bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 1 of 46 USC 105 and is also made available for use under a CC0 license.

1 NMDAR-mediated transcriptional control of gene

2 expression in the specification of interneuron subtype

3 identity

a b c a a 4 Vivek Mahadevan , Apratim Mitra , Yajun Zhang , Areg Peltekian , Ramesh Chittajallu , b,1 d c a 5 Caraoline Esnault , Dragan Maric , Christopher Rhodes , Kenneth A. Pelkey , Ryan b c a,∗ 6 Dale , Timothy J. Petros , Chris J. McBain

a 7 Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health 8 and Human Development (NICHD), Bethesda, 20892, MD, USA b 9 Bioinformatics and Scientific Programming Core, NICHD, Bethesda, 20892, MD, USA c 10 Unit on Cellular and Molecular Neurodevelopment, NICHD, Bethesda, 20892, MD, USA d 11 Flow and Imaging Cytometry Core Facility, National Institute of Neurological Disorders and Stroke 12 (NINDS), Bethesda, 20852, MD, USA

13 Abstract Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+ and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecu- lar, anatomical and physiological properties. However, the molecular mechanisms regulat- ing their diversity remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates subtype-specific transcriptional regulation of gene expression in MGE-derived interneurons, leading to altered subtype identities. Notably, MGE-specific conditional Grin1 loss results in a systemic downregulation of diverse transcriptional, synaptogenic and membrane excitabil- ity regulatory programs. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders, particularly schizophrenia. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

14 Keywords: Interneurons, Medial ganglionic eminence, PV, SST, Neurogliaform, NMDA 15 receptor, Transcriptional regulation, Neurodevelopmental disorders, Schizophrenia, 16 NMDA-hypofunction, Hippocampus, Frontal Cortex, Mouse model, scRNAseq

Email addresses: [email protected] (Vivek Mahadevan), [email protected] (Ryan Dale), [email protected] (Timothy J. Petros), [email protected] (Chris J. McBain) ∗Corresponding author.

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 2 of 46 USC 105 and is also made available for use under a CC0 license.

17 Introduction

18 Medial ganglionic eminence (MGE)-derived forebrain GABAergic interneurons com- 19 prise the parvalbumin-containing (PV) and somatostatin-containing (SST) subpopulations 20 throughout the entire forebrain accounting for approximately 60% of all cortical interneu- 21 rons [1, 2]. In addition, approximately half of all hippocampal neurogliaform-type cells 22 (NGFCs), the so called Ivy cells, originate from the MGE [3, 4]. Interestingly, though only 23 rarely found in rodent neocortex such MGE-derived NGFCs are significantly more popu- 24 lous in primate neocortex, including humans [5]. While PV neurons exert robust somatic, 25 and proximal dendritic inhibition, the SST and NGFCs mediate domain-specific dendritic 26 inhibition on their downstream pyramidal neuron targets [6]. Collectively these classes of 27 interneurons shape diverse aspects of cortical and hippocampal circuit maturation during 28 development, and critically regulate information processing in mature circuits by maintain- 29 ing appropriate excitation-inhibition (E-I) balance [7]. Recent evidence indicates a critical 30 role for activity, particularly through ionotropic glutamate receptors (iGluRs), in driving 31 the morpho-physiological maturation of MGE-derived interneurons [8–12]. Unlike mature 32 interneurons where iGluRs differentially contribute towards synaptic transmission, imma- 33 ture and migrating interneurons express different glutamate receptor subunits including the 34 NMDA-type iGluR (NMDAR) and AMPA/Kainate-type iGluR (AMPAR/KAR) [13–15] 35 prior to the expression of any functional synapses. This becomes particularly important 36 as the developing brain contains higher ambient glutamate levels than the adult brain [16]. 37 Collectively, higher ambient glutamate, developmental expression of iGluRs and recruitment 38 of glutamatergic signaling is considered to be trophic [8, 17, 18] and thought to engage mech- 39 anisms to regulate various aspects of interneuron development including morphological and 40 electrical maturation to promote appropriate circuit integration [9, 11, 14, 16, 19–22]. 41 Interneuron-specific impairments are increasingly considered central to the etiology of

42 multiple neurodevelopmental and circuit disorders [23]. The importance of interneuron-

43 expressed iGluRs is most notable in psychiatric disorders exhibiting impaired NMDAR-

44 associated systems [24, 25]. In the adult brain, acute pharmacological NMDAR blockade re-

45 sults in circuit disinhibition and psychotic symptoms [26], mediated in-part, by the enhanced

46 sensitivity of interneuronal NMDARs to their antagonists [27]. Indeed, direct blockade of in-

47 terneuron activity also precipitates distinct behavioral deficits relevant to schizophrenia [28].

48 In particular, ablation of the obligate NMDAR subunit gene Grin1 in interneuron-specific

49 early postnatal mouse [29], but not PV-specific [30], or glutamatergic neuron-specific Grin1

50 ablation [31], resembles global Grin1 -mutants [32] in their constellation of schizophrenia-like

51 behavioral aberrations. This indicates that Grin1 dysfunction across multiple interneuron-

52 subtypes precipitates schizophrenia-like abnormalities [33]. In addition, this interneuron-

53 specific NMDAR-hypofunction model is sensitive to developmental age, since adult-onset

54 Grin1 loss does not result in the same phenotypes [29]. Despite the importance of develop-

55 mental NMDAR function in interneurons, and its relevance to human neurodevelopmental

56 disorders, a comprehensive interrogation of the impact of developmental NMDAR ablation

57 in interneurons, particularly across MGE-derived interneurons, is lacking.

58 It is clear that during the developmental window between embryonic day (ED) 13.5 and

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 3 of 46 USC 105 and is also made available for use under a CC0 license.

59 postnatal day (PD) ~10 [34], a combination of innate genetic programs, external environ- 60 ment, and neuronal activity shapes interneuron subtype specification leading to remark- 61 able diversity [2, 21, 35, 36] The NMDAR signaling complex comprises an essential node 62 for regulating gene expression via excitation-transcription (E-T) coupling in mature cir- 63 cuits [37–39]. Moreover, different NMDAR subunits are widely expressed in the developing 2+ 64 brain [40] where they provide a critical source of Ca -entry via trophic glutamate signaling 65 prior to synaptogenesis [15, 19, 41]. However, it is not clear whether the NMDAR-mediated 2+ 66 Ca cascades in nascent and developing MGE-derived interneurons engage transcriptional 67 programs necessary for MGE-derived interneuron diversity. To investigate this, we con- 68 ditionally deleted Grin1 in MGE progenitors that give rise to cortical and hippocampal 69 PV, SST, and NGFC subsets, using the Nkx2-1-Cre mouse line [3, 4, 42]. In this model, 70 Nkx2-1 -driven Cre expression is reported in cycling/proliferating MGE cells, well before the 71 cells become postmitotic, allowing for assessment of the developmental impact of embry- 72 onic loss of Grin1 activity across all subsets of MGE-derived interneurons. Applying high- 73 throughput single-cell RNA sequencing (scRNAseq), we establish that NMDAR-mediated 74 transcriptional cascades promote MGE subtype identity, by regulating the expression of di- 75 verse transcriptional, synaptogenic and membrane excitability genetic programs. Notably, 76 we identify numerous disease-relevant genes that are misexpressed in MGE-derived interneu- 77 rons upon Grin1 -ablation, providing a broad road map for examination of MGE-subtype 78 specific regulation via NMDAR signaling.

79 Results

80 scRNAseq recapitulates cardinal MGE subtypes and a continuum of 81 molecular profiles 82 To examine the molecular heterogeneity of MGE-derived GABAergic interneurons

83 by scRNAseq, we microdissected frontal cortex (CX) and hippocampus (HPC) from

84 fresh brain slices obtained from PD18-20 Nkx2.1 -Cre:Ai14 mouse (Figure1A, Figure1- + 85 Supplement1A). Ai14-TdTomato (TdT ) single-cell suspensions were harvested by

86 fluorescence-activated cell sorting (FACS) using stringent gating constraints including via-

87 bility and doublet discrimination (Figure1-Supplement1B) as previously described [43–

88 45], and subsequently processed through the 10X Genomics Chromium controller. 9064 and + 89 9964 TdT cells were recovered from cortex and hippocampus respectively across 3 biolog-

90 ical replicates. To minimize the effect of excitotoxicity and stress-related transcriptional

91 noise, the tissue processing, FACS, and sample collection steps were performed in buffers

92 supplemented with Tetrodotoxin (TTX), DL -2-Amino-5-phosphonopentanoic acid (APV)

93 and Actinomycin-D (Act-D) [46]. Because we observed concordant cell clustering across the

94 replicates during preliminary analysis by Seurat v3 [47, 48] (Figure1-Supplement2A),

95 the replicates were pooled for in-depth analysis. Subsequent clustering and marker gene + 96 analyses revealed that ~ 62% and 33% of the TdT MGE-sorts from cortex and hippocam-

97 pus respectively, express classical GABA markers including Gad1 / Gad2, Lhx6 ; and the

98 MGE-subclass markers Pvalb, Sst, and Lamp5, marking PV and SST, NGFC subsets respec-

99 tively (Figure1B, Figure1-Supplement3A). While we did not recover cells expressing

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity (which wasnotcertifiedbypeerreview)istheauthor/funder.ThisarticleaUSGovernmentwork.Itsubjecttocopyrightunder17 bioRxiv preprint esslg odcag F)btenec fteMEcria ls,rpeetn h o nihdmarkers enriched top the representing class, of enrichment cardinal change MGE cortex-specific the fold of a each between at (FC) change subtypes. fold MGE log2 the11 versus across genes marker cell-type-enriched subtypes. tative hippocampus the subtype. and each from in (MGE.CX) Oligodendrocyte). numbers enriched cortex OLIGO, cell from of Mural; subtypes recovery Gad1/Gad2 MUR, interneuron the MGE-derived Astrocyte; and 11 Glutamatergic; (MGE.HPC), ASTR, and of GLUT, Microglia; coded visualization Somatostatin; MG, color UMAP SST, were Plexus; Hydroxylase; clusters hip- Tyrosine Choroid Cell from CP, TH, populations. 9,964 Neurogliaform; MGE and NGFC, cardinal cortex valbumin; the frontal showing from brains), (9,064 annotated mouse transcriptomes 6 single-cell of 19,028 pocampus of reduction dimensional A, 1: Figure Nkx2.1 Lamp5 Prox1 Htr3a Pvalb Gad2 Gad1 Vip Lhx6 vriwo h xeietlworkflow. experimental the of Overview C B A Sst i Th CP FRONTAL CORTEX

SST.1 PVALB dnicto fMEdrvditrernsbye ntecre n hippocampus. and cortex the in subtypes interneuron MGE-derived of Identification othoc post doi: TH + SST.2 MGE.CX.HPC.Merged SST el eoee nec G utp rmtecre n ipcmu,adtedfiiggenes defining the and hippocampus, and cortex the from subtype MGE each in recovered cells

SST.3 MG https://doi.org/10.1101/2020.06.10.144295 SST.4 GLUT . n D <10e-25. FDR and ≥0.5

SST.5 HIPPOCAMPUS

ae ntertasrpinlpol dniis(eltp brvain:PAB Par- PVALB, abbreviations: type (Cell identities profile transcriptional their on based SST.6 ASTR NGFC

TH.1 MUR PVALB.1 C, PVALB.2 USC 105andisalsomadeavailableforuseunderaCC0license. OLIGO Pthlh ilnpo hwn h itiuino xrsinlvl fwl-nw represen- well-known of levels expression of distribution the showing plot Violin NGFC.1 NGFC.2 P20 P20 Nkx2.1 epesn VL. utp hti o bevdi h hippocampus. the in observed not is that subtype PVALB.2 -expressing (TTX, APV, APV, (TTX, Dissociate, D E B ♂ ii , ♀

UMAP2 - Cre UMAP1 Slice, Slice, ActD

Unc5b Fgf13 C1ql1 Pthlh Ai14 ) B E, MGE.CX WT.MGE.CX i , MPrpeetto fPABcutr ihihigthe highlighting clusters PVALB of representation UMAP nfr aiodApoiainadPoeto (UMAP) Projection and Approximation Manifold Uniform ; this versionpostedJune12,2020. PVALB.2 TH.1 WT.MGE.HPC PVALB.1 SST.1 Cell suspension Cell MGE.HPC SST.4 (TTX, APV, APV, (TTX, NGFC.1 Ai14 + Ai14 PVALB.2 SST.5 D, SST.3 ve ActD SST.2 PVALB.2 B NGFC.2 SST.6 ) o1 as icvr ae(FDR) Rate Discovery False −log10 iii Necab2 Crabp1 Pcp4 Cnr1 , Single al niaigtenme of number the indicating Table Bioinformatic analyses, analyses, Bioinformatic WT.MGE.CX 10X GENOMICS V2, V3 B PVALB.2 PVALB.1 NGFC.2 NGFC.1 subset ofPVALB.2 SST.6 SST.5 SST.4 SST.3 SST.2 SST.1 - Cortex iii TH.1 cell The copyrightholderforthispreprint Seurat v3 Seurat barcoding,RNAseq - 1121 enriched 191 141 825 416 300 939 908 377 317 111 CX HPC 542 498 102 249 216 128 399 505 248 92 23 Elfn1, Elfn1, Grm1, Th, Th, Elfn1, Grm1, Lamp5, Lamp5, Sema3c, WT.MGE.HPC Lamp5, Lamp5, Sema3c, Chodl Elfn1, Elfn1, Grm1, Crh Pthlh Definingmarkers Syt2, Tac1,Syt2, , Nr4a2, , Nr4a2, Rasgrp1, Bcl6 , Tacr1,Nos1, Zbtb20, Zbtb20, , , C1ql1, Fgf13 , C1ql1, Snhg11 Reln Reln Reln Nfix , Myh8, Reln , Myh8, Pld5 Reln Lpl , Nppc +, Penk - , B Ngf Ngf Nppc , ii - + , bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 5 of 46 USC 105 and is also made available for use under a CC0 license.

