Regulatory Networks Specifying Cortical Interneurons from Human

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Regulatory networks specifying cortical interneurons PNAS PLUS from human embryonic stem cells reveal roles for CHD2 in interneuron development

Kesavan Meganathana, Emily M. A. Lewisa, Paul Gontarza, Shaopeng Liua, Edouard G. Stanleyb, Andrew G. Elefantyb, James E. Huettnerc, Bo Zhanga, and Kristen L. Krolla,1

aDepartment of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110; bMurdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, VIC 3052, Australia; and cDepartment of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA, and approved November 14, 2017 (received for review July 17, 2017)

Cortical interneurons (cINs) modulate excitatory neuronal activity followed by differentiation, tangentialneuronalmigrationtotarget by providing local inhibition. During fetal development, several cIN locations, and maturation to acquire physiological properties. These subtypes derive from the medial ganglionic eminence (MGE), a processes are largely determined by cell-intrinsic activities (2). transient ventral telencephalic structure. While altered cIN devel- Notably, cIN development in rodents and primates differs signifi- opment contributes to neurodevelopmental disorders, the inac- cantly (6). During primate evolution, cINs increased in number, cessibility of human fetal brain tissue during development has complexity, and proportion, relative to cortical excitatory neurons hampered efforts to define molecular networks controlling this (7, 8). Likewise, the human cortex is twice as thick as that of the process. Here, we modified protocols for directed differentiation mouse, necessitating greater numbers of cIN progenitors. These of human embryonic stem cells, obtaining efficient, accelerated differentiate over an extended gestational period, increasing their sensitivity to perturbation (7, 8). production of MGE-like progenitors and MGE-derived cIN subtypes Study of the molecular networks controlling human cIN devel- with the expected electrophysiological properties. We defined

opment is limited by lack of access to fetal brain materials and BIOLOGY transcriptome changes accompanying this process and integrated inability to obtain expandable cIN progenitors or purify cINs from these data with direct transcriptional targets of NKX2-1, a trans- brain tissues. Therefore, approaches for modeling human cIN de- DEVELOPMENTAL cription factor controlling MGE specification. This analysis defined velopment in vitro are essential to define this developmental pro- NKX2-1–associated genes with enriched expression during MGE gram and understand its disruption in disease. Here, we addressed specification and cIN differentiation, including known and previ- these questions by refining protocols for directed differentiation of ously unreported transcription factor targets with likely roles in human embryonic stem cells (hESCs) into MGE-like progenitors MGE specification, and other target classes regulating cIN migra- and cIN-like neurons, obtaining efficient, accelerated production of tion and function. NKX2-1–associated peaks were enriched for con- mature cIN subtypes with the expected functional properties. We sensus binding motifs for NKX2-1, LHX, and SOX transcription used this model to define transcriptome changes accompanying factors, suggesting roles in coregulating MGE gene expression. MGE specification and cIN differentiation and direct targets of the Among the NKX2-1 direct target genes with cIN-enriched expression was CHD2, which encodes a chromatin remodeling protein Significance mutated to cause human epilepsies. Accordingly, CHD2 deficiency impaired cIN specification and altered later electrophysiological In the human cerebral cortex, activities of excitatory neurons cis function, while CHD2 coassociated with NKX2-1 at -regulatory are balanced by local inhibition provided by cortical interneu- elements and was required for their transactivation by NKX2-1 in rons (cINs). Although disrupted cIN development contributes to MGE-like progenitors. This analysis identified several aspects of neurodevelopmental disorders, molecular networks controlling gene-regulatory networks underlying human MGE specification this process were largely unknown. Here, we refined protocols and suggested mechanisms by which NKX2-1 acts with chromatin for differentiating human embryonic stem cells into functional remodeling activities to regulate gene expression programs under- cINs. We defined gene-expression programs underlying cIN lying cIN development. development and direct targets of the NKX2-1 transcription factor in this process, identifying potential regulators. These human embryonic stem cells | medial ganglionic eminence | included CHD2, a gene mutated to cause human epilepsies. cortical interneurons | NKX2-1 | CHD2 Accordingly, CHD2 deficiency impaired cIN development and altered later cIN function, while CHD2 and NKX2-1 could co- he mammalian cerebral cortex consists of two main cate- regulate cIN gene expression by cobinding shared genomic Tgories of neurons, excitatory neurons that convey information regulatory regions. This work defines key features of both to distant neurons within and outside the cortex and GABAergic normal and disrupted cIN development. cortical interneurons (cINs) that provide local inhibition to modulate excitatory neuron activity (1, 2). Interneuron loss or dysfunction Author contributions: K.M., and K.L.K. designed research; K.M., E.M.A.L., and J.E.H. performed research; S.L., E.G.S., and A.G.E. contributed new reagents/analytic tools; K.M., during development alters the balance between neuronal excitation E.M.A.L., P.G., J.E.H., B.Z., and K.L.K. analyzed data; and K.M., J.E.H., and K.L.K. wrote and inhibition, contributing to many neurodevelopmental disorders, the paper. including epilepsy (1–3). The authors declare no conflict of interest. cIN development begins with dorsoventral patterning of the This article is a PNAS Direct Submission. telencephalon through regional transcription factor (TF) activities. Published under the PNAS license. While the cerebral cortex derives from the dorsal telencephalon, Data deposition: The data reported in this paper have been deposited in the Gene Ex- in the ventral telencephalon three progenitor domains, the medial, pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. lateral, and caudal ganglionic eminences (MGE, LGE, and CGE, GSE99937). respectively) generate distinct neuronal types (1, 3–5). In rodents, 1To whom correspondence should be addressed. Email: [email protected]. the MGE provides the majority of cINs, which populate the cor- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tex, hippocampus, and striatum. MGE progenitor specification is 1073/pnas.1712365115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1712365115 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 NKX2-1 TF during this process. In mice, NKX2-1 controls both most cells by d12–15, and continued to be expressed through MGE specification and cIN differentiation, with conditional loss neuronal differentiation and maturation (d27–60) (Fig. S1C). of function resulting in MGE deficits, overproduction of alternate More than 80% of cells expressed NKX2-1–eGFP by d15, sug- cell fates (e.g., LGE), and spontaneous seizures (9–11). During gesting that specification to an NKX2-1–expressing progenitor both murine and human neurodevelopment, NKX2-1 exhibits had occurred by then, with NKX2-1 expression at similar fre- MGE-restricted expression (7, 8), while human NKX2-1 muta- quencies through d35 (Fig. 1B). tions cause chorea, hypotonia, and dyskinesia (3, 12). Although To further characterize neurons generated with this protocol, we NKX2-1 direct targets were recently defined in mouse MGE (13), performed immunocytochemistry (ICC) at d35. We observed high in human cIN development they remained unknown. frequencies of cells expressing NKX2-1 (86.2 ± 1.9%), the ventral Here, we integrated NKX2-1–associated genes in MGE-like telencephalic markers ASCL1 (83.8 ± 1.6%) and OLIG2 (85.9 ± progenitors with the MGE- and cIN-enriched transcriptome. This 1.6%), the ganglionic eminence (GE) marker DLX2 (83.9 ± 2.1%), defined NKX2-1 direct target genes encoding known and pre- the pan-telencephalon–enriched gene FOXG1 (67.4 ± 2.6%), and viously unreported regulators of cIN migration, differentiation, and the neuronal markers DCX (65.6 ± 3.7%) and TUBB3 (69.8 ± function and suites of transcriptional and epigenetic regulators that 3.8%). These cells also efficiently expressed the GABAergic neu- may comprise a gene-regulatory network mediating human MGE ron markers GAD65/67 (65.5 ± 4.1 %) and GABA (74.4 ± 2.8%) specification. Among these was CHD2, a member of the chro- (Fig. 1 C and D). We also performed ICC for CXCR4 (35.2 ± modomain helicase DNA-binding (CHD) family of chromatin- 2.7%), marking migrating neurons, CALB1 (38.8 ± 3.2%), marking remodeling enzymes (14). Human mutations resulting in CHD2 GABAergic interneurons, and parvalbumin (PV; 37.7 ± 2.1%) and haploinsufficiency cause pediatric epilepsies involving refractory somatostatin (SST; 34.2 ± 1.4%), which mark the two major MGE- seizures, cognitive decline, and poor prognosis (14). As CHD2 derived cIN subtypes (Fig. 1 C–E) (7, 18). By contrast, few cells expression increased during cIN differentiation, we examined its were immunopositive for calretinin (CR), which predominantly requirements for this process, determining that NKX2-1 regulates marks a CGE-derived cIN subtype (Fig. S1D) (10). Few or no cells CHD2 expression by binding a cis-regulatory element (CRE) in the expressed Ki-67, which marks proliferating cells, NKX2-2, a di- CHD2 gene promoter, while CHD2 and NKX2-1 cobind some of encephalon marker, RAX, a hypothalamic marker, or DARPP32, thesameCREs,andCHD2promotesNKX2-1–dependent target which marks LGE-derived striatal neurons (Fig. 1 C and D). Total gene transactivation. Accordingly, CHD2 knockdown (KD) and numbers of immunopositive and scored cells are given in Table S1. knockout (KO) revealed requirements in cIN differentiation and Together, these results suggest that our modified protocol pro- CHD2-dependent transcriptional programs, including regulation of motes the formation of MGE-like neural progenitors, which dif- human epilepsy genes, during this process. Together, these data ferentiate into GABAergic neurons expressing cIN markers, provide a foundation for beginning to define the molecular basis of including PV or SST, which mark the two major subtypes of human cIN specification and differentiation and to identify how its MGE-derived cINs. This constitutes higher percentages of cells dysregulation may contribute to neurodevelopmental disorders. expressing SST or PV than were previously observed without further coculturing (16, 17, 19). Results Directed Differentiation of hESCs into MGE-Like Progenitors and cIN- Engraftment and Functional Characterization of hESC-Derived cIN- Like Neurons. To generate hESC-derived ventral telencephalic- Like Neurons. We next tested the engraftment capacity and func- like neuroectoderm, MGE-like progenitors (hMGEs), and ma- tional properties of these hcINs. We prepared hcINs for trans- ture cIN-like neurons (hcINs) we used the hESC lines H9 and plantation into murine brain, using polysialic acid (PSA)–neural Hes3-NKX2-1GFP/W, which has a GFP reporter knockin at the cell adhesion molecule (NCAM) selection to remove residual NKX2-1 locus (15). Beginning with previously published pro- proliferating cells, transducing neurons with a synapsin (SYN)– tocols (16, 17), we tested many variables systematically, with eGFP transgene (Fig. 2A), and transplanting them into either the goals of generating high percentages of hESC-derived hMGEs adult hippocampus or neonatal cortex of NOD/SCID mice, ob- and driving their rapid, efficient differentiation into hcINs. serving engraftment 1 mo posttransplantation (Fig. 2 B and C and Protocol modifications were tested with the NKX2-1–eGFP line Fig. S1 E and F). to track frequencies of NKX2-1–expressing hMGEs, with fur- For electrophysiology, hcINs were prepared as above and then ther experimentation in H9 hESCs. Prior protocols differed in cultured on a rat cortical astrocyte feeder layer for ∼3 wk (Fig. several respects, using either embryoid body (EB) or monolayer 2D and SI Methods). hcINs developed voltage-gated and ligand‐ culture, inhibiting Wnt, BMP (ALK2/3), and TGF-β (ALK5) sig- gated currents typical of CNS neurons. Under current clamp, naling with either recombinant proteins or small-molecule inhibi- hcINs fired action potentials (Fig. 2 E and F) and received tors, and combining these manipulations with either continuous or spontaneous synaptic inputs (Fig. 2G), with most cells firing timed [day (d) 10–18] administration of the sonic hedgehog (SHH) numerous spikes during prolonged depolarizing current pulses agonist purmorphamine (16, 17). We tested these and other vari- (Fig. 2F). Under whole‐cell voltage clamp, depolarizing steps ables and obtained the highest frequency of NKX2-1–expressing elicited an inward TTX-sensitive sodium current (Fig. 2H) and progenitors by using EB culture, continuous purmorphamine treat- outward potassium currents that were blocked by a combination ment, and small-molecule inhibitors. In particular, EB approaches of tetraethyl ammonium and 4‐aminopyridine (20). Agonists for inducedbothNKX2-1–expressing progenitors and neurons express- excitatory and inhibitory neurotransmitter receptors, including ing mature cIN subtype markers more efficiently than monolayer kainate, NMDA, GABA, and glycine, evoked whole‐cell currents culture (Fig. S1A) and so were used for further experiments. when applied by local perfusion (Fig. 2I). Recordings revealed Other parameters tested included starting cell numbers, time of frequent spontaneous inhibitory postsynaptic currents (sIPSCs) Y-27632 withdrawal, time of EB plating for differentiation, and that were blocked by the GABA receptor antagonist bicuculline inclusion of a rosette selection purification step (Fig. S1B). The methiodide (Fig. 2G), while evoked inhibitory postsynaptic cur- modified protocol used for all work below is schematized in Fig. rents (IPSCs) were relatively infrequent (3 of 31 cells tested in 1A and is described in Methods and SI Methods. paired recordings) (Fig. 2J). Fig. 2K elaborates the electrophys- We used the NKX2-1–eGFP hESC line to assess the kinetics iological properties observed, which suggest that these differen- of producing NKX2-1–expressing progenitors. During human tiation conditions result in hESC-derived neurons with the fetal development, NKX2-1 is expressed specifically in the MGE expected functional properties of GABAergic cINs. from approximately gestational weeks 8–14, and NKX2-1– expressing GABAergic interneurons are also detected in the Transcriptome Changes Accompanying Differentiation of hESCs. Our cortex in the third trimester, potentially following migration from analysis above suggested that this directed differentiation pro- the MGE (7). During directed differentiation in vitro, NKX2-1– tocol generated MGE-like progenitors that differentiated into eGFP expression was visible by d4–6, was strongly expressed in neurons expressing GABAergic, cIN, and mature MGE-derived

