paragraphs of "Generation of Chd8 mutant mice" and "Western blot analysis" of Methods.

12. For animals housed in a vivarium, is the light/dark cycle reported? The environmental light conditions of the vivarium in which the animals used in the behavioral analysis is described within the text Where (section, paragraph #)? in paragraph 1 of "Behavioral Testing" in Methods.

13. For animals housed in a vivarium, is the housing group (i.e. number of The number animals per cage used in the behavioral analysis is animals per cage) reported? described within the text in paragraph 1 of "Behavioral Testing" in Methods. Where (section, paragraph #)?

14. For behavioral experiments, is the time of day reported (e.g. light or The environmental light of the behavioral testing conditions in dark cycle)? which the animals used in the behavioral analysis is described within the text in paragraph 1 of "Behavioral Testing" in Methods. Where (section, paragraph #)?

15. Is the previous history of the animals/subjects (e.g. prior drug Animals used in the behavioral analysis were not previously administration, surgery, behavioral testing) reported? subjected to other assays or analysis. This is described within the text in paragraph 1 of "Behavioral Testing" in Methods. Where (section, paragraph #)?

a. If multiple behavioral tests were conducted in the same Animals used in the behavioral analysis were subjected to a set group of animals, is this reported? sequence of behavioral assays. This is described within the text in paragraph 1 of "Behavioral Testing" in Methods. March 2016 Where (section, paragraph #)?

16. If any animals/subjects were excluded from analysis, is this reported? No data points were excluded from behavioral analysis. This is described within the text in paragraph 3 of Statistics in Methods. Where (section, paragraph #)?

12 nature neuroscience | reporting checklist a. How were the criteria for exclusion defined? N/A

Where is this described (section, paragraph #)?

b. Specify reasons for any discrepancy between the number of One animal died during behavioral testing. This happened during animals at the beginning and end of the study. Three-Chambered Social approach, in the middle of the behavioral battery. This is described in paragraph 3 of Statistics in Methods. Where is this described (section, paragraph #)?

} Reagents

1. Have antibodies been validated for use in the system under study Yes, except for Tbr2. (assay and species)?

a. Is antibody catalog number given? Yes, in methods.

Where does this appear (section, paragraph #)?

b. Where were the validation data reported (citation, Antibodies used in previous studies are cited in the methods. For supplementary information, Antibodypedia)? Tbr2, we cite the Abcam data sheet. The Tbr2 antibody exhibits similar histology staining patterns to other Tbr2 antibodies. Where does this appear (section, paragraph #)?

2. Cell line identity NA a. Are any cell lines used in this paper listed in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample?

Where (section, paragraph #)?

b. If yes, include in the Methods section a scientific NA justification of their use--indicate here in which section and paragraph the justification can be found.

c. For each cell line, include in the Methods section a NA statement that specifies: - the source of the cell lines - have the cell lines been authenticated? If so, by which method? - have the cell lines been tested for mycoplasma contamination? Where (section, paragraph #)? March 2016

13 nature neuroscience | reporting checklist } Data availability

Provide a Data availability statement in the Methods section under "Data Data availability reported in methods with DOIs. availability", which should include, where applicable: • Accession codes for deposited data • Other unique identifiers (such as DOIs and hyperlinks for any other datasets) • At a minimum, a statement confirming that all relevant data are available from the authors • Formal citations of datasets that are assigned DOIs • A statement regarding data available in the manuscript as source data • A statement regarding data available with restrictions

See our data availability and data citations policy page for more information.

Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Microarray data

Deposition is strongly recommended for many other datasets for which structured public repositories exist; more details on our data policy are available here. We encourage the provision of other source data in supplementary information or in unstructured repositories such as Figshare and Dryad.

We encourage publication of Data Descriptors (see Scientific Data) to maximize data reuse.

Where is the Data Availability statement provided (section, paragraph #)?

} Computer code/software

Any custom algorithm/software that is central to the methods must be supplied by the authors in a usable and readable form for readers at the time of publication. However, referees may ask for this information at any time during the review process.

1. Identify all custom software or scripts that were required to conduct Described in methods. All analysis software was previously the study and where in the procedures each was used. published.