100 the CGE-markers Prox1, Htr3a or Vip, we recovered a minor fraction of cells corresponding + 101 to glutamatergic neurons, astrocytes and microglia. In addition, ~25% and 71% TdT MGE-

102 sorts from cortex and hippocampus respectively were enriched in oligodendrocytes marked

103 by Olig1 expression across all replicates (Figure1-Supplement 2B,2C). However, we fo-

104 cused our subsequent analyses on the 5656 and 3002 Gad1 / Gad2 positive cortical and

105 hippocampal MGE-derived interneurons.

106 Unbiased cell clustering by Seurat v3 identified six subtypes of SST, two subtypes of PV,

107 two subtypes of NGFCs, and one subtype of Tyrosine hydroxylse (TH) expressing interneu-

108 rons, expressing the markers Sst, Pvalb, Lamp5 and Th respectively, across the two brain

109 regions examined (Figure1C). Notably, all but two subtypes (SST.5 and SST.6) expressed

110 high levels of Lhx6, and 2 clusters corresponding to PV.2 and NGFC.1 expressed Nkx2.1

111 at this developmental time. While the PV- SST- and NGFC- clusters clearly exhibited ro-

112 bust gene expression differences among each other (Figure1D), the TH cluster appeared

113 to express genes that correspond to both PV: SST clusters, including Sst and Pvalb expres-

114 sions (Figure1C, Figure1-Supplement3B). Particularly, at this developmental window

115 we could not observe robustly different gene expression variances between the cortical and

116 hippocampal counterparts, barring a few marginal, but significant differences (Figure1-

117 Supplement4B) . This gave us sufficient rationale to perform subsequent analyses using

118 the MGE-derived interneurons pooled from cortex and hippocampus.

119 Among the SST sub clusters, SST.1-5 uniquely expresses Chodl, Igf2bp3, Cdh7,

120 Pld5 and Nfix respectively, while SST.6 expresses only markers that are common with

121 other SST clusters (Figure1-Supplement3B). With the exception of SST.6 the remain-

122 ing SST-expressing subclusters are described in previous scRNAseq assays (Figure1-

123 Supplement5A). For example, the Chodl-expressing SST.1 cluster co-express high Nos1,

124 Tacr1, Penk, and Npy, and it has been previously described as putative GABAergic long-

125 range projections neurons [49, 50]. Clusters SST.2/3/4 express Elfn1, Reln and Grm1

126 characteristic of putative cortical martinotti and their hippocampal counterpart, oriens-

127 lacunosum/moleculare (O-LM) [43, 51, 52](Figure1-Supplement5B). Lastly, Zbtb20 -

128 expressing SST.5 is predicted to be septal-projecting interneurons [43]. Among the PV sub

129 clusters, while both PVALB.1&2 coexpresses several common markers including Pvalb,

130 Kcnip2, Tcap and Kcnc1 there are several notable differences between the two clusters.

131 PVALB.1 appears to contain continuous, but non-overlapping populations expressing Syt2

132 representing putative fast-spiking basket cells or Rbp4/Sst containing putative bistrati-

133 fied cells [1, 43, 53](Figure1-Supplement5D) . PVALB.2 contains cells that uniquely

134 expresses Pthlh, C1ql1, Fgf13 and Unc5b representing putative axo-axonic chandelier

135 cells [43, 49, 54]. We also observed a TH cluster, which, in addition to expressing several

136 genes common to the SST: PV clusters, expresses several unique genes including Rasgrp1,

137 Bcl6, Myo1b that segregated into mutually exclusive cluster space expressing Crh or Nr4a2

138 (Figure1-Supplement3B, Figure1-Supplement5B) . This cluster is also described pre-

139 viously as putative bistratified-like cells [43, 53] . Among the NGFC sub clusters, while

140 both NGFC.1&2 coexpress several common markers including Lamp5, Hapln1, Cacna2d1,

141 Sema3c and Id2, the NGFC.1 cluster uniquely expresses several genes like Reln, Ngf, Egfr,

142 Gabra5 that are not expressed by NGFC.2. (Figure1-Supplement3B). While the Reln+

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 6 of 46 USC 105 and is also made available for use under a CC0 license.

143 represents MGE-derived neurogliaforms, the Reln-population may represent putative ivy

144 cells [43] (Figure1-Supplement5C).

145 While the majority of the UMAP space aligns well between the cortical and hippocam-

146 pal MGE-derived interneurons, we observed some regional differences as well. (Figure1- 147 Supplement3AiAiii). (i) First, we observed an increase in the HPC-expressed NGFC.1&2 148 in comparison to their cortical counterparts, consistent with preferential localization of

149 MGE-derived NGFCs to HPC over CX in rodents [1, 3, 4]. (ii) Next, the Pthlh-expressing

150 PVALB.2 subcluster splits into two islands, only in the cortex and distinctly lacking from

151 the hippocampus. Only one of the PVALB.2 islands expresses C1ql1, while the other cortex-

152 enriched island expresses unique markers Etv1, Cnr1, Pcp4, Crabp1, Necab2, Epha4, Crabp1

153 and Hapln1 (Figure1E, Figure1-Supplement5D) . Whether this represents a novel sub-

154 class of chandelier cells remains to be determined. (iii) Lasty, we also observed a distinc-

155 tion in the hippocampal SST.3 corresponding to a subset of O-LM interneurons (Figure1- 156 Supplement3Aii). The overall MGE cell numbers indicate that the SST cells account for 157 the majority of MGE cell population recovered in the scRNAseq assay from both brain re- 158 gions (Figure1-Supplement3Aii, Aiii). The PV and TH clusters accounted for a greater 159 share of MGE-derived interneurons in the CX than in the HPC. While it is plausible these

160 relative cell proportions may be skewed by differential survivability of these subtypes during

161 tissue dissociation, sorting and single-cell barcoding, these relative percentages were similar

162 across biological replicates. 163 NMDAR signaling maintains MGE identities and subtype diversity 164 Because neuronal activity and glutamatergic signaling are known to regulate multiple 165 facets of interneuronal development [2, 21] [11, 36, 55] , we hypothesized that the key ob-

166 ligate subunit Grin1 and the NMDAR signaling complex may play an instructive role in

167 determining MGE subtype identities. To test whether NMDAR signaling impact the devel-

168 opment and function of MGE-derived interneurons, we ablated them in MGE progenitors by

169 crossing floxed-Grin1 mice with the Nkx2.1 -Cre mouse line [42]. The Earliest expressions of

170 Nkx2.1 and Grin1 in the developing rodent brains occur around ~embryonic day (ED) 10.5 2+ 171 and ~ED14 respectively [56–58]. Moreover, NMDAR-mediated Ca signaling in migrating

172 interneurons are reported by ~ED16 [15]. Because the expression and activity of Nkx2.1 pre-

173 cedes Grin1 expression, we rationalized that utilizing Nkx2.1 -Cre mouse will ablate Grin1

174 and NMDAR signaling in MGE progenitors from the earliest developmental point. We + fl/fl 175 sorted TdT cells from the cortex and hippocampus of Nkx2.1 -Cre:Grin1 :Ai14 mice and

176 performed scRNAseq using the 10X platform. The scRNAseq experiments were performed

177 using juvenile mice (PD18-20) of both sexes and from the same litters as the wildtypes (WT) fl/fl 178 to enable subsequent direct comparison. Similar to the WT-datasets, the MGE-Grin1 + 179 mutants also revealed an enrichment of TdT oligodendrocytes (Figure2-Supplement3B),

180 however, we again focused our attention on the Gad1/2 positive interneurons. wt fl/fl 181 We next performed integrated analyses of the MGE-Grin1 and MGE-Grin1 cortical

182 and hippocampal scRNAseq datasets. Applying similar unbiased clustering parameters used wt 183 for the MGE-Grin1 analyses, we observed a total of twelve Gad1/2 positive clusters in the

184 integrated dataset (Figure2A, B). As a robust control, Grin1 appeared to be absent or fl/fl 185 vastly reduced in all MGE subsets in both brain regions from MGE-Grin1 (Figure2C),

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 7 of 46 USC 105 and is also made available for use under a CC0 license.

A B i C Grin1fl/fl wt/wt SST.5 Grin1 PVALB.1 PVALB.2 PVALB.3 NGFC.1 NGFC.2 SST.6 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 TH.1 TH.1 SST.1 Gad1

PVALB.1 Gad2

SST.2 Grin1 PVALB.2 Lhx6

PVALB.3 SST PV NGFC Nkx2-1 CX Sst SST.4 SST.3 NGFC.1 Pvalb NGFC.2 Lamp5 Grin1

UMAP2 Prox1 UMAP1 SST PV NGFC Htr3a HPC SST.all VipPVALB.all SST.all D E �2 = 11.6 �2 = 4.07NGFC.all E SST.all i ii �2 = 286 �2 = 209 p = 0.003 p = 0.13, ns CX.Integrated p < 0.0001 p < 0.0001 100 100 SST.all 100 7.6 1.7 2.0 SST.1 PVALB.all 20.1 16.7 20.9 SST.2SST.1 24.4 51.7 50.6 NGFC.all SST.3SST.2 72.2 71.0 18.6 25.5 SST.3 16.4 SST.4 50 50 34.3 100 10.7 SST.5SST.4 scRNAseq 13.5 15.5 50 9.1 18.8 9.0 5.6 SST.5 % all MGE SST.6 scRNAseq

from from SST.6 3.9 12.2 TH.1 22.7 21.5 % all SST 34.8 33.9 2.3 32.3 15.1 5.1 TH.1 from from 20.9 0 5.1 7.5 8.4 0 17.2 fl/fl 50WT Null WT Null 8.5 7.0 7.5 CX.Grin1 0 CX.Grin1wt/wt CX HPC WT Null WT Null CX HPC

�2 = NGFC.all13 �2 = 232 NGFC.1 HPC.Integrated 0 �2 = 236 �2 = 8.43 PV.allPV.all p = 0.0003 p < 0.0001 Eiii p < 0.0001 p = 0.014 Eiv NGFC.2 100 100100 NGFC.all NGFC.1 PVALB.1PVALB.1 NGFC.2 PVALB.2PVALB.2 45.6 50.3 PVALB.3PVALB.3 100 66.5 65.2 85.0 81.6 73.3 5050 87.9 50

scRNAseq 27.3 scRNAseq 31.5 54.4 % all PVALB % all NGFCs 50 from from from 34.8 22.4 9.1 26.4 15.0 18.4 fl/fl HPC.Grin1 00 0 HPC.Grin1wt/wt 3.0 0 2.1 WT Null WT Null WT Null WT Null CX HPC CX HPC 0

Figure 2: Altered interneuron subtype proportions upon Grin1 -ablation A, Integrated UMAP visualization of 12 subtypes of MGE-derived interneurons obtained from cortex (CX) and hippocampus (HPC) of Grin1 wt/wt and Grin1 fl/flmice B, Violin plot showing the distribution of expression levels of well-known representative cell-type-enriched marker genes across the 12 interneuron subtypes. C, Violin plot from both genotypes indicating the expression of Grin1 in the cardinal of MGE- derived interneuron subtypes. D, UMAP representation colored by brain-region, highlighting the differential enrichments of cells (brown arrows) within interneuron subsets in Grin1 -WT and Grin1 -null from CX and HPC. E, Stacked-barplots representing the proportions of recovered cell numbers within Ei, pooled cardinal MGE subtypes, Eii, SST subtypes; Eiii, PVALB subtypes and Eiv, NGFC subtypes in Grin1 -WT and Grin1 -null from cortex or hippocampus. χ2, Chi-square test of proportions; ns, not significant.

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 8 of 46 USC 105 and is also made available for use under a CC0 license.

186 but not in the Slc17a7 expressing glutamatergic neurons (Figure2-Supplement3A). Over-

187 laying the WT and NULL datasets from the brain regions revealed differential enrichments

188 among the recovered cells between the genotypes (Figure2D). Intriguingly, Grin1 -ablation

189 did not seem to alter the SST or PV recovery percentages, with the exception of a mod- 2 190 est increase in the cortical NGFCs (χ = 11.6, p = 0.003), but not hippocampal NGFCs 2 191 (χ = 4.07, p = 0.13) (Figure2Ei, Figure2-Supplement2Ai,B). However, we observed a 192 marked change in the recovery percentages of the subsets of SST, PV and NGFCs from both 193 cortex and hippocampus (Figure2Eii−iv, Supplement2Aii,C). Particularly, we observed 194 a robust increase in the Chodl-expressing cortical SST.1 population, and a decrease in hip- fl/fl 2 195 pocampal SST.6 population in MGE-Grin1 (CX, HPC: χ = 286, 209; p-value = 2.2e-16

196 for both regions). In addition, we found a reduction in the cortical PVALB.1 population, fl/fl 2 197 and a compensatory increase in PVALB.2/3 populations in MGE-Grin1 (CX, HPC: χ

198 = 236, 8.4; p-value = 2.2e-16, 0.14). Finally, we observed an increase in the NGFC.1 along 2 199 with a compensatory decrease in NGFC.2 in both cortex and hippocampus (CX, HPC: χ

200 = 13, 232; p-value = 0.0003, 0.14).