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712365115 Meganathan et al. Downloaded by guest on October 1, 2021 cIN subtype markers and exhibiting electrophysiological properties PNAS PLUS A hESC NKX2.1 TUBB3 GABAergic neuron expected for cINs. Therefore, to comprehensively define the transcriptome changes accompanying this specification and dif- d0 d4 d15 d25 d35 ferentiation process, we performed RNA-sequencing (RNA-seq) SB431542 analysis at d0 (hESCs), d4, d15, d25, and d35. Stage-specific LDN193189 DAPT cAMP differentially expressed genes (DEGs) were defined by compar- Y-27632 XAV939 Purmorphamine BDNF/ Asc. acid ison with normalized expression at the prior time point, as described (SI Methods and Dataset S1), and were visualized by a A d0 4 10 15 25 27 31 35 multidimensional scaling plot (Fig. 3 ). Replicates exhibited 4 minimal variability, with the greatest change in DEGs from d0–4, 3x10 cells/well Shaker on Matrigel Dissociated NKX2-1 V-bottom plate on Matrigel when expression increased 280-fold, while the pluripotency marker NANOG decreased >1,000-fold (Dataset S1). B 100 d15 100 d25 100 d35 Significant changes also occurred from d4–15 and d15–25, while 80 80 80 fewer DEGs were observed from d25–35, suggesting potential 60 81.9±1.6 60 79.2±7.1 60 70.5±7.3 stabilization of the neuronal state (Fig. 3A). 40 40 40 To assess whether this differentiation approach elicited gene- %Max of 20 %Max of 20 %Max of 20 expression changes that accompany MGE specification and cIN 0 n=3 0 n=6 0 n=4 differentiation during mouse and/or human development in vivo, 3 4 5 3 4 5 3 4 5 01010 10 01010 10 01010 10 we examined the temporal expression of genes that mark and Non-expressing control NKX2-1GFP regulate the formation of different forebrain regions and cell C ICC DAPI merge ICC DAPI merge lineages in these data. During murine MGE specification, SHH NKX2.1 GAD65/67 signaling induces NKX2-1, LHX6,andLHX8 predominantly in the MGE. This expression continues during cIN differentiation, while NKX2-1 expression diminishes in maturing cINs (21). We observed a similar temporal sequence during hcIN differentiation: ASCL1 GABA NKX2-1 expression peaked at d15, with continued expression to d35, while LHX6/8 expression began at d15 and increased through B ASCL1 OLIG2 GABAergic neurons d35 (Fig. 3 ). and mark the ventral forebrain in BIOLOGY mouse and were most highly expressed from d15–25 and were OLIG2 CXCR4 reduced at d35 here. DLX1, DLX2,andDLX5 mark the GEs in DEVELOPMENTAL vivo and exhibited enriched expression from d14–35, as did the general anterior neural or forebrain markers FOXG1, OTX2, SIX3 SIX6 SYP TUBB3 DLX2 ,and and neuronal markers and .Bycon- CALB1 trast, the dorsal telencephalic markers EMX1, EMX2,andPAX6 were never expressed during differentiation (Fig. 3 B and C).