2. If computer code was used to generate results that are central to the Code availability: paper's conclusions, include a statement in the Methods section

under "Code availability" to indicate whether and how the code can All custom scripts and TrackHubs used for data processing and March 2016 be accessed. Include version information as necessary and any analysis will be available at the Nord Lab Git Repository (https:// github.com/NordNeurogenomicsLab/). restrictions on availability.

} Human subjects

14 nature neuroscience | reporting checklist

1. Which IRB approved the protocol? NA

Where is this stated (section, paragraph #)?

2. Is demographic information on all subjects provided? NA

Where (section, paragraph #)?

3. Is the number of human subjects, their age and sex clearly defined? NA

Where (section, paragraph #)?

4. Are the inclusion and exclusion criteria (if any) clearly specified? NA

Where (section, paragraph #)?

5. How well were the groups matched? NA

Where is this information described (section, paragraph #)?

6. Is a statement included confirming that informed consent was NA obtained from all subjects?

Where (section, paragraph #)?

7. For publication of patient photos, is a statement included confirming NA that consent to publish was obtained?

Where (section, paragraph #)?

} fMRI studies

For papers reporting functional imaging (fMRI) results please ensure that these minimal reporting guidelines are met and that all this information is clearly provided in the methods:

1. Were any subjects scanned but then rejected for the analysis after the NA data was collected?

a. If yes, is the number rejected and reasons for rejection NA described?

Where (section, paragraph #)?

2. Is the number of blocks, trials or experimental units per session and/ NA or subjects specified?

Where (section, paragraph #)? March 2016

3. Is the length of each trial and interval between trials specified? NA

4. Is a blocked, event-related, or mixed design being used? If applicable, NA please specify the block length or how the event-related or mixed design was optimized.

15 nature neuroscience | reporting checklist 5. Is the task design clearly described? NA

Where (section, paragraph #)?

6. How was behavioral performance measured? NA

7. Is an ANOVA or factorial design being used? NA

8. For data acquisition, is a whole brain scan used? NA

If not, state area of acquisition.

a. How was this region determined? NA

9. Is the field strength (in Tesla) of the MRI system stated? NA

a. Is the pulse sequence type (gradient/spin echo, EPI/spiral) NA stated?

b. Are the field-of-view, matrix size, slice thickness, and TE/TR/ NA flip angle clearly stated?

10. Are the software and specific parameters (model/functions, NA smoothing kernel size if applicable, etc.) used for data processing and pre-processing clearly stated?

11. Is the coordinate space for the anatomical/functional imaging data NA clearly defined as subject/native space or standardized stereotaxic space, e.g., original Talairach, MNI305, ICBM152, etc? Where (section, paragraph #)?

12. If there was data normalization/standardization to a specific space NA template, are the type of transformation (linear vs. nonlinear) used and image types being transformed clearly described? Where (section, paragraph #)?

13. How were anatomical locations determined, e.g., via an automated NA labeling algorithm (AAL), standardized coordinate database (Talairach daemon), probabilistic atlases, etc.?

14. Were any additional regressors (behavioral covariates, motion etc) NA used?

15. Is the contrast construction clearly defined? NA March 2016

16. Is a mixed/random effects or fixed inference used? NA

a. If fixed effects inference used, is this justified? NA

17. Were repeated measures used (multiple measurements per subject)? NA

16 nature neuroscience | reporting checklist a. If so, are the method to account for within subject NA correlation and the assumptions made about variance clearly stated?

18. If the threshold used for inference and visualization in figures varies, is NA this clearly stated?

19. Are statistical inferences corrected for multiple comparisons? NA

a. If not, is this labeled as uncorrected? NA

20. Are the results based on an ROI (region of interest) analysis? NA

a. If so, is the rationale clearly described? NA

b. How were the ROI’s defined (functional vs anatomical NA localization)?

21. Is there correction for multiple comparisons within each voxel? NA

22. For cluster-wise significance, is the cluster-defining threshold and the NA corrected significance level defined?

} Additional comments

Additional Comments Efforts have been made to report specific methods in the manuscript or in the methods section. We will make our raw data, processed data, and analysis codes available via our GitHub site. March 2016

17