201 To independently examine whether Grin1 ablation promotes changes in interneuron + 202 abundances, we conducted immunostaining experiments to probe total TdT MGE-derived

203 interneurons and the PV / SST subtypes. First, we observed no significant change in total + 204 TdT hippocampal MGE-derived interneuron density between postnatal day (PD) 30-210 205 (Figure2-Supplement1Ai) similar to what was indicated in the scRNAseq cell recoveries + 206 (Figure2Ei). Next, we observed a modest decrease in cortical TdT numbers at PD30, 207 which became progressively greater by PD210 (Figure2-Supplement1Bi), indicating dif- 208 ferent effects of Grin1 -ablation on total MGE cell numbers in cortex and hippocampus.

209 We observed no change in hippocampal expressed total PV/SST cell type counts at PD30 210 (Figure2-Supplement1Aii), but we noted a modest reduction in cortical PV cell type 211 counts along with an increase in cortical SST cell type counts at the same age Figure2- 212 Supplement1Bii). 213 Among the differentially enriched subclusters, Pthlh-expressing PVALB.3 is quite no-

214 table (Figure2B, Figure2-Supplement3A,B). This cortex-enriched cluster lacking in

215 the hippocampus was identified within the PVALB.2 putative-chandelier cells in the MGE- wt/wt 216 Grin1 (Figure1E, Figure1-Supplement5D), However, subsequent to integration of fl/fl 217 the MGE-Grin1 scRNAseq dataset, it segregated as a unique cluster, far from other

218 PVALB clusters in the UMAP space. We observed Prox1 expression in PVALB.3, which

219 is uncharacteristic of MGE-derived interneurons, additional to robust expressions of genes

220 associated with NGFCs such as Hapln1 and Reln (Figure2-Supplement3A,B). More-

221 over, we observed an increase in recovery of the cortical PVALB.3 cell numbers, including

222 the emergence of these cells in the hippocampus subsequent to Grin1 -ablation (Figure2-

223 Supplement2A,B). It is unclear whether the changes in marker expression reflect a true

224 change in cell identity or whether this is reflective of alterations in relative interneuron

225 subtype proportions. Nevertheless, these data demonstrate clear changes in MGE-derived

226 interneuron subtype diversity following loss early embryonic loss of Grin1 function. 227 NMDAR signaling shapes the transcriptional landscape in MGE-derived 228 interneurons

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 9 of 46 USC 105 and is also made available for use under a CC0 license.

Log2 normalized differential fl/fl A expression in MGE-Grin1 ; FDR<0.01 Bcl6, Rgs12, Epha7, Npas3, Grin1, Neto1, Epha5, Sema5a, Cdh9, S100a10 # of Gabra4 Olfm1, Cdh2, Scn2b Gabrd Grm1 Penk1 Fgf13, Elmo1 Avp DEGs 354 SST CX 359 Gpm6b Hcrtr2 PVALB 336 Apoe Nog Hapln1 NGFC Nkain3 359 SST HPC 423 Wnt5a PVALB 280 Kcns3 NGFC Erbb4

Common DEGs Unique DEGs

Bi Bii C HPC CX

Synaptogenesis signaling # of # of Regulon NES Endocannabinoid signaling targets motifs CREB signaling Rest 4.7 97 10 Synaptic LTP signaling Tia1 4.6 242 8 GABA receptor signaling Opioid signaling Nfix 4.0 205 5 NFAT signaling

SST, Irf1 3.7 78 1 Huntingtin signaling

CX & HPC & CX Clk1 3.6 234 6 Glutamate receptor signaling 2+ Parp1 4.8 249 4 Intracellular Ca signaling cAMP signaling Tia1 4.7 217 5 CRH signaling Rest 4.2 97 10 Synaptic LTD signaling Trove2 3.8 262 7 14-3-3 signaling PVALB, G signaling

CX & HPC & CX �� Odc1 3.4 350 4 Purinergic receptor signaling Tia1 4.6 220 4 nNOS signaling Rest 4.6 67 10 CDK5 signaling Trove2 4.0 249 5 Melatonin signaling IL-1 signaling

NGFC, Pax4 3.2 113 4 CXCR4 signaling CX & HPC & CX Pou3f3 3.2 113 1 Gap junction signaling CCK signaling G� signaling Predicted master regulators upstream of the 603 DEGs PKA signaling based on common transcriptional motifs -Log.FDR 603 target DEGs co-regulated (among all 802) 0 7.5 15 Regulons Regulons Regulons Regulons common to In PVALB In SST In NGFC SST / PVALB / NGFC

Figure 3: Cell-autonomous transcriptional changes subsequent to MGE-specific developmental Grin1 -ablation A, Combined heatmap representing the 802 differentially expressed (DEGs) in the cor- tical and hippocampal MGE-derived interneurons upon Grin1 -ablation, at a FDR<0.01 and FC>10%, as determined by MAST analysis (see details in Methods). Bi, iRegulon in silico analysis identifying high- confidence master upstream transcriptional regulators(indicated in red) of the DEGs (indicated in lavender). Representative DNA-binding motifs are indicated next to the transcriptional regulators. Bii, Top five tran- scriptional regulators predicted by iRegulon, associated normalized enrichment score (NES) and number of predicted targets and motifs associated with each transcription factor cluster, indicated for the three in- terneuron subtypes (CX and HPC pooled). C, Ingenuity Pathway Analysis of significantly overrepresented molecular pathways in each MGE-subtype.

229 It is now well-established that transcriptional signatures defines the subtype identities

230 of GABAergic interneurons [59]. To examine the full range of transcriptional impairments

231 triggered by Grin1 ablation in MGE-derived interneurons, we next performed differential

232 gene expression testing by pooling the SST / PVALB / NGFC subtypes into their cardinal

233 MGE classes to identify the genes that are differentially expressed between the genotypes.

234 For instance SST1-7 and TH.1 are pooled together as SST; PVALB1-3 are pooled together

235 as PVALB, and NGFC1-2 are pooled together as NGFC cardinal classes for this assay . At

236 a stringent false-discovery rate (FDR) <0.01, 802 genes passed the 10%-foldchange (FC)

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 10 of 46 USC 105 and is also made available for use under a CC0 license.

237 threshold across the MGE subtypes from both brain regions (Figure3A, Supplemental

238 Table1). Several interesting features were observed in the differentially expressed gene

239 (DEG) pattern upon MGE-specific Grin1 -ablation. (i) Among all DEGs only ~10% and 1%

240 are upregulated in the cortex and hippocampus respectively, while the remaining genes were 241 all downregulated (Figure3-Supplement1Aii,B,C). (ii) While Grin1 ablation resulted in 242 several unique DEGs between the MGE classes, ~10 and 27% of the DEGs are common

243 within cortex and hippocampus respectively (Figure3A, Figure3-Supplement2). For

244 instance, while S100a10, Hapln1, Hcrt2 are uniquely upregulated in cortical SST, PV and

245 NGFC respectively (Figure3A), Apoe, Kcns3, Wnt5a were uniquely altered in hippocampal

246 SST, PV and NGFC respectively. In contrast, Grin1 ablation induced common changes in

247 Penk1 and Erbb4 expression patterns across all MGE-derived interneuron classes in the

248 cortex and hippocampus respectively. (iii) ~27-43% of all DEGs were shared by MGE 249 classes across brain regions (Figure3-Supplement2Aii ). For example, Npas3, Cdh9, 250 Grm1 are commonly downregulated in all SST subclasses; Bcl6, Epha7, Gabra4 common

251 to PV class; and Rgs12, Gabrad, Sema5a common to NGFCs from both brain regions. (iv)

252 Lastly, 28 genes are commonly differentially expressed across both brain regions, across all

253 MGE subtypes. For example, Grin1, Neto1, Cdh2, Scn2b are commonly downregulated

254 across the board, while Epha5, Olfm1 are commonly downregulated across all, but cortical

255 PV cells (Figure3A).

256 Gene expression co-regulation is intrinsic to cellular diversity [60, 61]. Since the ma-

257 jority of DEGs are downregulated across the MGE subtypes, we examined whether they

258 correspond to clusters of coordinated co-regulation. We applied the iRegulon in silico frame-

259 work [62], which identifies transcription factor binding motifs that are enriched in genomic

260 regions of the DEGs upon Grin1 -ablation, and predicts the transcription factors that bind

261 to the motifs. This in silico analysis predicted 51 significantly enriched motifs (normalized

262 enrichment score > 3) that clustered into 10 groups by similarity, 33 of which were asso-

263 ciated with transcription factors (Figure3B, Supplemental Table2). Put together, 10

264 transcription factors were predicted to bind with the motifs with high confidence, strongly

265 supporting targeted co-regulation of 617 among the 802 DEG genes upon Grin1 -ablation.

266 Notably, the RE1-silencing transcription factor (Rest) is a master transcriptional repressor

267 that mediates the transcriptional accessibility for several synaptic genes [63], including NM-

268 DAR subunits themselves [64]. It is intriguing to observe that the downregulation of the

269 DEGs upon MGE-specific Grin1 -ablation are, in part, predicted to occur via Rest-mediated

270 transcriptional repression.

271 To examine the broad biological impact of the DEGs, we performed Gene Ontology

272 (GO) analyses. Broad GO analyses on all DEGs indicates that these genes serve to regu-

273 late multiple molecular functions in interneurons, including regulation of GABAergic and

274 glutamatergic synapses, additional to biological pathways related to addiction and circadian

275 entrainment (Figure3-Supplement2B). Further classification of DEGs based on their cel-

276 lular functions within the MGE subtypes revealed genes critical for regulation of membrane

277 excitability, gene expression, synaptic partnering and assembly, as well as major intracellu- 2+ 278 lar Ca signaling cascades and second messengers (Figure3C, Figure3-Supplement2C,

279 Supplemental Table3) .

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

A CX HPC B Legends: Log2 normalized differential fl/fl CX HPC expression in MGE-Grin1 ; FDR<0.01

SST PVALB NGFC expressed TFs, TFs, expressed

- C CX HPC TFs Interneuron Broadlyexpressed TFs, TRs dependant Regulatorsof subtype differentiation, function - Subset of activity of Subset

Figure 4: Grin1 -signaling in MGE-derived interneurons are highly dedicated to the transcrip- tional control of interneuron identity Heatmap of log2 FC of significant DEGs in cortical and hip- pocampal MGE cardinal subtypes, showing a subset of Ai, Transcription factors (TFs) that are previously established to regulate MGE subtype identity and function; Bi, broadly expressed TFs and transcriptional regulators (TRs) that are not currently known to regulate MGE function; Ci, neuronal activity-regulated TFs. Clusters of commonly differentially expressed genes in cortex and hippocampus are indicated in yellow or green boxes.

280 Transcription factor expression is a key component of NMDAR-mediated

281 MGE regulation

282 Because transcriptional regulation underlies numerous fundamental processes including

283 the expression of other classes of genes, we next examined the DE-transcriptional regulators

284 in detail. We first examined the 67 genes that are differentially expressed upon Grin1 -

285 ablation and are known to mediate transcriptional regulation of gene expression. Of these,

286 35 genes are previously established to be expressed in different GABAergic interneuron 287 classes including some notable MGE-expressed transcription factors (Figure4Ai). The re- 288 maining 32 are broadly expressed TFs (Figure4Bi), that include a small subset of 15 genes 289 that are regulated by neuronal activity (Figure4Ci ). Barring a few genes, we observed 2+ 290 the majority of TFs to be down regulated in both brain regions. Intracellular Ca sig- bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 12 of 46 USC 105 and is also made available for use under a CC0 license.

291 naling cascades and second messenger systems are key mediators of NMDAR signaling to

292 the nucleus for transcriptional regulation. Theoretically, an early first wave impairment of 2+ 293 Ca signaling in Grin1 -lacking MGE progenitors could result in transcriptional silencing 2+ 294 of the mediators of Ca signaling cascades and second messenger systems, which would

295 sustain the transcriptional impairments. Indeed, we also observed a downregulation of var- 2+ 296 ious Ca homeostasis-regulators, kinases / phosphatases and second messengers that are

297 activated downstream of Grin1 (Figure4-Supplement1A,B,C). Furthermore, we noted

298 that hippocampal MGE neurons had a greater proportion of DE-TFs and kinase signaling

299 cascade effectors that were downregulated across all 3 subtypes compared to their corti-

300 cal counterparts. Together, this suggests that hippocampal MGE-derived interneurons may 2+ 301 be more vulnerable than cortical MGE-derived interneurons towards Grin1 -mediated Ca

302 transcriptional silencing at this age.

303 Interestingly, among the early TF cascades in the progenitors that sequentially deter-

304 mine and maintain MGE fate, several members appear to be expressed at ~P20, and starkly

305 downregulated upon Grin1 -ablation. For instance, Lhx6, Maf, Arx, Myt1l, Dlx1 are among

306 the genes broadly downregulated across all hippocampal MGE subtypes and within spe- 307 cific class(es) in their cortical parallels (Figure4Aii). Other MGE fate-determining TFs, 308 Nkx2-1, Mafb, Satb1, Nr2f1 (CoupTf1), Sp9, also appear to be downregulated in discrete

309 populations. This also includes a downregulation of Bcl11b (Ctip2) in both hippocampal

310 and cortical NGFCs, a gene recently linked to regulation of NGFC morphology and func-

311 tion [65]. Among the few transcriptional regulators upregulated are Sirt2, Elmo1, Zcchc12, 312 none of which have been characterized in the context of MGE function (Figure4Bii ). Sirt2 313 is an established transcriptional repressor [66, 67] that may regulate the repression of sev-

314 eral target genes in an MGE-specific manner, and Elmo1 has been previously characterized

315 during the activity-dependent migration of CGE subtypes [21]. Finally, a recent study has

316 predicted that the expression of Zcchc12 correlates with slower intrinsic firing among hip-

317 pocampal CA1 interneurons [43]. This suggests that increased Zcchc12 expression might

318 regulate the expression of synaptic genes enabling reduced intrinsic excitability in the MGE

319 subsets. Related to such putative decreased excitability in the MGE-derived interneurons,

320 among the activity-regulated TFs, we observe broad downregulation of Jun, Egr1, Fos, Fosb, 321 Arc, Satb1, Arnt2 across all classes of MGE in both brain regions (Figure4Cii ). While 322 most of these are well-established activity-regulated TFs, Arnt2 has been recently described 2+ 323 to partner with Npas4, downstream of Ca signaling in response to neuronal activity [68].