Telencephalic/GE/MGE markers Telencephalic/GE/MGE We also examined markers of later cell lineages. At d35 neurons expressed markers of GABAergic neurons (GAD1/2 and SLC32A1/ FOXG1 PV SLC6A1, required for GABA transport) (Fig. 3C)andmaturecIN subtype markers, including the neuropeptides SST and NPY,but not VIP, which is expressed in CGE-derived interneurons (Fig. 3D),

mature cortical interneuron SST while expression of genes associated with neural or cIN migration DCX (CXCR4, ARX, DCX, NRN1, RELN) was enriched from earlier time points. We also evaluated markers of other cell lineages, including RAX, a prospective forebrain and hypothalamic marker, NKX2-2 DARPP32 TUBB3 RAX , a diencephalic marker, and , a striatal neuron marker. These exhibited transient, low-level expression during neural induction, which did not persist to later stages (Fig. 3D). Based upon marker expression, this in vitro differentiation pro- Ki-67 DARPP32 tocol yields ventral neuroectoderm of forebrain character by d4, neural progenitors expressing MGE markers most highly at d15 alternate fates Proliferation/differentiation (with some persisting to later stages), and neurons expressing markers of cIN differentiation, function, and MGE-derived cIN DEsubtype identity at d35. Therefore, we comprehensively character- 100 100 Unstained IgG ized gene-expression profiles accompanying these developmental 80 80 SST+ transitions by k-means clustering, based upon z-scores of expression 60 across all data (Fig. 3E), to define major gene-expression changes 60 40 35.6±4 accompanying the transition from a ventral telencephalic-like state. 40 This analysis yielded nine clusters of DEGs (Fig. 3F and Fig. S2A). % Marker+

20 %Max of 0 20 Of particular interest were clusters 1 and 7, in which genes exhibited greater than fourfold increased expression from d4–15 (Fig. 3F). PV n=3 DCX SST Ki67 0 DLX2 OLIG2 GABA 3 4 5 Based upon marker gene expression, genes in these clusters sig- NKX2-1ASCL1 FOXG1 TUBB3 CALB1 CXCR4NKX2-2 01010 10 GAD65/67 nificantly increased in expression during specification of ventral telencephalic neuroectoderm to an MGE progenitor-like state. Fig. 1. Differentiation of hESCs into hcINs. (A) Scheme used, with representative Cluster 4 also exhibited greater than fourfold enrichment at later markers or light microscopic images and days of treatment. (B) Representative time points relative to d4, but this was only seen by d25–35 (Fig. 3F). FACS plots measuring cells expressing NKX2-1–eGFP reporter (red) and non- expressing cells (blue); the average percentage of eGFP-expressing cells ± SEM and biological replicates (n) are shown. (C and D) ICC at d35 with antibodies shown (C) Gene Expression Signatures of MGE-Like Progenitors and cIN-Like and quantified (mean ± SEM) (D). (E) Representative FACS plot in d35 hcINs stained Neurons and Comparison with Gene Expression in Human Fetal with SST antibody (yellow) or IgG control (blue) or unstained (red). The percentage Brain. We performed gene ontology (GO) analysis on the k-means of SST-immunopositive cells (mean ± SEM) is shown; n = 3. (Scale bars: A, 200 μmin clusters highlighted above. Clusters 1 and 7 are enriched for GO brightfield images and 50 μm in fluorescence images; C,50μm.) terms related to early neurodevelopment (neurogenesis, axonal

Meganathan et al. PNAS Early Edition | 3of10 Downloaded by guest on October 1, 2021 A B C MGE and less similarity with other 8–9 wk or second–third tri- d0 d33 ~d35 Adult transplantation Neonatal Transplantation mester fetal brain structures (Fig. 4E). Together, these data sug- cIN PSA-NCAM SYN-eGFP gest that hMGE specification mimics many aspects of regionally differentiation selection transduction B’ C’ enriched gene expression in wk 8–9 MGEs in vivo, providing an experimentally manipulable context for studying molecular regulation of this developmental program.

D FE NKX2-1 Direct Targets Regulating Specification of hMGE-Derived d0 d33 d35 d38 d60 20 mV 20 mV rat cortical Cortical Interneurons. NKX2-1 is expressed in the MGE and cIN PSA-NCAM SYN-eGFP feeder 40 msec 100 msec differentiation selection transduction culture MGE-derived cINs in both mouse and human and is required for both MGE specification and cIN differentiation in mouse (9–11). To define gene regulatory networks through which NKX2-1 may directly control these processes during human MGE specification, GHI Jwe identified its direct targets by performing NKX2-1 ChIP- bicuculline +50 mV K NMDA GABA gly pre 0.5 nA Methods SI Methods at -20 mV -80 sequencing (ChIP-seq) in d15 hMGEs ( and ). NKX2-1 chromatin enrichment was compared with IgG controls, 300 pA 3 pA post 10 msec 50 msec 200 pA 60 pA at -80 mV control 5 sec 5 sec TTX K A B Parameters Mean+/-sem #cells Parameters Mean+/-sem #cells 120 Capacitance (pF) 34.4 +/- 1.5 168 Max # of spikes 5.9 +/- 0.3 109 Input Resistance (GOhm) 0.9 +/- 0.06 168 Max 1st frequency (hz) 16.1 +/- 0.6 97 Day: Resting Potential (mV) -41.4 +/- 0.8 141 Max instantaneous Freq (hz) 12.4 +/- 0.5 97 0

Rheobase (pA) 19.2 +/- 2.3 109 Peak I Na (nA) -2.0 +/- 0.1 134 2 4 RPKM 1st spike threshold (mV) -39.1 +/- 0.5 109 Vm for peak I Na (mV) -26.9 +/- 0.9 134 15 1st spike amplitude (mV) 28.6 +/- 1.8 109 I Na at 0 mV -1.2 +/- 0.08 134 25 0 Day0 35 Day4 1st spike halfwidth (msec) 5.5 +/- 0.4 109 I K at 0 mV 0.45 +/- 0.02 134 0 LHX6 LHX8 Day15

25 NKX2-1 Fig. 2. Electrophysiology and engraftment of hcINs into the murine adult Day25 Day35 – – – – Dimension 2 and neonatal brain. (A C) Scheme: SYN GFP expressing hcINs (after PSA -2

NCAM selection) (A) were transplanted into the hippocampus (CA3) of RPKM adult mice (B)(B′ shows the boxed region in B) or cortex of postnatal d2 NOD/SCID mice (C)(C′ shows the boxed region in C). Images in B and C were -2.5 0 2.5 Dimension 1 0 taken 30 d posttransplantation. The “scan slide” mode in the MetaMorph DLX1 DLX2 DLX5 PAX6 software of the Olympus confocal microscope was used to assemble a EMX1 EMX2 OLIG2 C 200 D ASCL1 composite view of the whole brain slice from several individual images. (D) 800 – – – Scheme: SYN GFP expressing hcINs (after PSA NCAM selection) were 500 cocultured on rat cortical astrocyte feeders for 3 wk before electrophysiology. 200 μ μ ′ RPKM

(Scale bars: 100 MinA, C,andD,50 MinB )(E) Sub- and suprathreshold RPKM 50 stimulation by injection of depolarizing current (52 and 56 pA for 40 ms). (F) 0 8 Multiple action potentials during injection of depolarizing current (56 pA for 0 SYP SIX3 SIX6 800 ms). (G) sIPSCs recorded at −80 and −20 mV are inhibited by exposure to OTX2 TUBB3 FOXG1 SST NPY ARX 25 DCX NRN1 100 μM bicuculline methiodide (black bars). (H) Whole-cell currents recorded RELN − + CXCR4 during a voltage step from 80 to 50 mV in control conditions (black trace) Day0 50 Day0 and with 0.5 μMTTX(redtrace).(I) Currents evoked by exposure to 100 μM Day4 Day4

kainate (K), NMDA, GABA, and glycine (gly). (J) Evoked IPSCs elicited by a RPKM Day15 Day15