324 Unsurprisingly, the Npas-family members Npas1/3/4 are also downregulated in discrete

325 MGE subtypes. 326 Impaired NMDAR signaling alters region-specific MGE subtype marker ex-

327 pression

328 Several GABAergic/MGE markers were mis-regulated upon Grin1 -ablation (Fig-

329 ure5A). For example, genes S100a0, Pthlh, Hcrtr2 that are normally expressed in

330 SST, PV and NGFCs respectively, are upregulated in the same clusters of MGE- fl/fl 331 Grin1 (Figure5Bi), indicating a misexpression in a subtype-specific manner. Next, 332 while certain genes such as Reln, Tenm1 are broadly downregulated across MGE classes,

333 some genes like Thsd7a show an upregulation in certain classes but a down regulation in

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 13 of 46 USC 105 and is also made available for use under a CC0 license.

B CX HPC iv A Bi Bii Biii CX HPC CX HPC CX HPC CX HPC Putative misexpression within subtypes within misexpression Putative Putative misexpression unique to subtypes to unique misexpression Putative Putative misexpression of key interneuron markers interneuron key of misexpression Putative Putative misexpression commonly across subtypes across commonly misexpression Putative

Legends: fl/fl SST HPC.Grin1 Log2 norm. DEG-FC HPC.Grin1wt/wt in MGE-Grin1fl/fl; FDR<0.01 PVALB CX.Grin1fl/fl wt/wt NGFC CX.Grin1

Figure 5: Fig. 5 | Differential expression of interneuron marker genes across subtypes upon Grin1 -ablation A, Heatmap of log2 FC of significant DEGs in cortical and hippocampal MGE cardinal subtypes, showing a subset notable MGE marker genes. Representative box-violin plots of top differentially fl/fl expressed genes from the above that represent Bi, a misexpression unique to a single MGE-Grin1 fl/fl subtype; Bii, a misexpression across all MGE-Grin1 subtypes; Biii, a misexpression between MGE- fl/fl Grin1 subtypes. Biv, Representative box-violin plots of fundamental interneuron markers.

334 the other classes (Figure5Bii). Interestingly, a few genes that are normally abundant in 335 one MGE class, appear to be misexpressed in another MGE class where they are not abun-

336 dant. For instance, Tcap, that is normally expressed in PV cells, in addition to being

337 decreased in PV cells, is upregulated in NGFCs in both cortex and hippocampus. Simi-

338 larly, Hapln1 expression which is typically limited to NGFCs, is upregulated in PV subsets 339 (Figure5Biii ). Lastly, we observed an upregulation in the Gad1 and Slc32a1 (vesicular + ¯ 340 GABA transporter, vGAT) and a downregulation in Gad2 and Slc6a1 (Na -Cl dependent

341 GABA transporter, GAT1), corresponding with GABA synthesis and reuptake machineries 342 respectively (Figure5Biv ). Taken together, these data indicate that Grin1 -ablation alters 343 region-specific MGE subtype numbers, and subtype marker expression indicative of altered

344 subtype identities. 345 NMDAR signaling regulates MGE subtype-specific expression of neurodevel- 346 opmental disorder risk genes 347 Interneuron-centric disease etiology is an emerging centrality in multiple psychiatric dis-

348 orders [23]. Thus, we questioned whether the Grin1 ablation induced DEGs presently iden-

349 tified correlate with disease etiology. Disease-ontology based Ingenuity Pathway Analysis of

350 the DEGs showed significant over-representation of genes implicated in ‘Schizophrenia’, ‘Psy-

351 chiatric disorders’ and ‘Movement disorders’, among other cellular impairments involving

352 aberrant morphology of neurons (Figure6A, Supplemental Table4). To independently

353 examine the DEGs for potential enrichment for neurodevelopmental disorders, we obtained

354 the risk genes for schizophrenia (Sz) and autism spectrum (As) from the SZDB [69] and

355 SFARI [70] databases respectively. These databases curate and rank disease-relevant gene

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 14 of 46 USC 105 and is also made available for use under a CC0 license.

MPPED1 MPP6 MOG MT1 MOBP MTFP1 MME

MTURN MMD2

MUSTN1 MGST3

MXRA7 MGP

MYH8 MGAT4C

MYL12A MEGF11

MYO1B MDH1

NAP1L5 MARCKS

NAPB MAPK10

NCALD MAN1A1

NCDN MAL2

NCS1 MAL

NDN MAG

NDNF LYPD6B

NDST4 LYPD6

NEB LY6H

NECAB1 LRRTM1

NECAB2 LOR

NEFH LMO3

NEFL LIN7B

NEGR1 LHX6

NEK7 LGI1

NFIX LDHB

NKX2-1 LDHA

NNAT LAYN

NOG KRT12

NOS1 KLHL13

NPAS1 KIT

NPAS3 KIF5C

NPNT KIF5A

NR2F1 KIAA1549L

NRCAM KIAA1217

NRIP3 KCTD6

NRSN2 KCTD12

NRTN KCNMB2

NSF KCNMA1

NSG1 KCNK3

NSG2 KCNK1

NT5DC3 KCNJ4

NTNG1 KCNJ3

-Log.FDR NTRK1 KCNIP4

NUDT4 KCNIP2

NXPH1 KCNIP1

NYAP2 KCNH7

OAZ2 KCNF1

OCIAD2 KCNC3

HPC CX OGFRL1 KCNC2

OPN3 KCNC1

OSTN KCNB2

OXCT1 KCNAB3

PACSIN2 KCNAB1

PAM JUNB

B PCDH11X JUN A i PCDH15 INA

PCDH17 ILDR2

PCDH7 IL1RAPL2

PCDH8 IGFBP5

PCP4 IGFBP4

PCP4L1 IGFBP2

PDCD4 IGF1

PDE11A IFI27

12.5 25 PDE1A IDI1

PDE3A ID2

0 PDGFA HTRA1

PEG10 HTR2C

PEG3 HSPA1B

PELI1 HSPA1A

PENK HSPA12A DEGs exclusive to top-ranked Schizophrenia (SZDB) gene sets (164)

PGBD5 HS6ST2

PGM2L1 HS3ST4

PHLDA1 HS3ST2

Schizophrenia PITPNC1 HRK

PKIB HPRT1

PLCB4 HPCAL1

PLCXD2 HOPX

PLCXD3 HMGCR

PLD5 HLF

PLEKHA1 HLA-A

PLIN3 HCRTR2

Psychiatric disorders PNCK HAPLN4

PODXL2 HAP1

PPFIBP1 GSTO1

PPM1E GSN DEGs exclusive to top-ranked Autism (SFARI) gene sets (25)

PPM1L GRM5

PPP1R14A GRM1

PPP1R14B GRB14

PPP1R14C GPX3 GRIA3 CPLX2 OXR1 PLEKHO1 SEZ6 GLUL CA10 KCNK2 EFNA5 SCG2 RAP2A SYNM Development of neurons PPP1R1A GPRASP2

PPP2R2B GPR83

PRKACB GPR22

PRKAR1B GPR158

PRKCE GPM6B

PRKCG GOT1

PRLR HMGCS1 GAD1 LMO4 DCLK1 PACSIN1 ADARB2 NFIB PGRMC1 GABRA4 TENM1 GRIK3 MEF2C GNG3

PRUNE2 GNG2

Neurotransmission PTHLH GNG13

PTPRE GNB4 DEGs common with top-ranked SZDB and SFARI gene sets (21)

PTPRT GNAL

PTPRU GM1673

PVALB GLTP PSD GABRG2 RAB27B GRIN3A HAPLN1 GABBR2 UGT8 NTRK3 RAB6B SERPINI1 NINJ1 KCND2

PYGM GFRA2

QDPR MYO5A LAMB1 GDA

Disorders of basal ganglia RAB3B GAP43 MYT1L KCNS3

RAMP3 GALNT18

RAPGEF4 GALNT14 NRXN3 KCNQ3

RASD2 GAL NRGN SOCS2 OLFM1 EGR1 TIMP2 ADCY1 PPM1H CDH2 SCRT1 RGS4 MAFB ALCAM

RASGEF1B GADD45G

RASGRF1 GADD45B

RASGRF2 PLXNA4 GABRG3 GAD2 Movement disorders RASGRP1 GABRG1 DEGs not mapped with top-SZDB and SFARI gene sets (592)

RASGRP2 GABRA2

RBFOX3 FXYD7 OSBPL3 PARM1 CHGA NRP1 TAC3 EFNB3 SYNGR3 CADM1 NFASC PTPRZ1 GABRA1 LPL

RBMS3 FSTL5 ROBO2 DPP10

RBP4 FSTL1

RCAN2 FRMD4A

Neuromuscular disease RCN1 FOSB

RELL1 FNDC5

RELL2 SATB1 DLGAP1 FLYWCH2 PCSK2 YWHAG UTRN TOX3 SNAP91 B3GAT1 NETO1 NETO2 GRIA4 NDRG4 SOX4 NREP

REPS2 FKBP3

RGCC FJX1 SZDB Both SFARI

RGS10 FGF9

RGS16 FGF13

Abnormal morphology of nervous system RGS17 SCN1A CTNND2 FGF12

RGS2 THY1 CAMK4 LGALS1 BCL6 CALB2 NPTN OPCML ITPR1 CPLX1 HPCAL4 NTM HPCA FBLL1

RGS6 FAM81A

RGS8 FAM49A B

RND2 FAM3C SCN8A CNTNAP4 ii

RNF122 FABP7 Neuritogenesis RNF13 FABP5 RNF152 PPARGC1A TENM3 ENHO RORA ATP1A3 AHI1 SLC1A2 MAP4K4 CIT FOS SPOCK2 CYP51A1 FABP3

RPH3A ETV1

RPL23A SEMA5A CNTN5 ERMN

RPP25 EPS8

RPS4L EPHX4

RSPO3 EPHA7 GABRD SLC25A1 ENC1 RAMP1 BTG2 KCNC4 SLIT2 SLC32A1 YWHAH YWHAQ STK32C SYP SLC12A5 CACNA2D3 Organization of cytoskeleton RTN1 EPHA5

RTN4RL1 EPHA4

RUNX1T1 ENPP2 SLC7A3 BCL11A

RXFP1 ENO2

S100A10 SPARCL1 ATP2B2 ELMOD1 ARX

S100A6 ELMO1 RGS12 TSPAN13 FYN OLFM3 RGS7BP KCNA1 PRDX6 DPYSL3 SRRM4 FLRT2 SYT1 GPM6A

SCG5 ELFN1

Microtubule dynamics SCN2B ELAVL4

SECISBP2L ELAVL2

SEMA3E EIF4A2

SEMA6A EGFR

SEPTIN4 QKI RAB3C IGSF8 EEF1A2 ERBB4 MBP SPP1 SNAP25 NPY CLU SLC4A10 CBLN2 EFHD2

SERP2 DZANK1 Quantity of neurons SERPINE2 DUSP1 SERTM1 DOCK10

SH3BGRL DNM1

SH3BP1 DNER

SH3GL3 CSPG5 CALB1 CAMK2A MAF RRAS2 APOE PTN KCTD8 FUT9 MPPED2 SLC38A1 SCN1B DLX6

SH3GLB1 DLX1

SHISA4 DISP2

Familial encephalopathy SHISA6 DGKG

SHISA9 DGKB

SIRT2 DDAH1 NR2F2 RIT2 PRKCA NRSN1 PLXNA2 TMEM108 IMPACT MT3 CPNE5 UCHL1 NFIA NPAS4

SLC24A2 DBI

SLC2A13 CYGB

SLC30A3 CX3CL1

SLC4A4 CTSZ

Synaptic transmission SLC5A7 CTSV

SLC7A14 CTSD KCND3 NELL2 NDRG2 FRMPD4 RAP1A

SMAD7 CST3

SMC2 CRYAB

SNAP47 CRTAC1

SNCA CRISPLD1

SNCB CRHBP

Dyskinesia SNHG11 CRH SNHG4 Grin2b CREG2 SNX10 CRABP1

SNX18 CPOX

SOX11 CPNE7

SPATS2L CPLX3

SPHKAP CPE

SPOCK1 Grin2a COX6A2

Potentiation of synapse SPOCK3 COX5A

SPON1 CORT

SST CORO6

SSTR2 COL25A1

ST3GAL6 NR4A2 COL19A1

ST6GALNAC5 PCDH10 GRIN2B CNTNAP5

ST8SIA4 Grin1 CNTNAP1 Huntington Disease STAC2 PCDH19 GRIN2A CNP

STMN2 CNNM1

STMN3 CLSTN3

STX1B PLCB1 GRIN1 CLIC4

STXBP6 CLDN11

SYN1 CITED2

SYN2 CHRM2

SYNPR CHODL

SYT10 PRKCB GRIA1 CHN2

Long term potentiation SYT2 CHN1

SYT4 CHL1

SYT7 CHGB

TAC1 CHCHD10

TACR1 RBFOX1 CNTNAP2 CENPA

TAGLN3 CEND1 SZDB Both SFARI

TALDO1 CEMIP

TBC1D9 CELF6

Abnormal morphology of the brain TCAP CDKN1A

TCEAL5 CDH9

TCEAL6 RELN CNTN4 CDH7

TENM2 CDH4

TESC CDH12

TF CD9

THRSP CD63

THSD7A CCNE1

TIAM1 RIMS1 CNR1 CCND2

TIMP3 CCK

Cell-cell contact TMEFF1 CCDC136 TMEFF2 CARTPT Avg. number of neighbors 4.119 3.754 2.14

TMEM132B CAMKV

TMEM132C CAMK2N2

TMEM141 SLC6A1 CNKSR2 CAMK2N1

TMEM44 CALY

TMEM47 CALN1

TMEM59L CALCB

TMEM65 CADPS

TMEM91 STXBP1 CELF4 CADM2

TMOD1 CACNG2

TMSB4X CACNA2D2

Progressive encephalopathy TNNT1 CACNA2D1 ZNF804A CDH13 TNR ACHE CABP1

TOX CA4

TOX2 C1QTNF4

TPI1 C1QL1

TRHDE BTBD3 Clustering coefficient 0.109 0.306 0.08

TRIM9 BTBD17

TRNP1 BTBD11

TRPC3 BRINP3

TRPC5 BRINP1

TRPS1 BIRC2

TSC22D1 BIN1

TSC22D4 BEX1

TSPAN2 BEND6

Progressive neurological disorder TSPAN7 BCL11B

TTC3 BCAS1

TTC39B BASP1

TTC9B BARX2

TUBA1B BAIAP3

TUBA4A B4GALT6

TUBB AW551984

TUBB2A AVP

TUBB2B ATXN1

TUBB3 ATRNL1

TUBB4B ATP6V1G2

UBASH3B ATP1B2

UBE2QL1 ARPP21

UBL3 ARNT2

UNC5C ARL4C

UNC80 ARHGDIG

VAMP2 ARF4

VAT1 ARAP2

VAT1L APLP1

VCAN AP1S2

VGF ANKRD34B

VIP ANKRD29

VSNL1 ANK1

VSTM2A AMN

VSTM2L ALDOC

VWC2 AKAP7

WNT5A AKAP5

XIST AK5

YPEL2 AFAP1

ZCCHC12 ADCY2

ZCCHC18 ADARB1 Plasticity of synapse ZDBF2 ADAMTS5 ZIC1 ACSL6 ZMAT4 ACSL3 ZWINT ABAT ACLY PPIs between disease-annotated DEGs with other DEGs Synaptic depression PPIs between genes not annotated with As/Sz Dendritic growth/branching

CX HPC Trophic/growth factor Secreted factors C Ci complex Legends: Log2 normalized differential expression in MGE-Grin1fl/fl; FDR<0.01 CX HPC Misc. ligands CX HPC SST Sz: Schizophrenia risk Slit, Netrin complex Sz Sz PVALB As: Autism risk CX HPC Semaphorin complex NGFC : Both Sz and As CX HPC * Sz Sz Sz *

Cii

As Ephrin complex Sz Sz As CX HPC As Sz Protocadherins, Sz Sz Sz Cadherins, IgCAMs Sz Sz Ciii * Contactin, Tetraspanin Neurexin and others Receptor tyrosine LRR- domain family ECM components kinase/phosphatase * As Sz As Sz Sz Sz * Sz Sz Sz As* Sz Sz CX HPC

CX HPC Sz Sz Sz Civ Sz Sz Sz Sz As Sz Guidance and migration CX HPC Intracellular effectors CAM modifying Sulfo/sialyl-transferase CX HPC Sz* Membrane-expressed adhesion complexes * Sz

Sz CX HPC CX HPC CX HPC

Figure 6: Aberrant Grin1 -signaling result in misexpression of high-risk Sz genes A, Ingenu- ity Pathway Analysis of significantly overrepresented disease pathways in each MGE-subtype. Bi, Global protein-protein interaction (PPI)map among all differentially expressed genes (DEGs). Red circles indi- cate the DEGs annotated to be top-ranked Sz-risk genes; Blue circles indicate the DEGs annotated to be top-ranked As-risk genes; Yellow circles indicate the DEGs annotated with both Sz and As-risk genes. Black circles in the periphery indicate the DEGs not annotated with high-risk Sz/As genes. The PPIs between DEGs indicated in grey lines. Bii, PPIs between Sz / As / dually enriched clusters, and other genes. The PPIs between disease-annotated DEGs and other disease-annotated DEGs or with other non- annotated DEGs are indicated in black lines. ThePPI between non-annotated DEGs indicated in grey lines. C, Heatmap of log2 FC of significant DEGs in cortical and hippocampal MGE cardinal subtypes, showing a subset of Ci, secreted trophic factors and secreted ligands and guidance cues. Cii, membrane-bound synaptogenic receptors and cell adhesion molecules (CAMs) Ciii, Extracellular Matrix (ECM) components and matrix modifying enzymes. Civ, Intracellular effectors of guidance and synaptogenic cues.

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 15 of 46 USC 105 and is also made available for use under a CC0 license.

356 sets, based on multiple evidence sources including genome-wide association studies, copy-

357 number variations, known human mutations and other integrative analyses. In particular,

358 we mapped the DEGs with the top-ranked genes from these disease datasets (see methods for

359 details). While 592 DEGs could not be mapped with either disease genes, 25 genes mapped

360 exclusively with the SFARI-AS gene list, 164 genes mapped exclusively with the SZDB-Sz 361 gene list and 21 genes mapped with both datasets (Figure6Bi, Supplemental Table5 ). 362 It is now well-established that several neurodevelopmental disorders exhibit a high degree of

363 converging molecular pathways employing proteins that exist in physical complexes [71–74].

364 Therefore, we examined whether these disease-associated DEGs are known to form pro-

365 tein complexes between each other, by mapping curated protein-protein interaction (PPI)

366 datasets for all 802 DEG products. Indeed, we observed that >95% of disease-annotated

367 DEG products are known to exist with PPIs, while only ~75% of DEG products not anno-

368 tated with Sz/As are known to exist with PPIs (Supplemental Table5C). Interestingly,

369 despite not mapping directly with the high-ranked disease gene sets, the remaining 592

370 genes are observed to exist in tightly-knit PPIs with the disease annotated genes. How-

371 ever, the PPIs mapped with SZDB form the most interconnected clusters in comparison to 372 the SFARI-mapped PPI network (Figure6Bii), as indicated by relatively higher clustering 373 coefficient. This indicate that members of the DEGs here identified share physical, and

374 functional pathways in MGE-derived interneurons, contributing towards disease etiology.

375 Among the 210 DEGs mapped with to Sz and As, 45 genes are established regulators

376 of axon path-finding, synapse formation and assembly, while 38 members are established

377 regulators of membrane excitability and neuronal firing. Because both of these gene classes

378 are intimately associated with interneuron function, we examined these classes in detail. We

379 observed multiple classes of secreted ligands and cognate receptor families corresponding

380 to semaphorin, netrin, slit, chemokine and growth factors, and their intracellular effectors 381 that are downregulated upon MGE-Grin1 -ablation (Figure6Ci,iv). These include Ntng1, 382 Sema3e, Slit2, Cx3cl1, and some of their receptors, Unc5c, Nrp1, Neto1/2, Robo2 that are

383 decreased in a MGE-class-specific manner. We observed Fgf13 that was recently demon-

384 strated to mediate MGE-subtype specific synapse assembly [75], to be upregulated in cortical

385 PV cells, but downregulated in cortical SST, while Apoe to be upregulated in hippocam-

386 pal SST cells. In addition to synaptic assembly molecules, we observed DE in a variety

387 of synaptic adhesion molecules, corresponding to protocadherin, cadherin, ephrin and con- 388 tactin families (Figure6Cii ). Notably, we also observed a downregulation of Erbb4 across 389 all hippocampal MGE-subtypes. Lastly, we observed increased expression of extracellular

390 matrix components Mgp, Ndst4, Hapln1, Adamts5, Mxra7, Thsd7a and the matrix modify- 391 ing enzymes Hs3st2/4 in cortical SST/PV subtypes (Figure6Ciii ). 392 Among the regulators of neuronal excitability, we observed a downregulation of multiple

393 members of postsynaptic glutamate receptor subunits, GABA receptors and their associ- 394 ated partners (Figure6-Supplement1Bii). Interestingly, while we noted a broad down- 395 regulation of several members of potassium and sodium channel subunits, a few discrete

396 members of the Kcn-families were upregulated in cortical PV and NGFC subtypes. Finally,

397 we also observed multiple members of presynaptic GABA synthesis, release and uptake

398 machineries including Gad1, Syt2/10, and Slc6a1 differentially expressed in discrete MGE

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 16 of 46 USC 105 and is also made available for use under a CC0 license.

399 subtypes (Figure6, Supplement1Bi ). Collectively, these findings highlight the centrality 400 of MGE-expressed Grin1 -signaling during synapse formation and connectivity, which when 401 aberrantly expressed, can lead to neurodevelopmental disorders.

402 Discussion

403 Centrality of MGE-derived interneuron-expressed NMDARs from juvenile 404 brain

405 NMDARs serve as critical activity dependent signaling hubs for myriad neuronal func-

406 tions due to their innate ability to directly link network dynamics to cellular calcium events,

407 and associated transcriptional coupling. Such NMDAR-dependent excitation-transcription

408 coupling is widely established in glutamatergic neurons [76], and in specific interneurons

409 using candidate approaches [77] within mature circuits. However, the detailed unbiased

410 evaluation of the transcriptional landscape of NMDAR signaling within interneurons in de-

411 veloping circuits undergoing refinement is lacking. Our study provides the first systematic

412 “fingerprinting” of the transcriptional coupling associated with NMDAR signaling, exclusive

413 to MGE-derived interneurons, providing a road map for examining NMDAR regulation of

414 MGE-derived interneurons in a subtype specific manner.

415 Our unbiased transcriptional profiling approach indicates that developmental NMDAR

416 signaling participates in MGE-derived interneuron specification by regulating the expres-

417 sion of transcription factors (67 genes), synaptogenic (53 genes) and connectivity fac-

418 tors/adhesion molecules (61 genes), and regulators of membrane excitability (78 genes),

419 among the 802 DEGs in interneurons (Figure7). We employed bioinformatic analy-

420 ses to examine whether system-wide downregulation of target genes can be attributed to

421 transcription-repression elements. Indeed, we identify a set of 10 transcriptional regulators

422 that commonly recognize the DNA-motifs present in the identified DEGs, including the

423 master-repressor Rest. Future studies are needed to examine the role of these putative re-

424 pressor and repression motifs that have not been previously associated with MGE-specific

425 transcription. However, based on broad transcriptional downregulation of target genes, we

426 can make several predictions that should guide future investigations. 427 Shaping interneuron identity and granularity amongst subtypes 428 Interneuron development from MGE is replete with combinatorial expressions of numer- 429 ous transcription factors, leading to diversity [78, 79]. Several transcription factors that are

430 impacted by Grin1-signaling are established regulators of MGE fate, subtype abundances

431 and identities (34 genes) including Nkx2-1, Lhx6, Dlx1, Dlx6, Maf, Mafb, Mef2c, Etv1,

432 Npas1, Npas3 and Sp9 [34, 56, 80–83] [84]. While the scRNAseq landscapes of interneurons

433 predict several transcriptomic features that would classify them as distinct ‘cell-types’ or

434 cell-states’ [49, 85, 86] [50] the precise mechanisms responsible for such granularity is still

435 emerging [2]. It is possible that NMDAR signaling in the developing interneuron progenitors

436 may provide a combinatorial cue that will couple with innate genetic programs to gener-

437 ate the diversity in interneuron subtypes. Indicating that the Grin1 -lacking MGE-derived

438 interneurons have impaired subtype identities, we observe differential recoveries of the sub-

439 types within SST, PV and NGFC in the scRNAseq assay (Figure2-Supplement2A,B,C).

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 17 of 46 USC 105 and is also made available for use under a CC0 license.

Legends: MGE NMDARs progenitors NGFC Ca2+ ions

Circuit maturation Interneuron Synapse refinement progenitors PV PCD, Synaptogenesis Laminar positioning Tangential migration Grin1wt/wt SST Nkx2-1 Grin1-Ca2+ A expression @ signaling @ ~E16 ~E10.5 P0 P10 P20 P30 Adult ~E9.5 scRNAseq

fl/fl B Grin1

Notable aberrations in gene expressions resulting from MGE-specific developmental NMDAR ablation Synaptic partnering MGE-TFs Excitability, LiGC and subunits Apoe, Cx3cl1, Efna5, Efnb3, Arx, Dlx1/6, Etv1, Lhx6, Maf, Cnr1, Gabbr2, Gabra1/a2/a4/d/g1/g2/g3, Epha4/5/7, Neto1/2, Nrp1, Nrtn, Mafb, Myt1l, Nkx2-1, Nr2f1, Shisa4/6/9, Gria1/a3/a4, Grik3, Ntng1, Ntrk1/3, Plxn12/4, Reln, Satb1, Sp9 Grin1/n2a/n2b/n3a, Grm1/5, Cacng2, Robo2, Sema3e/5a/6a, Slit2, Neto1/2 Tenm1/2/3, Unc5c/5d, Utrn, Wnt5a Synaptic assembly Alcam, Cadm1/2, Cdh2/3/7/9/12/13, Cntn4/5, Cntnap1/2/4/5, Activity-TFs Excitability, VGCCs, subunits and transporters Elfn1, Erbb4, Lrrtm1, Nptn, Nrcam, Arnt2, Btg2, Egr1, Elmo1, Fos, Cacna2d1/d2/d3, multiple subunits Pcdh7/8/10/11x/15/17/19, Fosb, Jun, Junb, Npas1/3/4, of the Kcn family, Scn1/1b/2b/8a, Trpc3/5, Ptchd4, Ptpre/t/u/z1, Nr4a2 Nptn, Slc4a10, Slc12a5

Figure 7: Transcriptional control of MGE development, synaptic partnering and excitablity are mediated by NMDAR signaling A, Nkx2.1 expression appears at ~ED10.5, driving MGE subtype fate in interneuronal progenitors [42] [56]. Subsequently, their sequential developmental milestones towards circuit refinement appears to be under a combination of innate genetic mechanisms and neuronal activity. While the earliest Grin1 expression is reported at ~ED14 in developing brain [58] [57] , MGE-specific Grin1 - mediated Ca2+ is recorded at ~ED16 [15]. However, the broad role played by interneuron-expressed NMDAR signaling during interneuron development until now is not well delineated. B, By driving Grin1 -ablation using Nkx2.1 -driven Cre-recombinase, we report the earliest developmental loss of NMDAR signaling, across MGE-derived interneuron subtypes. In particular, by performing scRNAseq assay in MGE-derived interneu- rons from the cortex and hippocampus of the mouse brain, we report a broad transcriptional aberration subsequent to loss of NMDAR-signaling. Notably, this expression abnormality involves numerous tran- scriptional factors, synaptogenic and regulators of interneuron excitability, that collectively estabish MGE subtype identities. ED, Embryonic day; PD, Postnatal day; PCD, Programmed cell death; LiGC, Ligand-gated channel; VGCC, Voltage-gated calcium channel

440 Particularly, we find in the Grin1 -ablated MGE-derived interneurons, the presence of diver-

441 sified PVALB.3 populations that express marker genes such as Prox1, otherwise not robust wt 442 in our scRNAseq screen from the MGE-Grin1 . Additionally, by independent immunos-

443 taining experiments, we observe a modest increase in cortical SST cell numbers along with

444 a modest decrease in cortical PV cells, in a manner similar to Maf:Mafb-mutants recently

445 reported [82]. However, detailed future studies are necessary to uncover whether/how the

446 NMDAR-dependent combinatorial transcriptional code works with innate mechanisms to

447 generate the diversity within PV/SST/NGFC subclasses. It would be intriguing to examine

448 whether these MGE-Grin1 -null mice exhibit aberrations in the expression of these master

449 MGE-regulators such as Nkx2-1 and Lhx6 earlier in development.

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 18 of 46 USC 105 and is also made available for use under a CC0 license.