presynaptic voltage step. (K) Electrophysiological properties of hESC-derived Day25 RPKM Day25 0 hcINs (mean ± SEM). Day35 Day35 0 GAD1 GAD2 VIP RAX SLC6A1 SLC32A1 z-score guidance) (Fig. 4A). Among these genes, the GO gene subset for E F NKX2-2 nervous system development (181 genes) includes many TFs with 04 DARPP32 6 roles in neurogenesis and ventral telencephalon/GE/MGE specifi- 123 cation (Fig. 4B). These clusters also include long noncoding RNAs 0 with known or potential roles in neurodevelopment and genes with -4 roles in axonal guidance and neuronal migration (examples are 6 456 given in Table S2; all cluster 1 and 7 genes are given in Dataset S1F). Together, these data support an MGE-like character for genes 0 with this enrichment profile and encourage future efforts to mine -4 these data to identify regulators of MGE specification. Cluster log2 (Fold change) 6 789 4 genes exhibited increased expression at d25–35 but not at d15, 0 reflecting expression of known genes involved in cIN differentiation, -4 function, and subtype identity. This cluster was enriched for GO d0 d4 d15 d25 d35 day: 4 15 25 35 4 15 25 35 4 15 25 35 terms associated with synaptogenesis, synaptic neurotransmission, and psychiatric disorders (Fig. 4C), including a distinct group of Fig. 3. Transcriptome changes accompanying hESC–hcIN differentiation determined by RNA-seq during differentiation, with samples collected as indicated. genes involved in neuronal guidance and genes that control – GABAergic neurotransmission and maintain a mature neuronal (A) Multidimensional scaling plot. (B D) Average reads per kilobase of transcript D G per million reads mapped (RPKM) values for the expression of genes marking state (Fig. 4 and Table S2 and Dataset S1 ). MGE, ventral or dorsal telencephalic neuroectoderm (B), anterior neuroecto- We also compared z-scores of transcript enrichment in our derm and neurons/GABAergic neurons (C), and cIN migration/maturation and d15 hMGEs with relative regional transcript enrichment in alternate cell lineages (D). (E) k-Means clustered data based upon expression postconception week 8–9 human brain (Fig. 4E). This revealed z-scores. (F) k-Means clusters, as a log2 change versus d4, defining enriched substantial similarity of d15 hMGE-enriched transcripts and fetal clusters accompanying hMGE specification and hcIN differentiation.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712365115 Meganathan et al. Downloaded by guest on October 1, 2021 PNAS PLUS MGE-like expression clusters (1/7) suggesting a possible role for NKX2-1 in promoting their ex- A B 10 Networks # p-val GAP43 TUBB3 pression, while the rest (410 genes; 41%) had diminished ex- Neuropeptide signaling pathways 25 4.6E-11 pression (Fig. 5C). As shown previously, NKX2-1 is expressed Neurogenesis in general 19 2.3E-05 SCN3A ARX SYT4 ECM remodeling 12 2.5E-05 DLX1 from ∼d4, peaks at d15, and is maintained during differentiation. Axonal guidance 20 9.1E-05 LHX8 EPHA3 Skeletal muscle development 13 1.1E-03 LHX6 ZIC1 As NKX2-1 can act as either a transcriptional activator or re- MEF2C DLX2 Processes VAX1 pressor in other contexts (22, 23), NKX2-1–associated genes with nervous system development 181 7.2E-50 OTP system development 245 6.0E-48 0 DLX5 OLIG1 increased expression from d4 to d15 or d35 could potentially be single-multicellular organism proc. 279 2.6E-46 DLX6 HES5 multicellular organism development 258 5.7E-45 – anatomical structure development 263 3.2E-41 direct targets of NKX2-1 dependent transactivation, while some Diseases NKX2-1–associated genes with low or diminished expression Schizophrenia 97 7.2E-50 Nervous system Schizophrenia, psychotic disorders 97 6.0E-48 Expression d35: log2(RPKM) development (Cluster 1, 7) here may be targets of NKX2-1 repression (e.g., to block gene Psychiatry and psychology 175 2.6E-46 Mental disorders 174 5.7E-45 0510expression associated with alternate fates, such as LGE- or Brain diseases 161 3.2E-41 Expression d4: log2 (RPKM) dorsal telencephalon-enriched genes). However, these changes C Cortical interneuron-like expression cluster (4) D in expression could also be independent of direct NKX2-1 regu- Networks # p-val 10 lation. GO analysis supports distinct roles for these sets of Transmission of nerve impulse 24 2.7E-08 SLC6A17 Synaptic vesicle exocytosis 17 2.5E-05 GAD2 NKX2-1–associated genes, with NKX2-1–bound genes with in- Synaptic contact 16 1.6E-04 NRN1 SYT1 creased expression from d4–15 involved in neurogenesis, syn- Cell-matrix interactions 16 7.7E-04 MYT1L Synaptogenesis 14 1.3E-03 NRXN2 aptogenesis, axonal guidance, and psychiatric disorders, while Processes RELN nervous system development 157 3.5E-30 GABRB1 SYT16 NRP1 single-multicellular organism proc. 260 4.9E-28 0 MAPT GABRB2 NCAM2 system development 220 1.4E-27 SEMA3D SYT16 neurogenesisis 121 7.4E-26 GABRA1 GABRA2 EPHA10 response to endogenous stimulus 125 4.6E-25 Nervous system Diseases A E

development (Cluster 4) 0 Mental disorders 171 2.1E-28 Trans synaptic sigaling 0 10 Psychiatry and psychology 171 3.6E-28 Pathological conditions, symptoms 191 7.0E-27 Expression d35: log2(RPKM) (Cluster 4) Neuroectodermal tumors 260 2.5E-25 0510 Neoplasms, Germ cell, embryonal 261 5.1E-25 Expression d4: log2 (RPKM) SOX2 AMY DFC CGE LGE MGE SO X3

E Day15_R1 z-score # of peaks HES1 Day15_R4 SX5O ZNF704 Day15_R3 08 -3 30 5 GBX2 Day15_R2 kb:<1 10 100 1000 CHD9 OTX2 BIOLOGY Cerebellum.19week Distance to TSS VAX1 ZHX1 CHD2 REST

Cerebellum.37week RAI1 CITED1 FOXK1 LMO4 DEVELOPMENTAL Parietal lobe.24week B TET2 OTP MBD2 MBD4 Frontal cortex.20week EYA1 MAML3 Temporal lobe.20week OLIG2 PHF21A Day15 expression: log2 (RPKM) VAX2 Temporal lobe.22week ZFHX3 ZNF536 MECP2 Frontal cortex.22week TSHZ2 EBF2 PHC3 Parietal lobe.22week MEF2C Occipital lobe.22week 0 EBF1 R UNX1T1 % of Peaks SIX1 HOPX

Occipital lobe.20week 060 0510 F Day0 expression: log2 (RPKM) Fig. 4. Gene-expression profiles for d15 hMGEs and d35 hcINs. (A–D) k-Means exon

clusters1and7(A and B) and cluster 4 (C and D) exhibited more than fourfold promoter intergenic 10 gene body increased expression from d4–15 (clusters 1 and 7) or d4–35 (cluster 4). CpG island

Enriched GO terms (A and C) and genes (B and D) in these clusters are shown. super enhancer = – (E) Z-scores of relative expression at d15 (replicates R1 4) compared with C DEGs associated regional expression in fetal brain at 8–9wk(x axis) and 19–37 wk (y axis) with NKX2-1 binding postconception. AMY, amygdala; DFC, dorsolateral prefrontal cortex. 590 up-reg. 410 down-reg. SYT1 5 EFNB2 KCNH2 EPHB2 CACNA1D EPHB3 CNTN6 STXBP1 KCNH5 KCNB1 EFNA5 NTM GABRG3 defining 4,867 peaks with significant, reproducible enrichment EPHA7 KCNA5 (Dataset S2). About one quarter of these NKX2-1–bound sites SCN1A Log2 (RPKM) SYT2 SLITRK3 01234567 02468 SCN7A (23%) were within 2 kb of the transcription start site (TSS), another Day35 expression: log2 (RPKM) KCNA4 KCNH3 quarter (23%) were 2–10 kb from the TSS, and the remainder 0 KCNN3 Day 4 Day 4 0510 Day 15 Day 25 Day 35 > A Day 15 Day 25 Day 35 mapped to locations 10 kb from the nearest TSS (Fig. 5 and Fig. Day0 expression: log2 (RPKM) B S2 ), with 62% of peaks overlapping the gene body, over 20% at D NKX2-1-associated up-regulatedpg d35 vs d4 the promoter, 18% in CpG islands, and 9% in brain-specific Networks # in DEG p-val Processes # in DEG p-val B Synaptic contact 17 1.9E-07 anatomical structure dev 198 1.1E-19 superenhancers (Fig. 5 ). To test whether d15 was an appropri- Synaptogenesis 12 3.2E-04 developmental process 204 6.1E-19 ate time point for ChIP analysis, we analyzed the temporal ex- Neurogenesis in general 11 2.0E-03 multicellular organism dev 188 6.4E-19 Cartilage development 6 2.3E-03 single organism dev process 200 4.4E-18 pression of MGE and alternate fate markers more precisely (Fig. Cytoplasmic microtubules 8 2.5E-03 nervous system dev 115 1.2E-17 S2C). Optimal expression of many GE/MGE markers was observed G H after d12, while expression of alternate cell fate markers was re- % of % of Motif position NKX2-1 duced after d15, suggesting that d15 is an appropriate time point to Motif Consensus P-value target bkgd. weight matrix identify NKX2-1 direct targets involved in MGE specification. We Nkx2.1 RSCACTYRAG 1.0E-80 46.6 33.4 SOX1490 LHX – Sox3 CCWTTGTY 1.0E-48 25.9 17.5 also validated several NKX2-1 enriched peaks by ChIP-qPCR at Lhx3 ADBTAATTAR 1.0E-26 24.0 17.8 406 250 d4, d15, and d35. All genes were enriched for NKX2-1 binding at 562 372 Sox6 CCATTGTTNY 1.0E-37 22.9 15.8 212 d15, but enrichment varied at the other time points (Fig. S2D). Lhx2 TAATTAGN 1.0E-31 16.7 11.1 118 Therefore, while NKX2-1 targets relevant to hcIN differentiation Sox2 BCCATTGTTC 1.0E-55 14.9 8.1 (e.g., SYT1)(Fig. S2D) may also be enriched at later time points, d15 NKX2-1 enrichment appears to select for many targets in- Fig. 5. Integrating NKX2-1 genome-wide association and enriched gene volved in MGE specification. expression in hMGEs and hcINs. (A) Distance from NKX2-1 peak locations to the nearest TSS. (B) NKX2-1 peaks intersected with annotated genomic To understand the relationship between NKX2-1 binding in features. (C) Integration of NKX2-1 binding with log2 fold changes in ex- hMGEs and regulation of gene expression, we assigned each peak pression for DEGs from d4–35 vs. d0. (D) Top GO terms for NKX2-1–associ- to the nearest TSS (Dataset S2) and assessed the expression of the ated genes with greater than twofold increased expression at d35 vs. d4. (E associated genes. Of the 1,000 NKX2-1–associated genes that and F) NKX2-1–associated genes with enriched expression at d15 (E) or d35 changed expression by more than fourfold from d4 to d15, 59% (F). (G) Top consensus binding motif sequences enriched in NKX2-1–associ- (590 genes) increased in expression during MGE specification, ated peaks. (H) Numbers of NKX2-1–bound peaks with each motif.

Meganathan et al. PNAS Early Edition | 5of10 Downloaded by guest on October 1, 2021 those with increased expression through d35 are also involved in A D later aspects of neuronal function (synaptic transmission, synaptic NKX2-1 peak Day0 NKX2-1 track plasticity) (Fig. 5D and Dataset S2). Conversely, many NKX2-1– 500 Day4 SOX2 peak Day15 SOX2 track associated genes with diminished expression from d4 to d15 or day 0 SPRY2 d35 were involved in general cellular and developmental processes Day25 day 4 Day35 day 14 (e.g., cell cycle regulation, epithelium development, chromosome 100 day 25 day 35 RPKM 5 organization) (Dataset S2). NKX2-1 peak We focused on NKX2-1–associated genes with hMGE-enriched NKX2-1 track SOX2 peak expression. These include many TFs that promote anterior or SOX2 track general neural identity, ventral telencephalon specification, cIN 0 day 0 SOX5 development, neurogenesis, or other neurodevelopmental func- day 4 LHX2 LHX3 LHX6 SOX2 SOX3 SOX6 day 14

tions, as well as TFs with no known role in neurodevelopment NKX2-1 E B day 25 (Fig. 5 and Table S2). These data support a major role for NKX2-1-SOX2 co-bound up >2-fold hESC-cIN day 35 NKX2-1 in regulating other TFs and suggest a transcriptional Networks # p-val NKX2-1 peak Synaptic contact 6 1.1E-03 NKX2-1 track regulatory network downstream of NKX2-1 in MGE specification. Attractive/repulsive receptors 5 5.1E-03 SOX2 peak Some NKX2-1–associated genes exhibit increased expression Regulation of epithelial-to- 5 1.4E-02 SOX2 track mesenchymal transition day 0 LSAMP during both hMGE specification and hcIN differentiation. Many Spermatogenesis 5 1.5E-02 day 4 Platelet aggregation 4 1.8E-02 day 14 of these control cIN migration, axonal guidance, synaptogenesis, Processes day 25 and synaptic neurotransmission and plasticity (Fig. 5F and Table regulation of cell development 27 2.9E-09 day 35 regulation of neurogenesis 24 1.5E-08 NKX2-1 peak S2). These include GABAA receptors, sodium, potassium, and regulation of nervous system dev 25 3.0E-08 NKX2-1 track generation of neurons 33 8.1E-08 SOX2 peak calcium channels, and other genes involved in synaptic develop- anatomical structure development 70 9.9E-08 ment and function (Table S2). Mutations in several of these genes C SOX2 track KCNB1 SCN1A STXBP1 F NKX2-1-SOX2 co-bound down >2-fold hESC-cIN result in epilepsy ( , , )(Fig.5 and Dataset day 0 NR2F2 S2). This supports a role for NKX2-1 in regulating targets involved Networks # p-val day 4 Chromatin modification 3 1.4E-03 day 14 in cIN differentiation and function. NKX2-1 also associates with S phase 3 2.2E-03 day 25 Iron transport 2 1.6E-02 day 35 many other gene classes, including natural antisense transcripts DSB repair 2 1.8E-02 NKX2-1 peak and other long noncoding RNAs (examples are given in Table S2). Wnt signaling 2 4.0E-02 NKX2-1 track SOX2 peak These represent putative regulators of cIN development to be Processes SOX2 track innate immune response in mucosa 4 3.7E-07 day 0 SIRT4 explored in future studies. mucosal immune response 4 1.5E-06 day 4 tissue specific immune response 4 1.9E-06 day 14 regulation of ectodermal cell fate 2 3.0E-06 day 25 TF Consensus Binding Motif Enrichment in NKX2-1–Associated CREs. negative reg. of ectodermal cell fate 2 3.0E-06 day 35 Using NKX2-1–associated peak sequences, we performed de EF100 Day0 Day4 4 IgG novo motif discovery to identify the most highly enriched TF Day15 * 3 ** SOX2 ChIP consensus binding motifs (Methods). An NKX2-1 consensus 20 Day25 Day35 binding motif was the most frequently found (47% of peaks) 3 2 ** *** RPKM % input *** (Fig. 5 G and H). This motif frequently co-occurred with motifs 1 ns ns predicted to be bound by LHX (LHX2/3) or SOX (SOX2/3/6) 0 0 – – 2 TFs, which were each present in 15 25% of the NKX2-1 bound P 2 T4 AM IR Gene G SOX5 S SPRY2 SOX5 NR2F SIRT4 peaks (Fig. 5 ); 1,264 peaks contained more than one type of SPRY2 LS NR2F LSAMP desert motif (Fig. 5H). TFs that recognize these motifs and can regulate Non-target neural or cIN gene expression are expressed during hMGE specification and hcIN differentiation (Fig. 6A). SOX2/3 can Fig. 6. NKX2-1 and SOX2 coassociate with putative CREs in hMGEs. (A) Expression profiles of TFs that recognize NKX2-1 peak-enriched consen- transactivate neural gene expression, while NKX2-1 promotes sus motifs (average RPKM). (B and C) Intersecting NKX2-1–bound peaks in MGE specification, and NKX2-1, LHX6, and SOX6 promote SOX2/3 hMGEs and SOX2-bound peaks in human NPCs (GSE69479) were mapped to cIN differentiation. Accordingly, expression is most the nearest TSS. Putative cobound-peak–associated genes with greater than highly enriched in d4 ventral neuroectoderm, while NKX2-1 twofold increased (B) or decreased (C) gene expression on d35 vs. d0 were peaks at d15 and persists through d35, and SOX6 and LHX6 are analyzed for GO enrichment. (D) Examples of intersecting NKX2-1 and expressed from d15–35 (Fig. 6A). SOX2 peaks. (E) Expression profiles of these genes. (F) SOX2 enrichment, SOX2/3/6 consensus motifs were highly enriched in NKX2-1– detected by ChIP-qPCR, in hMGEs at predicted cobound locations in D. Data bound peaks, suggesting that SOX TFs might cobind these genomic are the average of n = 3 independent biological replicates. *P < 0.05, **P < locations with NKX2-1. Therefore, we compared our NKX2-1– 0.01, ***P < 0.001 (unpaired Student’s t test); ns, not significant. binding data for hMGEs to ChIP-seq data for SOX2 in human neural progenitor cells (NPCs) (24). Approximately 20% of our NKX2-1–bound peaks intersected with sites of SOX2 binding (991 cobind some CREs in hMGEs (Fig. 6F). We also found that co- SOX2-bound peaks intersecting the 4,867 NKX2-1–bound peaks), overexpression (co-OE) of NKX2-1 and SOX2 could regulate supporting a potential coregulatory role. We focused on the subset reporter expression driven by these CREs in a luciferase assay, of 299 peaks predicted to be cobound by both NKX2-1 and while this activity was lost upon mutation of the NKX2-1 con- SOX2 and associated with genes with a greater than twofold ex- sensus binding motifs in each CRE (Fig. S3A). Therefore SOX pression change between d0–35 of hcIN differentiation. Of these, TFs could potentially contribute to NKX2-1–mediated regula- 81% (243) exhibited increased expression, and this gene set was tion of hMGE gene expression through NKX2-1 and SOX2/3/ highly enriched for genes involved in neural development (Fig. 6B). 6 coassociation at these CREs. The much smaller set of predicted cobound genes with diminished expression was instead involved in general cellular functions (sig- CHD2 Is a Direct Target of NKX2-1, and CHD2 Deficiencies Dysregulate naling, cell cycle, chromatin) (Fig. 6C). the Program That Specifies cIN-Like Neurons. Interneuron dysfunc- We tested whether SOX2 and NKX2-1 could cobind the same tion during development contributes to many neurodevelopmental putative CREs in hMGEs by selecting five CREs predicted to be disorders, including epilepsy (1–3). Therefore we sought to utilize bound by both TFs that also contained NKX2-1 and SOX con- this cIN model to identify regulators of cIN development impli- sensus motifs (Fig. 6D). Expression of each CRE-associated gene cated in neurodevelopmental disorders. As NKX2-1 controls cIN increased during hESC–hMGE specification (Fig. 6E). SOX2 specification and differentiation, we examined the overlap be- binding was highly enriched at each NKX2-1–bound putative tween NKX2-1 direct targets with hcIN-enriched expression and CRE, supporting the hypothesis that NKX2-1 and SOX TFs may annotated human epilepsy genes (Epi4K database; epi4kdb.org/).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712365115 Meganathan et al. Downloaded by guest on October 1, 2021 Among these, we focused on CHD2, as haploinsufficiency following reduced expression in the CHD2 KD include TFs involved in PNAS PLUS mutation or deletion of one allele of CHD2 causes epileptic en- neurogenesis and hcIN specification, regulators of synaptogenesis, cephalopathies and, in some cases, autism spectrum disorders. sodium channels, potassium channels, and genes required for Our ChIP-seq data defined an NKX2-1–bound putative CRE in potassium channel clustering (CNTN2, CNTNAP2) (Fig. 7F and hMGEs near the CHD2 gene promoter (Fig. 7A). This CRE could Table S2 and Dataset S3). All CHD2-dependent synaptic and drive luciferase reporter expression in hMGEs (Fig. 7B). NKX2- channel-related genes shown in Table S2 are also human epilepsy 1 overexpression (OE) further increased reporter expression, while genes (Dataset S3), suggesting that their altered expression upon mutating the NKX2-1 motif in this CRE reduced reporter to CHD2 deficiency could contribute to disrupted neuronal devel- baseline levels, even in the presence of overexpressed NKX2-1 opment or function underlying the epileptic phenotype. (Fig. 7B). These data suggest that NKX2-1 binding to this CRE To further test CHD2 requirements in hcIN differentiation, we could promote CHD2 expression (Fig. 7B). CHD2 expression in- generated hESC clonal lines with biallelic CHD2 gene disruption creased during hcIN differentiation (Fig. 7C), and CHD2 protein (CHD2 KO) (Methods, SI Methods,andFig. S3 C and D). These was enriched in nuclei of hcINs (Fig. 7D), supporting a role in this hESC lines did not exhibit altered expression of pluripotency, process. Therefore, we used two lentiviral shRNA constructs that proliferation, or apoptotic markers or changes in EB size or mor- reduce CHD2 levels to ∼50% of wild-type (CHD2 KD) to mimic phology during early neural specification (d0–4) (Fig. S4). We haploinsufficiency seen in human patients (Fig. S3B). assessed whether CHD2 loss affected cIN differentiation and gene To assess CHD2 requirements for hMGE specification, we expression by RT-qPCR and ICC in d15 hMGEs, focusing on genes conducted CHD2 KD in hESCs and performed RNA-seq analysis with reduced expression in the KD RNA-seq data. Consistent with in d15 hMGEs. Differentially regulated genes in CHD2 KD versus CHD2 KD, CHD2 KO reduced the levels of many of the same nontarget control hMGEs are shown in Dataset S3. CHD2 KD genes encoding TFs, potassium channels, and GABAergic neuronal reduced the expression of genes involved in neurogenesis, syn- markers (Fig. 7G). TUBB3 immunostaining at d15 suggested that aptogenesis, and neurotransmission, including potassium and so- some of these gene-expression changes involved delayed or im- dium channels, and genes associated with neurological disorders, paired differentiation, as the CHD2-KO cells exhibited fewer while genes up-regulated upon CHD2 KD were instead involved TUBB3-expressing neurons with shorter neurites (Fig. 7 H–J)and in adhesion and nonneural fate acquisition (Fig. 7E). Genes with reduced expression of the channel gene HCN4 (Fig. 7 H and I). BIOLOGY