450 Shaping interneuron subtype-specific synaptic assembly and connectivity

451 What is the biological context of differential expression of the TFs, in the juvenile fore-

452 brain when MGE-derived interneuron fate is assumed to be already sealed, and subtype

453 identities established? It is emerging that some of these TFs are continually required for the

454 maintenance of MGE fate, post development [87]. One of the ways the TFs maintain MGE

455 subtype fate into adulthood, is by controlling the expression of genes that are essential for

456 ongoing interneuron function. Accordingly, we predict that NMDAR-dependent expression

457 of synaptogenic and synaptic partnering molecules regulate the assembly of synapses with

458 appropriate targets. Secreted semaphorin, ephrin, slit, netrin and neurotrophin-based sig-

459 naling systems have been investigated in GABAergic neurons, during axonal pathfinding,

460 and cell migration [88–95]. However, only recently have inroads been made into delineat-

461 ing their expression, and interaction with appropriate receptor systems in target synapses

462 during accurate synaptogenesis. In addition, the NMDAR-dependent expression of synap-

463 tic adhesion molecules will further promote stability of newly formed synapses. Here, the

464 mis-expression of diverse secreted cues, their receptors and adhesion molecules by MGE sub-

465 types during Grin1 -ablation, provides unique insight into the molecular diversity employed

466 during synapse establishment. Our findings also reveal numerous candidates for examining

467 subtype specific synapse assembly, which are centrally regulated by NMDAR signaling. Of

468 particular interest are the family of protocadherins that are reported recently to be com-

469 monly downregulated in cortical interneurons generated from Sz patient-derived induced

470 pluripotent stem cells [96].

471 Subsequent to synapse formation nascent connections remain susceptible to strength

472 modifications according to neuronal activity. Again, NMDAR-signaling in MGE-derived in-

473 terneurons seems to regulate this process by the transcriptional regulation of the expressions

474 of both presynaptic and postsynaptic members, including excitatory and inhibitory synap-

475 tic molecules and their auxiliary subunits, as well as presynaptic GABA release machinery

476 molecules such as Cplx1/2, Stx1b, Rab3c. However, most dramatic is the massive down reg-

477 ulation of several members of the potassium channel subunits and their auxiliary subunits

478 across MGE subtypes, with the exception of an upregulation of a few Kcn-genes in cortical

479 PVs and NGFCs. While the precise impact of the diverse changes in these genes on MGE

480 firing are currently unclear, the pattern of expression of the activity-dependent transcription

481 factors provides us an indication.

482 Notable activity-dependent TFs such as Jun, Egr1 are downregulated across all MGE

483 subtypes, while Fosb, Fos, Arx are down regulated across all hippocampal MGEs, and

484 Satb1, Arnt2 are downregulated across all cortical MGEs. In addition, Npas4, an established 2+ 485 early-response TF [97–99] activated upon neuronal activity and Ca influx in MGE-derived

486 interneurons [100], was downregulated in cortical NGFCs upon Grin1 -ablation. Etv1 was 2+ 487 previously demonstrated to be an activity-dependent TF that inversely correlates Ca in-

488 flux, regulating the identity of a subset of PV-interneurons [80]. Remarkably, we observe an

489 increase in Etv1 expression in cortical PV cells. Lastly, Ostn was recently established as an

490 activity-regulated secreted factor [101], and we observed Ostn to be downregulated specifi- 491 cally in cortical PV subtypes (Figure4C ii ). Together, these changes are consistent with 492 reduced neuronal activity in MGE subtypes upon Grin1 -ablation, consistent with previous

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 19 of 46 USC 105 and is also made available for use under a CC0 license.

493 reports indicating that NMDAR-antagonists can directly reduce the activity of GABAer-

494 gic interneurons in adult mice [27]. Interpreting the differential expressions of activity-

495 dependent genes during scRNAseq has been challenging, particularly, when these genes

496 could get activated by the very process involved in cell dissociation and sorting [102, 103]. wt 497 However, our use of activity-blockers and actinomycin-D throughout our MGE-Grin1 and fl/fl 498 MGE-Grin1 scRNAseq pipelines [46], gives confidence that the differential expressions

499 of activity-dependent TFs reflect biological relevance. 500 NMDAR signaling in NGFCs 501 Among the MGE subtypes, the PV and SST interneurons are traditionally widely stud- 502 ied in comparison to the dendrite-targeting NGFC subtypes (that include the Ivy cells). In

503 the present study we provide the first detailed molecular insight into the cortical and hip-

504 pocampal NGFCs, subsequent to NMDAR ablation. We anticipated that these cell types

505 could be particularly susceptible to loss of NMDARs, since we previously reported that

506 NGFCs exhibit the most robust synaptic NMDAR conductances among the MGE sub-

507 types [12]. Intriguingly, while the cortical NGFCs had comparable numbers of both total

508 and unique DEGs with respect to other cortical MGE-derived interneurons (Figure3B), we

509 observed far fewer total and NGFC-specific DEGs in the hippocampus, compared to other

510 hippocampal MGEs. However, based on the scRNAseq cell type recoveries, we predict an

511 elaboration of NGFC.1, and a reduction in the NGFC.2 subtype upon Grin1 -ablation. Fi-

512 nally, NGFCs exhibited dendritic arborization impairments subsequent to impaired NMDAR

513 signaling [9, 11]. Indeed, we observe 49 genes among the DEGs (Supplemental Table1

514 ) that have established roles in regulating neuronal cytoskeleton and associated signaling,

515 likely mediating the observed dendritic impairments in NGFCs. 516 Developmental NMDAR ablation in interneurons and schizophrenia 517 Impaired NMDAR function observed during human NMDAR gene mutations [104], and 518 anti-NMDAR-encephalitis [105] results in a wide range of neuropsychiatric disorders in-

519 cluding autism spectrum disorders [106, 107], intellectual disability [108], psychosis [109],

520 epilepsy and associated comorbidities [110, 111]. While broadly aberrant NMDAR signaling

521 in neurons is thought to underlie a wide range of these neurological disorders, an interneuron-

522 centric developmental NMDAR aberration is emerging central to schizophrenia-related syn-

523 dromes. Indeed, in the present study, disease mapping of the DEGs using high-ranked

524 SZDB-Sz and SFARI-As datasets indicate that many more DEGs map with the Sz than

525 the As database. Moreover, these disease-relevent DEGs exist in physical and functional

526 complexes with other DEGs that are not directly mapped to the Sz database. We used only

527 stringent, high-ranked disease genes from the database that pass several disease-relevant cri-

528 teria. However, there are other DEGs that still map to lower-ranked Sz and As datasets that

529 are ‘non-annotated’ in present study. While our study can be argued as an ‘extreme’ case

530 of NMDAR hypofunction in MGE-derived interneurons, it provides a starting point high-

531 lighting the centrality and broad range of interneuronal NMDAR-transcriptional pathways

532 during development.

533 A multitude of studies implicate NMDAR-hypofunction specific to PV cell types as a

534 central underlying feature of schizophrenia etiology [112, 113]. However, the measurable NM-

535 DAR conductances within PV interneurons are relatively small in comparison to other MGE

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 20 of 46 USC 105 and is also made available for use under a CC0 license.

536 subtypes [12] . Additionally, NMDA signaling in non-PV interneuron subtypes drives robust

537 dendritic inhibition in pyramidal neurons [114, 115]. Moreover, while NMDAR-ablation in

538 Pvalb-Cre lines produces other behavioral deficits unrelated to the Sz-like phenotypes [30, 33]

539 , a developmental, but not adult-onset Grin1 -ablation in Ppp1r2 -Cre line [29] that targets

540 a subset of PV interneurons among other subtypes [116], recapitulates core Sz-like pheno-

541 types. Lastly, studies that map interneuron subtypes to Sz-like phenotypes indeed support

542 the role of different interneuron classes beyond PV cells towards disease etiology [28, 33].

543 Integrating these ideas and based on findings from the present study, we propose the

544 following: (i) Despite a smaller NMDAR conductance in PV interneurons, we observe a ro-

545 bust transcriptional coupling via NMDARs, as observed by several distinct gene expression

546 abnormalities in this cell type relevant to human Sz. Therefore, PV-expressed NMDARs pri-

547 marily serve to regulate transcriptional coupling, mediating the abundances of PV-subtype

548 abundances. (ii) The developmental window for NMDAR loss of function is particularly

549 important because, its transcriptional regulation maintains the correct synaptogenic and

550 assembly cues, which when lost, lead to disease causing-impaired connectivity. Perhaps, fl/fl 551 in the Grin1 : Pvalb-Cre mouse line, the Grin1 -ablation occurs only at a developmen-

552 tal window when synaptic connectivity is sufficiently complete, explaining why the animal

553 model does not lead to profound Sz-like impairments. (iii) The dendrite targeting SST and

554 NGFC interneurons also exhibit robust NMDAR signaling and transcriptional coupling.

555 During aberrant NMDAR-transcriptional coupling, it is therefore likely that impaired den-

556 dritic connectivity and inhibition onto pyramidal neurons also contributes towards disease

557 etiology. Therefore, our dataset provides credence to interneuronal subtype-specific granu-

558 larilty, connectivity and excitability, all playing combinatorial and mutually-supporting roles

559 during disease etiology.

560 Taken together, our study presents a rich resource, laying the road map for systematic

561 examination of NMDAR signaling in interneuron subtypes, by providing multiple molecular 562 targets for examination in both normal and impaired circuits.

563 Materials and methods

564 Contact for Reagent and Resource Sharing 565 Further information and requests for resources and reagents should be directed 566 to and reasonable requests will be fulfilled by the Lead Contact, Chris McBain

567 ([email protected]).

568 Animals 569 All experiments were conducted in accordance with animal protocols approved by the Na- 570 tional Institutes of Health. The Nkx2.1-Cre driver line (C57BL/6J-Tg(Nkx2 -1-Cre)2Sand/J; tm2Stl 571 Cat. No. 008661 | Nkx2.1-Cre, Floxed Grin1 mouse line (B6.129S4-Grin1 /J; Cat. No. tm14(CAG−tdT omato)Hze 572 005246 | fNR1) and Ai14 reporter mouse (B6.Cg-Gt (ROSA)26Sor /J;

573 Cat. No. 007914 | Ai14, Ai14D or Ai14(RCL-tdT)-D), purchased from the Jackson Labora- fl/fl 574 tory, were used to generate the MGE-derived interneuron-specific Grin1 line. Littermate wt/wt 575 MGE-Grin1 controls, and both male and female mice were used during this study. Mice

576 were housed and bred in conventional vivarium with standard laboratory chow and water in

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 21 of 46 USC 105 and is also made available for use under a CC0 license.