ACB D DEVELOPMENTAL 6 * *** CHD2 chr15 20 92900100 9290090 ** NKX2-1 track 15 NKX2-1 peak CHD2 10 d0 0 Merge RLU (fold/ vector) d4 RPKM Ave. 5 CRE CRE

d14 mCRE mCRE CHD2 Fig. 7. Regulation of CHD2 expression by NKX2- d25 0 d35 day: 0 4 15 25 35 1 and CHD2 requirements for cIN development. (A)An +NKX2-1 OE +NKX2-1 OE empty vector NKX2-1–bound putative CRE was defined at the CHD2 E down in CHD2 KD up in CHD2 KD F Networks # DEG p-val Networks # DEG p-val promoter. (B) This CRE drives luciferase expression in Transmission of nerve 31 1.9E-12 Platelet-endothelium- 24 1.3E-09 15 hMGEs, while NKX2-1 OE further stimulates expres- impulse leucocyte interactions Potassium transport 28 3.4E-11 Muscle contraction 23 6.2E-09 sion, and mutating the NKX2-1 consensus motif Neuropeptide signaling 23 1.2E-09 Cartilage development 12 1.0E-06 ● KCNB1 Neurogenesis in general 24 1.8E-08 Blood vessel 23 1.1E-06 (mCRE) reduces expression to baseline. Average fold morphogenesis change of relative luciferase units (RLU) versus empty Synaptogenesis 23 2.4E-08 Actin filaments 18 1.4E-05 = Processes Processes 10 vector (n 3; each replicate was performed in quin- nervous system dev 187 1.6E-47 antigen processing, 44 1.3E-53 tuplicate). (C) CHD2 expression from d0–35. (D) MHC class I multicellular organism 294 4.6E-45 type I interferon 47 3.1E-44 CHD2 and DAPI (blue) ICC at d35. (E and F)RNA-seq process signaling pathway ● KCNQ3 analysis of CHD2-KD versus control shRNA-transduced multicellular organism 264 1.6E-39 cellular response to 47 4.7E-44 CACNA1G ● HCN4 CNTN2 GRIK1 ● development type I interferon ● GNAO1 hMGEs. (E) GO analysis of genes with greater than anatomical structure 273 6.3E-39 interferon-gamma 50 1.2E-43 - log10 (FDR) CNTNAP2 GAD2 ● 5 GRID2 ● ● SYT1 development signaling pathway ATP1A2 CHRNA4 twofold decreased (down) or increased (up) expres- system development 240 9.1E-37 response to type I 47 3.5E-43 HCN2 KCNJ10 SEZ6● sion in CHD2-KD versus control hMGEs. (F) Differen- interferon MAPT ●● Diseases Diseases ●●● tially regulated genes, with some highlighted. (G) PCDH19 SCN2A ●●● Schizophrenia, psychotic 88 1.1E-29 Lung diseases 180 2.3E-57 STX1B ●● ● SYT4 Genes with reduced expression in the CHD2-KD RNA- disorders SYN1 GRIN1●●● ATN1 SYNGAP1 ARX●● seq were also diminished in hMGEs with biallelic Schizophrenia 87 3.5E-29 Pathologic processes 192 3.1E-53 0 NR2F1 KCNJ2 Psychiatry, psychology 169 1.1E-28 Osteoporosis 57 1.8E-52 CHD2-KO vs. wild-type hMGEs. (H–J) CHD2 KO re- Mental disorders 168 2.1E-28 Melanoma 117 3.0E-51 −10 −50510 Fatigue, chronic 24 2.5E-25 Joint diseases 164 4.1E-51 log2 FC: CHD2-KD vs. WT duced TUBB3 and HCN4 immunopositive cells (H and 1.0 I) and neurite length (J). (K) Currents mediated by GHTUBB3 HCN4 I TTX-sensitive sodium channels during steps from a *** holding potential of −80 mV to test potentials WT CHD2 KO WT CHD2 KO 15 WT CHD2 KO ranging from −60 to +35 mV. Traces were obtained 10 *** by subtracting currents recorded during exposure to relative

expression TTX from currents recorded in control external so- (CHD2 KO/wt) 5 0.0 % Marker + lution without TTX. Peak inward current is plotted as a function of test potential for wild-type (black 0 LHX6 DLX2 HCN4 GBX2 GAD2 NR2F1

KCNJ3 traces) and KO (red traces) cells. (L) Phase plots of

KCNQ3 TUBB3 HCN4 action potentials from 13 wild-type (gray traces) and -60 to +35 mV JK1500 -60 -40 -20 20 40 L nine KO (red traces) neurons. The initial increase in *** -80 120 10 Vm (mV) WT dV/dt occurs at more negative membrane potentials KO 0

( μ M) -0.4 in KO cells, indicating a difference in action potential -35 -10 I (nA)

-30 dV/dt -45 -30 threshold. (Upper Right) The inset is plotted on ex- -0.8 0 20 mV panded scales. (Lower Right) The action potentials

Neurite Length -45 0 5 msec are aligned at the point of maximal upslope. In B, I, 0 0.3 nA WT 5 msec -1.2 -60 < < < WT CHD2 KO -40 KO -60 0 60 and J,*P 0.05, **P 0.01, ***P 0.001 (unpaired Vm (mV) Student’s t test). (Scale bars in D and H:50μM.)

Meganathan et al. PNAS Early Edition | 7of10 Downloaded by guest on October 1, 2021 We also characterized the electrophysiological properties of SI A B 50 both cortical excitatory neurons (generated as described in NKX2-1 track Day 0 Methods) and wild-type hcINs with versus hcINs with biallelic NKX2-1 peak Day 4 Day 15 CHD2 KO. By d35, CHD2-KO hcINs did not exhibit gross defi- Day 25 Day 35 ciencies in neuronal marker or cIN gene expression, suggesting day 0 that CHD2 loss delayed but did not block differentiation. How- day 4 day 14 Ave. RPKM day 25 ever, while the excitatory neurons did not exhibit significant elec- day 35 trophysiological differences upon CHD2 KO, CHD2-KO hcINs NKX2-1 track displayed a significantly lower threshold for action potential firing, NKX2-1 peak 0 ZIC1 SPRY1 PAX2 larger peak sodium current, and more negative voltage for peak day 0 sodium current than wild-type cells (Fig. 7 K and L and Table S5), day 4 C 1.5 day 14 IgG suggesting developmental and/or continued CHD2 requirements day 25 CHD2 ChIP day 35 for normal hcIN electrophysiological function. * NKX2-1 track NKX2-1 peak ** *

Coregulation of Gene Expression by NKX2-1 and CHD2 in MGE-Like % Input Progenitors. About 1/10th (9.6%; hypergeometric P value = 2.0 × day 0 −61 day 4 10 ) of the CHD2-regulated genes were also NKX2-1–associated day 14 day 25 0.0 in hMGEs, suggesting that CHD2 might coregulate some of the day 35 ZIC1 SPRY1 PAX2 GD NT NKX2-1 transcriptional program. During myogenesis, CHD2 can 1.0 interact with the MyoD TF and contributes to MyoD-dependent D EFempty vector 8 CRE 8 CHD2 KO activation of muscle differentiation gene expression (25), suggesting CRE+NKX2-1 OE empty vector the potential for analogous CHD2 roles during neural development. * * mCRE CRE To test whether such coregulation could occur, we focused on mCRE+NKX2-1 OE CRE + NKX2-1 OE * * units – A units several candidate NKX2-1 associated putative CREs (Fig. 8 ). The (CHD2 KO/ WT) nsns ns ns ns * ns ns

relative expression ns ns associated genes exhibit distinct temporal expression, with ZIC1 and 0.0 ** Relative luciferase SPRY1 Relative luciferase expression enriched in hMGEs and cINs relative to hESCs, ZIC1 0 PAX2 0 while PAX2 expression is enriched in ventral telencephalic neuro- SPRY1 ZIC1 SPRY1 PAX2 ZIC1 SPRY1 PAX2 ectoderm at d4 but is reduced in hMGEs (Fig. 8B). ChIP-qPCR – – Fig. 8. Coregulation of gene expression by NKX2-1 and CHD2. (A) NKX2-1 revealed CHD2 enrichment at all three NKX2-1 bound putative bound peaks. (B) Associated genes exhibiting increased (ZIC1/SPRY1) or little CREs but not at unrelated genomic locations used as controls [gene (PAX2) expression from d0–35. (C) NKX2-1–bound peaks coenrich for CHD2; desert (GD) and nontarget (NT) in Fig. 8C]. CHD2 KD reduced control amplicons (GD and NT) are not enriched. Shown is average enrich- the expression of each gene in hMGEs in our prior RNA-seq ment; n = 3 independent biological replicates. (D) Expression of these genes analysis, and expression of all three genes was also reduced in is reduced in CHD2-KO hMGEs (d15). (E) NKX2-1 OE promotes reporter ex- hMGEs upon CHD2 KO, suggesting that CHD2 could directly or pression from the ZIC1/SPRY1 CREs but not the PAX2 CRE, which represses indirectly regulate their expression (Fig. 8D). basal luciferase expression in hMGEs. Mutating the NKX2-1 consensus motif Since NKX2-1 and CHD2 coassociate with these genomic lo- (mCRE) eliminates the response to NKX2-1 OE. (F) In CHD2-KO hMGEs, NKX2- cations, we tested whether they could regulate the expression of 1 OE does not promote ZIC1 or SPRY1 CRE-reporter expression. In E and F, the putative CREs by luciferase reporter assay in hMGEs. Re- the average fold change of RLUs versus the empty vector is shown; n = 3or4 porter expression from the ZIC1 and SPRY1 CREs was induced by per CRE, each performed in quintuplicate. For C, E, and F,*P < 0.05, **P < ’ NKX2-1 OE, while luciferase expression from the PAX2 CRE was 0.01 (unpaired Student s t test); ns, not significant. instead repressed relative to basal levels from the empty minimal promoter vector and was not induced by NKX2-1 OE. These ef- fects of NKX2-1 OE were lost in CREs upon mutation of the suspension versus monolayer culture conditions, defined optimal NKX2-1 consensus binding site (Fig. 8E). By contrast, in hMGEs time windows for adding small-molecule signaling inhibitors and generated from the CHD2-KO hESC line, NKX2-1 OE did not other supplements, and purified the progenitors by neural ro- induce reporter expression driven by the ZIC1 or SPRY1 CREs sette selection. This promoted very efficient specification of (Fig. 8F). Together, these data support a role for NKX2-1 binding NKX2-1–expressing hMGEs by d15 (80–90%), with most dif- to these CREs in regulating gene expression and suggest that ferentiating into hcINs that expressed general GABAergic, cIN, CHD2 cobinding to some NKX2-1–activated CREs may facilitate and mature MGE-derived cIN subtype markers (PV or SST) by transactivation of the associated genes during hMGE specifica- d35. This approach yields higher percentages of hcINs expressing tion. This may account, at least in part, for the impaired expres- either PV or SST by 35 d than did prior protocols (16, 17). sion of MGE and neuronal genes that we observed under At d35, negligible numbers of cells expressed the proliferative conditions of CHD2 deficiency (Fig. 7). Together, these data cell marker Ki-67 or alternate cell-lineage markers, indicating support the utility of this model to define transcriptional events that this directed differentiation approach yields a largely post- that underlie human MGE specification and cIN differentiation mitotic population of GABAergic, cIN-like neurons derived and to understand how their disruption alters cIN development from MGE-like progenitors. These hcINs engrafted into the and may contribute to neurodevelopmental disorders. postnatal or adult murine brain and were still present more than Discussion 1 mo after transplantation. In vitro electrophysiological characterization demonstrated that these hcINs exhibit the expected Accelerated Differentiation of Mature cIN-Like Neurons from hESCs. electrophysiological properties: formation of voltage-gated and Combining directed differentiation of human pluripotent cells ligand-gated currents, action potential firing with most cells fir- with genome-wide analyses enables analysis of otherwise in- ing numerous spikes during prolonged depolarizing current accessible regulatory events underlying aspects of human fetal development. Here, we began to elucidate gene regulatory net- pulses, and spontaneous inhibitory postsynaptic currents blocked works that control specification of hMGEs and their differenti- by the GABA receptor antagonist bicuculline, resembling hcINs ation into hcINs. We characterized molecular requirements for derived in prior work (16, 17). As previously described (16, 17), human cIN specification and differentiation by modifying di- coculture of d35 hcINs with rat cortical astrocyte feeders elicited rected differentiation approaches from prior work (17), with the maturation of the action potential firing pattern and observation goals of accelerating and improving the differentiation efficiency. of frequent spontaneous inhibitory postsynaptic currents. These Use of an EB step can accelerate hESC differentiation into results corroborate prior findings that coculturing increases cIN mature neuronal cell types (26, 27). Therefore, we compared EB functional maturation by an unknown mechanism (17).

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1712365115 Meganathan et al. Downloaded by guest on October 1, 2021 Molecular Programs Regulating Specification and Differentiation of bility but does not have apparent neurological phenotypes or PNAS PLUS Human cIN-Like Neurons. In the mouse, ∼60% of cINs are MGE- exhibit seizures (29, 30). Therefore, we thought it would be derived, while ∼30% and ∼10% derive from the CGE or preoptic informative to use hcINs to define molecular pathways sensitive area, respectively (18). Although relative contributions to the hu- to CHD2 deficiency and their relationship to NKX2-1. man cIN population have been harder to ascertain, a significant We found that NKX2-1 promotes CHD2 expression by binding a proportion also appear to be MGE-derived (7, 18). Based upon CRE at the CHD2 promoter during MGE specification, while molecular marker expression, this approach predominantly gener- CHD2-deficient hMGE cells had reduced expression of many ates MGE-like progenitors: ∼80% of d15 cells express NKX2-1, an human epilepsy genes. These frequently encode sodium and po- MGE-restricted marker, and other GE markers. We also observed tassium channels, while CHD2 loss altered hcIN sodium channel molecular similarity between d15 hMGEs and MGEs in human 8- current activity and action potential thresholds. These data suggest to 9-wk fetal brain. This protocol yields ∼70–80% GABAergic that dysregulated channel expression and activity may alter hcIN neurons, which express the MGE-derived cIN subtype markers SST function, contributing to epilepsy involving CHD2 deficiency. No- or PV. By contrast, markers expressed by other cIN subtypes tably, these results differ substantially from findings made in mu- originating from the CGE or preoptic area (CR/VIP) (18, 28) were rine cortical excitatory neurons (31). In this work, shRNA KD of not induced. Together, these findings suggest that this protocol CHD2 during mouse cortical neurogenesis promoted differentia- produces neurons resembling those derived from the MGE. tion of radial glial progenitors into TUBB3-expressing neurons. Transcriptome changes are much more limited from d25–35 than During hcIN specification and differentiation, we instead observed earlier, suggesting stabilization of the neuronal state. At this time, diminished and/or delayed production of TUBB3-expressing neu- hcINs express both mature subtype markers (SST or PV) and other rons and expression of synapse- and channel-related genes, which hallmarks of maturing neurons (e.g., enriched expression of genes could reflect either assessment of different neurodevelopmental involved in GABAergic synaptic neurotransmission). Therefore, processes and/or species-specific differences. while prior work describes stabilization of the transcriptome after d100 (19), some protocol modifications introduced here, such as Mechanisms of NKX2-1–Mediated Target Gene Transactivation. Cross- N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester species comparisons revealed that 9.4% (hypergeometric P value = − (DAPT)-mediated inhibition of Notch signaling and neural rosette 3.4 × 10 27) of human NKX2-1–associated genes associated with selection, enabled mature neurons to be isolated without coculturing an NKX2-1–bound CRE in mouse MGEs (13). Many human- by d35. This may enhance the utility of this system for modeling cell- specific CREs and target genes were found, which may reflect