577 standard animal cages under a 12hr circadian cycle. Genotyping of the mice were performed

578 as indicated in the appropriate Jackson Laboratory mice catalog. 579 Single-cell dissociation and FACS wt/wt + fl/fl + 580 P18-20 juvenile Nkx2 -1-Cre: Grin1 : TdT and Nkx2-1-Cre: Grin1 : TdT mice 581 were used for single-cell sequencing experiments. All mice were anesthetized with isoflu-

582 rane and then decapitated. Brain dissection, slicing and FACS sorting were carried out

583 as described [43, 44], with slight modifications. NMDG-HEPES–based solution was used

584 in all steps to enable better recovery of the cells [45] during FACS sorting and single-cell

585 bar coding. Briefly, the brain sectioning solution contained NMDG-HEPES–based high- 2+ 586 Mg cutting solution contained 93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM 587 NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 588 2mM Thiourea, 10 mM MgSO4*7H2O, and 0.5 mM CaCl2*2H2O; it was adjusted to pH 7.4 589 with 12.1N HCl, an osmolarity of 300-310 mOsm, and carbogenated (mix of 95% O2 and 590 5% CO2) before use. This solution was chilled and the process of sectioning were conducted 591 on a ice-chamber in the vibratome. wt/wt + fl/fl + 592 3-4, Nkx2-1 -Cre: Grin1 : TdT or Nkx2-1 -Cre: Grin1 : TdT mice were pro-

593 cessed on consecutive days for single-cell sequencing experiments. TdT negative animals

594 were processed in parallel for initially setting FACS gate for the Tomato-channel. Across

595 the replicates, 10XMGE-Grin1-WT and 6XMGE-Grin1-null animals were used for the scR-

596 NAseq. Coronal slices containing frontal cortex and hippocampus (350mM) were cut using 2+ 597 VT-1000S vibratome (Leica Microsystems) in cold NMDG-HEPES–based high-Mg cut- ◦ 598 ting solution. Slices were recovered in the same solution at 20 C for 30 minutes during

599 when, they were visually inspected under fluorescence microscope and micro dissected, all

600 under constant carbogenation. The recovery and microdissection were conducted in the 2+ 601 NMDG-HEPES high-Mg solution supplemented with 0.5µM tetrodotoxin (TTX), 50 µM

602 DL -2-Amino-5-phosphonopentanoic acid (APV) and 10 µM Actinomycin-D (Act-D).

603 Cell dissociation was performed using the Worthington Papain Dissociation System

604 (LK003150) according to manufacturer instructions with minor modifications. Briefly,

605 single-cell suspensions of the micro dissected frontal cortices or hippocampus were prepared

606 using sufficiently carbogenated dissociation solution (containing Papain, DNAse in Earle’s

607 Balanced Salt Solution, EBSS), supplemented with 1µM TTX, 100 µM APV and 20 µM ◦ 608 Act-D. After a 60 min enzymatic digestion at 37 C, followed by gentle manual trituration

609 with fire-polished Pasteur pipettes, the cell dissociates were centrifuged at 300g for 5 min- ◦ 610 utes at 20 C, and the supernatants were discarded. The enzymatic digestion was quenched

611 in the next step by the addition of ovomucoid protease inhibitor. Albumin density gradi-

612 ent was performed on the pellets, using a sufficiently carbogenated debris removal solution

613 (containing albumin-ovomucoid inhibitor, DNAse in EBSS). The resulting cell pellets were

614 resuspended in 1ml FACS buffer containing 10% FBS, 10U/µl of DNAse, 1µM TTX, 100

615 µM APV and 20 µM Act-D in a 50:50 mix of carbogenated EBSS: NMDG-HEPES–based 2+ 616 cutting saline (with 1mM MgSO4*7H2O, it is important to not use High-Mg in the FACS 617 buffer, as it interferes with the subsequent 10X scRNAseq reaction). Cells were placed in

618 polystyrene tubes (Falcon 352235) on ice during the FACS. + 619 For single cell sorting of TdT expressing cells by FACS, resuspended cell dissociates

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620 were filtered through 35mm cell strainer (Falcon 352235) to remove cell clumps. The single

621 cell suspensions were then incubated with 1mg/ml DAPI (1:500, Thermo Scientific 62248) ◦ 622 and 1mM DRAQ5 (Thermo Scientific 62251) at 4 C for 5 minutes to label dead cells and live

623 cells respectively. Samples were analyzed for TdTomato expression and sorted using a MoFlo

624 Astrios EQ high speed cell sorter (Beckman Coulter). TdT-negative cells were used as a con-

625 trol to set the thresholding FACS gate for the detection and sorting of the Ai14-TdTomato-

626 expressing cells, and the same gate was then applied for all subsequent experiments. Flow

627 data analysis and setting of sorting gates on live (DAPI-negative, DRAQ5-positive) and

628 Ai14-TdTomato-expressing cells were carried out using Summit software V6.3.016900 (Beck-

629 man Coulter). Per sample/session, 20,000 – 40,000 individual cells were sorted into a FBS-

630 precoated, Eppendorf LoBind Microcentrifuge tubes containing carbogenated 10ml FACS

631 buffer, that served as the starting material for 10X Genomics bar-coding. 632 10X Genomics Chromium 633 The cells were inspected for viability, counted, and loaded on the 10X Genomics 634 Chromium system, aiming to recover ~5000 cells per condition. 12 PCR cycles were con-

635 ducted for cDNA amplification, and the subsequent library preparation and sequencing were TM 636 carried out in accordance with the manufacturer recommendation (Chromium Single Cell

637 3’ Library & Gel Bead Kit v2 and v3, 16 reactions). Sequencing of the libraries were

638 performed on the Illumina HiSeq2500 at the NICHD, Molecular Genomics Core facility.

639 Replicate 1 of the scRNAseq were performed using 10X v2 reaction from which, the cell

640 estimates, mean reads per cell (raw), median genes per cell respectively, are as follows Cor-

641 tical WT: 1277, 149K, 4615; Cortical NULL: 181, 159K, 4826; Hippocampal WT: 2221, 92K,

642 2578; Hippocampal NULL: 404, 154K, 4903. Replicate 2 of the scRNAseq were performed

643 using 10X v3 reaction from which, the cell estimates, mean reads per cell (raw), median

644 genes per cell respectively, are as follows Cortical WT: 3851, 22.8K, 1536; Cortical NULL:

645 2898, 23.5K, 2759; Hippocampal WT: 4600, 23.6K, 850; Hippocampal NULL: 4436, 25.8K,

646 3143. Replicate 3 of the scRNAseq were performed using 10X v3 reaction from which, cell

647 estimates, mean reads per cell (raw), median genes per cell respectively, are as follows Cor-

648 tical WT: 3960, 24.8K, 2870; Hippocampal WT: 3159, 26.9K, 2956. Representative quality

649 metrics from Replicate 2 are indicated in Figure1-Supplement1B,C ,D,E. Demultiplexed

650 samples were aligned to the mouse reference genome (mm10). The end definitions of genes

651 were extended 4k bp downstream (or halfway to the next feature if closer), and converted

652 to mRNA counts using the Cell Ranger Version 2.1.1, provided by the manufacturer. 653 Data processing, analyses, visualization and differential expression testing 654 Processing (load, align, merge, cluster, differential expression testing) and visualiza- 655 tion of the scRNAseq datasets were performed with the R statistical programming environ-

656 ment [117] (v3.5.1) and Seurat package (v3.1.5, a development version of Seurat v3.1.5.9000

657 was used to generate violin plots in 2C and 5B) [47, 48] . Data set preprocessing, compari-

658 son of WT- and NULL-Ai14 cells, canonical correlation analyses, and differential expression 659 of genes (padj < 0.01) within the same cluster between WT- and NULL-Ai14 cells were 660 performed according to default Seurat parameters, unless otherwise mentioned. Quality

661 control filtering was performed by only including cells that had between 200-6000 unique

662 genes, and that had <30% of reads from mitochondrial genes. While the WT replicates

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663 had no cells above 30% mitochondrial genes, only NULL replicates from both brain regions

664 exhibited 7-12% of cells above this threshold. Suggestive of inherent biological impact of

665 Grin1 -ablation, we repeated the clustering and subsequent analyses without excluding any

666 cells. These analyses did not alter the clustering or skew the gene list. Clustering was per-

667 formed on the top 25 PCs using the function FindClusters() by applying the shared nearest

668 neighbor modularity optimization with varying clustering resolution. A cluster resolution

669 of 1.0 was determined to be biologically meaningful, that yielded all known MGE cardinal

670 classes. Initial analyses were performed on the WT datasets separately (WT.alone), and sim-

671 ilar set of analysis parameters were applied when the WT and NULL samples were merged

672 (WT.NULL.integrated) for subsequent differential expression testing. Phylogenetic tree re-

673 lating the ’average’ cell from each identity class based on a distance matrix constructed in

674 gene expression space using the BuildClusterTree() function. Overall, we identified 27, and

675 33 clusters using this approach in the WT.alone, and WT.NULL.integrated assays respec-

676 tively. The WT.alone correspond to 11 MGE.GAD1/2 clusters (Figure1&2), while the

677 WT.NULL.integrated assay correspond to 12 clusters (Figure5-Supplement.1). We first

678 searched for the top differential markers for each MGE subcluster using the FindAllMarkers()

679 function. The genes thus identified for the integrated data is presented in Supplemental

680 Table1b. Determination of MGE and non-MGE identities are performed based on existing

681 interneuron literature and other scRNAseq datasets [1, 43, 50, 53, 85, 87, 118–120]. The

682 labels from Figures 1 and 2 are matched with the top gene markers identified by the Find-

683 AllMarkers() function and the similarly named clusters in Figures 1 and 2 have the same

684 identities. Lastly, for the integrated analyses and differential expression testing, we first

685 merged the identities of the subclusters SST.1-SST.6 and TH.1, and relabelled as SST sub-

686 set; PVALB.1-3 relabelled as PVALB subset; and NGFC.1-2 relabelled as the NGFC subset

687 during subsequent analysis (Figure3).

688 Differential gene expression testing were performed using the MAST package within the

689 FindMarkers function to identify the differentially expressed genes between two subclusters.

690 MAST utilizes a hurdle model with normalized UMI as covariate to generate the differential

691 fold changes [121] , and is known to result in underestimation of the magnitude of fold

692 change (FC) [122]. Therefore, while applying a stringent false-discovery rate <0.01, we

693 determined the minimum FC based on the control gene Grin1, which is the target gene

694 knocked out in MGE-derived interneuron celltypes. Notably for Grin1, we had previously

695 demonstrated that the NGFCs which carry maximum NMDAR component among MGEs,

696 are devoid of NMDAR current at this comparable age [9] . In the present scRNAseq assay,

697 we observe a logFC for Grin1 ranging between -0.1 to -0.35 across both brain regions and

698 all MGE subtypes. Therefore, we determined a minimum logFC in our DEGs as ±0.1 to be

699 meaningful. Previous studies have demonstrated the MAST approach for DEG testing to

700 be powerful in determining subtle changes in highly transcribed genes, and among abundant

701 populations, additional to under representing changes among weakly transcribed genes [121,

702 122] . Volcano plots and Heat maps for the DEG were generated using EnhancedVolcano

703 package [123] and Morpheus package https://software.broadinstitute.org/morpheus within

704 the R framework. 705 Pathway analyses, PPI network mapping and disease mapping

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706 Ingenuity Pathway Analyses were conducted on the differentially expressed genes to gen-

707 erate the molecular functional annotation and to identify the biological pathways and dis-

708 ease pathways overrepresented. This tool was also used to annotate genes with their known

709 cellular functional classes. Additional Gene Ontology mapping and KEGG analyses were

710 conducted using ShinyGO [124]. Protein-protein interaction (PPI) mapping datasets from a

711 variety of curated databases [125–127] were conducted as previously described [72] [128] us-

712 ing the Cytoscape [129] platform (v3.8.0). Schizophrenia risk genes integrated from various

713 sources including genome-wide association studies (GWAS), copy number variation (CNV),

714 association and linkage studies, post-mortem human brain gene expression, expression quan-

715 titative trait loci (eQTL) and encyclopedia of DNA elements (ENCODE), were downloaded

716 from http://www.szdb.org/ [69] . Autism Spectrum Disorder risk genes integrated from var-

717 ious sources were downloaded from Simons Foundation https://gene.sfari.org/ [70]. SZDB

718 genes that had a integrated total score of 3-6 (1419 genes, 22% out of 6387) were consid-

719 ered ‘high-risk’ for DEG mapping (Supplemental Table5a). SFARI genes scored 1-2 with

720 accounting for a high strength of evidence (392 genes, 42% out of 943), were considered

721 ‘high-risk’ for DEG mapping (Supplemental Table5b). Transcriptional factor motif en-

722 richment search using the iRegulon [62] was also conducted using Cytoscape using default

723 parameters.

724 Immunostaining 725 All solutions were freshly prepared and filtered using 0.22µm syringe filters for parallel 726 treatments of wildtype and MGE-Grin1-null groups. Adult mice of postnatal day (PD)

727 30/60/210 were Mice were deeply anesthetized with isoflurane and perfused transcardially

728 with 1X phosphate buffer saline (PBS) and followed by the fixative 4% paraformaldehyde. ◦ 729 The brains were post-fixed in the same fixative for overnight at 4 C for the immunostaining

730 assays. Postfixed brains were serially dehydrated using 10%/20%/30% sucrose solutions at ◦ 731 4 C. Coronal sections (50 µm) were cut on a freezing microtome. Immunostaining was

732 performed on free-floating sections. Tissue sections were permeabilized and blocked in 1 ×

733 PBS + 1% bovine serum albumin + 10% normal goat serum + 0.5% Triton X-100 (Carrier

734 PB) at room temperature for 2 h, followed by incubation in primary antibodies, listed below,

735 diluted with 1 × PBS + 1% bovine serum albumin + 1% normal goat serum + 0.1% Triton ◦ 736 X-100 overnight at 4 C. Tissue sections were then incubated with secondary antibodies,

737 listed below, diluted in Carrier Solution (1:1000), and DAPI (1:2000) at room temperature

738 for 1–2 h and mounted on Superfrost glass slides, and coverslipped using Mowiol mounting

739 medium and 1.5 mm cover glasses.