intrinsic aspects of normal and altered cIN differentiation and both biological differences, including evolutionary divergence in BIOLOGY function. developmental mechanisms, CRE locations, sequences, and genome organization, and the use of divergent models (dissected DEVELOPMENTAL Integrating the Genome-Wide NKX2-1 Binding Profile with Enriched Gene mouse tissue versus hESC-directed differentiation). Enriched Expression During cIN-Like Neuron Specification and Differentiation consensus motifs for TF binding in human and mouse CREs also Reveals Direct Targets Involved in These Processes. In the mouse, exhibited both similarities and differences: Mouse CREs were NKX2-1 is required for MGE specification, acting at or near the coenriched for Nkx, Lhx, Oct4, and E-Box motifs (13), with top of regulatory networks controlling this process, while also Lhx6 and Nkx2-1 motifs associated with genes activated during promoting cIN differentiation (9–11). While NKX2-1 also ex- cIN differentiation. In human hMGEs, we likewise found NKX2-1 hibits MGE-restricted expression during fetal brain development and LHX motif enrichment, but we did not observe OCT4 or in humans, its direct targets had not previously been defined. E-BOX motifs and additionally found coenrichment for SOX2/3/ Here, we identified these targets by ChIP-seq analysis in hMGEs 6motifsin15–25% of the NKX2-1–bound peaks. and integrated these data with hMGE- and hcIN-enriched ex- Sox and homeodomain TFs cobind CREs to regulate multi- pression. This revealed several prominent classes of direct targets ple aspects of neural development (32). For example, ventral through which NKX2-1 could promote MGE specification and neural tube patterning involves CRE co-occupancy by SOXB1 cIN differentiation. In hMGEs, NKX2-1 associated with many TFs (SOX1–3), which broadly activate neural transcription, and TFs, including those with known roles in cIN development, in by NKX2-2 and NKX6-1, which repress alternate fates to ventral telencephalic neural identity, or other aspects of neuro- control regional patterning (33–35). During human fetal de- development, or with no previously defined neurodevelopmental velopment, SOX2 and NKX2-1 are coexpressed by MGE pro- role. These findings support a major role for NKX2-1 in regu- genitors until postconception week 28, supporting potential lating the expression of TFs, which may comprise a transcrip- cofunction (7, 8). Relative to murine development, the gener- tional regulatory network for MGE specification. NKX2-1 also ationofmorecINsoveranextendedperiodcouldfavortheuse associates with many other gene classes, such as genes encoding of coactivators such as SOX2/3, which promote MGE pro- natural antisense or long noncoding RNAs, which represent genitor maintenance. Iterative occupancy of the same binding potential regulators of cIN development (for examples, see Table sites by SOX2, SOX3, and SOX11 promotes gene expression S2). Other classes of NKX2-1–associated genes were highly during neural fate acquisition (36). Similarly, here SOX2/ expressed in differentiating hcINs and control later cIN devel- 3 might cooperate with NKX2-1 to promote the formation or opment and function. These include genes encoding GABA re- maintenance of MGE progenitors, with SOX6 regulating later ceptors, sodium and potassium channels, and other genes involved expression in cINs. Consistent with a potential role, SOX2/ in synaptic output. These data support a continued role for NKX2-1 3 levels peak in d4 ventral neuroectoderm and are diminished in directly controlling the expression of genes mediating cIN dif- by d35, while SOX6 expression initiates at d15 and continues in ferentiation and function. However, as association does not dem- cINs. Twenty percent of these NKX2-1–bound peaks inter- onstrate that NKX2-1 directly transactivates the expression of these sected with SOX2-binding profiles defined previously for hu- genes, functional assays will be used to address this question in man neural progenitors, and SOX2 and NKX2-1 cobound a set future work. of CREs at neural development-related genes that increase expression during MGE specification. CHD2-Dependent Pathways in cIN Development and Function. Mutation We also defined a potential role for CHD2 as a cofactor in of some NKX2-1–associated genes results in epilepsy, autism, and NKX2-1–dependent gene regulation. A subset of NKX2-1–associ- other neurodevelopmental disorders. We focused on one of these, ated genes had diminished expression following CHD2 deficiency. a gene encoding the chromatin-remodeling enzyme CHD2. In CHD2 coassociated with NKX2-1 at CREs for several of these genes humans, CHD2 haploinsufficiency causes epileptic encephalopa- in hMGEs, while CHD2 KO interfered with CRE-driven reporter thies, pediatric disorders involving refractory seizures and cognitive activation by NKX2-1. Both NKX2-1 and CHD2 deficiencies can decline (14). The CHD2-KO mouse model exhibits altered growth, cause epilepsy in humans and/or mice, and CHD2 KO in hcINs hematopoietic differentiation, renal function, and tumor suscepti- altered their electrophysiological properties and dysregulated the

Meganathan et al. PNAS Early Edition | 9of10 Downloaded by guest on October 1, 2021 expression of genes encoding sodium and potassium channels that RNA-Seq and RT-qPCR Analysis. Total RNA was collected from H9 hESCs and are mutated to cause human epilepsy. Therefore, it would be in- H9-derived neural derivatives using the NucleoSpin RNA II kit (Takara). Illumina teresting to test whether any of these epilepsy genes is coordinately library construction, sequencing, data analysis, and RT-qPCR are described in regulated by NKX2-1 and CHD2 binding to the same CRE. To- detail in SI Methods. qPCR primer sequences are given in Table S4. gether, these data demonstrate the utility of this directed differentiation approach to elaborate transcriptional events underlying ChIP/ChIP-Seq. hMGEs were used for NKX2-1 ChIP by modification of standard methods (SI Methods). human MGE specification and cIN differentiation, to define direct targets and mechanisms by which TFs and chromatin-modifying FACS. HES3 or H9 hESC-derived neurons were dissociated with Accutase activities contribute to this process, and to study how its dysregula- (STEMCELL Technologies), and a single-cell suspension was prepared. Cells were tion may contribute to neurodevelopmental disorders. washed once each with PBS and FACS buffer (2% BSA, 1 mM EDTA in PBS) and were resuspended in FACS buffer. For detailed protocols, see SI Methods. Methods hESC Maintenance and Differentiation. H9 and HES3 hESC lines were grown CHD2 KD and KO in hESCs. For KD, hESCs were stably transduced with vali- under feeder-free conditions on Matrigel (Corning) in mTeSR1 (STEMCELL dated lentiviral shRNA expression vectors directed against CHD2. CRISPR/Cas9 Technologies). Cortical excitatory and inhibitory neuronal differentiation was technology was used to generate clonal hESC lines with biallelic disrup- performed by modification of prior protocols (SI Methods)(16,17).Experiments tion of CHD2. For detailed information and shRNA/gRNA sequences, see adhered to protocols (#12-002) approved by the Washington University hESC SI Methods. Research Oversight Committee. As this work was designated “nonhuman subjects research” by Washington University’s Institutional Review Board, in- ACKNOWLEDGMENTS. We thank Matheus Victor and Andrew Yoo for formed consent was not required. assisting in cIN transplantations and for the SYN–GFP plasmid; David Mu for the NKX2-1 vector (Addgene); and Andrew Yoo and Harrison W. Gabel for constructive suggestions on the manuscript. This project was supported Immunocytochemistry. Primary antibodies are given in Table S3, and ICC and by NIH Grants GM66815 (to K.L.K.) and NS3088 (to J.E.H.); March of Dimes neurite outgrowth analysis is described in SI Methods. Grant 1-FY13-413 (to K.L.K.); grants from the American Epilepsy Society, the McDonnell Center for Cellular and Molecular Neurobiology at Washington Electrophysiology and cIN Transplantation. d35 PSA–NCAM–purified, SYN– University (WU), and the WU Center for Regenerative Medicine (CRM); WU eGFP–expressing hcINs were used for transplantation and electrophysiology Institute of Clinical and Translational Sciences Grant UL1 TR000448; the WU Animal Surgery Core; and the WU Gene Technology Access Center as described in SI Methods. Transplantation experiments were performed by (supported by Siteman Cancer Center Grants CA91842 and UL1 TR000448). the Animal Surgery Core at Washington University School of Medicine using K.M. was supported by a Postdoctoral Fellowship from the WU CRM, and protocols approved by the Institutional Animal Care and Use Committee of E.M.A.L. was supported by a National Institute of General Medical Science T32 Washington University in St. Louis. Training Grant.

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