740 Antibodies 741 The following primary antibodies were used: mouse anti-PV (1:1000; Sigma-Aldrich 742 Cat# P3088, RRID: AB_477329), rat anti-SST (1:1000; Millipore Cat# MAB354, RRID:

743 AB_2255365). Secondary antibodies were conjugated with Alexa Fluor dyes 488 or 633

744 (1:1000; Thermo Fisher Scientific). 745 Image acquisition and analysis 746 Mouse brains from 4–8 different animals were used for each condition, and section depth 747 were matched between the genotypes for parallel immunostaining. Fluorescent images were

748 captured using the 10X objective of a Nikon Widefield Fluorescence, Spinning Disk Confocal

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749 microscope. For all slices with immunostained or genetically reported signal, 50 µm thin 750 sections were imaged using 10x/0.45 CFI PlanApo objective (imaging settings: Numerical 751 Aperture 0.75, bit depth 16-bit, Exposure 100ms). Confocal stacks were stitched using NIS 752 Elements (Nikon) before importing them into Imaris software (Bitplane, version 9.2). Cell + 753 bodies were marked in Imaris software using the ‘Spots’ function. Nkx2-1 -Cre:TdT RFP+, 754 PV+ cell bodies were detected using the automatic function, with a signal detection radius 755 of 10 µm. The Imaris ‘Quality’ filter was set above an empirically determined threshold to 756 maximize the number of detected cells while minimizing observed false positives. SST+ cell 757 bodies were marked manually using the Imaris ‘Spots’ function. ROI 3D borders around 758 hippocampus or cortex, drawn manually using the Imaris function ‘Surfaces’. Spots were 759 then split within each ROI using the Imaris function ‘Split Spots’. Overlap of RFP+ cells 760 with other markers (PV, SST) was addressed by filtering the RFP+ Spots above an em- 761 pirically determined threshold intensity in the channel relative to the marker of interest. 762 Each image with an automatic analysis by Imaris was checked by an expert and incorrectly 763 identified cell bodies where refined if required. In Figure5A,B Error bars reflect standard 764 error of mean; Two-tailed unpaired t-test was performed using Prism8.

765 Author contributions

766 VM and CJM conceived the project. VM, TJP and CJM designed the experiments, DM 767 performed FACS sorting and analysis. VM, YZ, TJP performed 10X scRNAseq. VM, AM, 768 CJR, CE, RD performed 10X scRNAseq bioinformatic analyses. VM and AP conducted 769 immunuflourescent staining, imaging and analysis. CJM supervised the study. VM and 770 CJM wrote the paper and all authors edited the manuscript.

771 Acknowledgment

772 This work was supported by Eunice Kennedy Shriver NICHD Intramural Award to CJM. 773 We thank Steven L. Coon, Tianwei Li and James R. Iben at the Molecular Genomics Core, 774 NICHD, for RNA sequencing and bioinformatics support. We thank Vincent Schram and 775 Lynne Holtzclaw of the NICHD Microscopy and Imaging Core for imaging support, and 776 we thank Carolina Bengtsson Gonzales for assistance with improving cell viability during 777 dissociation and FACS. We also thank Xiaoqing Yuan, Steven Hunt, Daniel Abebe for 778 assistance with animal colony maintenance.

779 References

780 [1] K. A. Pelkey, R. Chittajallu, M. T. Craig, L. Tricoire, J. C. Wester, C. J. McBain, Hip- 781 pocampal GABAergic Inhibitory Interneurons, Physiological Reviews 97 (4) (2017) 1619–1747. 782 doi:10.1152/physrev.00007.2017. 783 URL https://dx.doi.org/10.1152/physrev.00007.2017 784 [2] B. Wamsley, G. Fishell, Genetic and activity-dependent mechanisms underlying interneuron diversity, 785 Nature Reviews Neuroscience 18 (5) (2017) 299–309. doi:10.1038/nrn.2017.30. 786 URL https://dx.doi.org/10.1038/nrn.2017.30

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 26 of 46 USC 105 and is also made available for use under a CC0 license.

A ♀ X ♂ Cre negative: Floxed.TomatoAi14/ Ai14 : Grin1flox/flox Nkx2.1Cre (Homozygous) (Homozygous)

♀ X♂ Nkx2.1Cre: Nkx2.1Cre: Grin1flox/WT: TomatoAi 14/ WT Grin1flox/WT: TomatoAi 14/ WT (Heterozygous) (Heterozygous) Neither used for experiments, nor Nkx2.1Cre Positive : for breeding F2 … TomatoAi14/ Ai14: Ectopic expression

Nkx2.1Cre Positive : Nkx2.1Cre Positive : TomatoAi14/ Ai14: TomatoAi14/ Ai14: Grin1Flox/WT Grin1Flox//Flox (MGE.Grin1.WT) (MGE.Grin1.Null)

Live:Dead-gating BiV Bi with DRAQ5:DAPI Singlet-gating Ai14-gating Bii Biii MR/cell 22.8K MG/cell 1536 - WT

CORTEX CX.WT

Ci Cii Ciii CiV MR/cell 23.5K MG/cell 2759 NULL -

CORTEX CORTEX CX.NULL

Di Dii Diii DiV MR/cell 26.9K MG/cell 2956 WT - HPC HPC

HPC.WT

Ei Eii Eiii EiV MR/cell 25.8K MG/cell 3143 NULL - HPC HPC HPC.NULL

Figure 8: (Figure1, Supp.1) | Schematic overview and quality control for scRNAseq A, Breed- ing strategy, Bi,Ci,Di,Ei, Representative FACS gates to sequentially isolate live: dead cells using DAPI: DRAQ5 staining, singlet-gating and TdT+-reporter gating to obtain reporter-positive MGE-derived in- terneurons from frontal cortex and hippocampus. Bii,Cii,Dii,Eii, Barcode Rank Plots for cells from WT and NULL mice, demonstrating separation of cell-associated barcodes and those associated with empty partitions. UMI, unique molecular identifier; MR, Mean Reads; MG, Median Genes. Biii,Ciii,Diii,Eiii, Distributions of the total number of genes, percentage of mitochondrial genes and UMIs per cell in control mice Biv,Civ,Div,Eiv , Pearson correlation coefficient of the distributions of the total number of genes and the UMI

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 27 of 46 USC 105 and is also made available for use under a CC0 license.

A B WT.MGE.CX1 WT.MGE.HPC1 WT.MGE.CX1 WT.MGE.HPC1 Olig1 Gad1 Olig1 Gad1

WT.MGE.CX2 WT.MGE.HPC2 WT.MGE.CX2 WT.MGE.HPC2 Olig1 Gad1 Olig1 Gad1

WT.MGE.CX3 WT.MGE.HPC3 WT.MGE.CX3 WT.MGE.HPC3 Olig1 Gad1 Olig1 Gad1

Biological replicates Biological replicates

Figure 9: (Figure1, Supp.2) | Biological replicates Representative UMAP plots of the 3 biological replicates from cortex and hippocampus indicates A, similar clustering, and B, similar expression pro- files of Nkx2-1 -derived, Gad1 -expressing MGE-derived interneurons and Nkx2-1 derived, Olig1 -expressing oligodendrocytes.

787 [3] L. Tricoire, K. A. Pelkey, B. E. Erkkila, B. W. Jeffries, X. Yuan, C. J. McBain, A Blueprint for the 788 Spatiotemporal Origins of Mouse Hippocampal Interneuron Diversity, Journal of Neuroscience 31 (30) 789 (2011) 10948–10970. doi:10.1523/jneurosci.0323-11.2011. 790 URL https://dx.doi.org/10.1523/jneurosci.0323-11.2011 791 [4] L. Tricoire, K. A. Pelkey, M. I. Daw, V. H. Sousa, G. Miyoshi, B. Jeffries, B. Cauli, G. Fishell, C. J. 792 McBain, Common Origins of Hippocampal Ivy and Nitric Oxide Synthase Expressing Neurogliaform 793 Cells, Journal of Neuroscience 30 (6) (2010) 2165–2176. doi:10.1523/jneurosci.5123-09.2010. 794 URL https://dx.doi.org/10.1523/jneurosci.5123-09.2010 795 [5] F. M. Krienen, M. Goldman, Q. Zhang, R. D. Rosario, M. Florio, R. Machold, A. Saunders, 796 K. Levandowski, H. Zaniewski, B. Schuman (2019). 797 [6] L. Overstreet-Wadiche, C. J. McBain, Neurogliaform cells in cortical circuits, Nature Reviews Neuro- 798 science 16 (8) (2015) 458–468. doi:10.1038/nrn3969.

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Subsets of NGFC1/2 enriched in the HPC

Ai Aii NGFC.1 NGFC.2

PVALB.1 PVALB.2

Subset of PVALB.2 SST.2 enriched in the CX SST.3 Subset of SST.3 TH.1 enriched in the HPC SST.6 Pie Chart MGE.HPC Pie Chart MGE.CX SST.4 SST.5

SST.1

Aiii MGE.CX MGE.HPC SST.3 (16%) SST.4 (13%) SST.3 (17%)

SST.4 (17%) SST.5 (4%) SST.2 (7%) SST.6 (3%) SST.2 (8%)

SST.5 (5%) SST.1 (6%) TH.1 (7%) SST.1 (1%) NGFC.2 (2%) SST.6 (7%) NGFC.1 (3%) PVALB.2 (2%) PVALB.1 (8%) NGFC.2 (18%) PVALB.2 (3%) TH.1 (15%) PVALB.1 (20%) NGFC.1 (17%)

Bi Bii Biii Biv TH.1 TH.1 TH.1 TH.1 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 PVALB.1 PVALB.2 NGFC.1 NGFC.2 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 PVALB.1 PVALB.2 NGFC.1 NGFC.2 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 PVALB.1 PVALB.2 NGFC.1 NGFC.2 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 PVALB.1 PVALB.2 NGFC.1 NGFC.2

Sst Th Pvalb Lamp5

Chodl Pvalb Kcnip2 Cacna2d1

Igf2bp3 Tcap Tcap Sema3c

Cdh7 Shisa8 Pthlh Nos1

Pld5 Sst C1ql1 Reln

Nfix Synpr Syt2 Ngf

Cit Crh Tac1 Egfr

Synpr Nr4a2 Me3 Gabra5

Grin3a Rasgrp1 Erbb4 Necab2

Grm1 Bcl6 Gabrg3 Hapln1

Npas1 Myo1b Cox6a2 Id2

SST-type TH-type PV- NGFC-type type

Figure 10: (Figure1, Supp.3) | Select marker gene expression across the subtypes of merged wt/wt cortical and hippocampal MGE-Grin1 Ai, UMAP representation of cardinal MGE markers genes in the cortical and hippocampal merged dataset. Aii, UMAP representation colored by region, highlighting the region-specific enrichments of MGE subsets. Aiii, Pie chart indicating the percentages of cells recovered across the interneuron subtypes from cortex and the hippocampus. B, Violin plot showing the distribution of expression levels of well-known representative cell-type-enriched marker genes across the 11 MGE subtypes. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 29 of 46 USC 105 and is also made available for use under a CC0 license.

Ai

Bi Bii

Biii Biv

Figure 11: (Figure1, Supp.4) | MGE subtype differences between cortex and hippocampus Volcano plot representing the −log10 False Discovery Rate (FDR) versus log2 fold change (FC) between A, TH-expressing MGE subsets and the remaining MGE subset SST, PVALB and NGFC; B, Differential expression of the cardinal MGE classes between cortex and hippocampus, at a fold change ≥0.5 and FDR <10e-5.

799 URL https://dx.doi.org/10.1038/nrn3969 800 [7] T. Klausberger, P. Somogyi (2008). 801 [8] G. Akgül, C. J. McBain, Diverse roles for ionotropic glutamate receptors on inhibitory in- 802 terneurons in developing and adult brain, The Journal of Physiology 594 (19) (2016) 5471–5490. 803 doi:10.1113/jp271764. 804 URL https://dx.doi.org/10.1113/jp271764 805 [9] R. Chittajallu, J. C. Wester, M. T. Craig, E. Barksdale, X. Q. Yuan, G. Akgül, C. Fang, D. Collins, 806 S. Hunt, K. A. Pelkey, C. J. McBain, Afferent specific role of NMDA receptors for the circuit integration

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity (which wasnotcertifiedbypeerreview)istheauthor/funder.ThisarticleaUSGovernmentwork.Itsubjecttocopyrightunder17 bioRxiv preprint niaigtesbp-enn akrgnsosre ntepeetsuyadterdsrpin nthe in descriptions subcluster). PVALB.2 their cortex-exclusive in and genes the subtype-enriched study in MGE and present expressed of genes the plots the UMAP in Representative (*indicates observed datasets genes scRNAseq marker previous subype-defining the indicating 12: Figure B

Elfn1 Nos1 WT.MGE.CX Grm1 SST.1 Sst UMAP2 SST.4 UMAP1 A D, PVALB.2 PVALB.1 NGFC.2 NGFC.1 SST.6 SST.5 SST.4 SST.3 SST.2 SST.1 TH.1 SST.2 SST.3 VL subclusters. PVALB doi: WT.MGE.HPC Fgr1 Supp.5) (Figure1, Pthlh SST.1 SST.4 D Tcap Kcnc1 Erbb4 Elfn1, Grm1, Kcnip2 Tac1:Pvalb, (Syt2, Th: Sst, Pvalb Sst,Pvalb Th:( PVALB .1 : (C1ql1, Fgf13) Fgf13) : (C1ql1, https://doi.org/10.1101/2020.06.10.144295 Elfn1, Elfn1, Grm1, Nos1, Nos1, Lamp5, Lamp5, Sema3c, Elfn1, Elfn1, Grm1, Lamp5, Lamp5, Sema3c, SST.2 WT.MGE.CX SST.3 Definingmarkers Chodl Snhg11, Snhg11, Meg3 Reln Zbtb20, Zbtb20, , Tacr1, PVALB.2 Reln Crh WT.MGE.CX Reln

Npy , Myh8, Pld5, Mgat4c Olfm3 Reln or SST.4 (Cnr1,Hapln1,Pcp4*) Lpl or Nfix , Myh8, Reln Reln , ) Nppc Nr4a2, Nr4a2, Tac1) Penk USC 105andisalsomadeavailableforuseunderaCC0license. or +, (Rbp4,Sst) SST - SST.2 PVALB.1 , SST.3 , Ngf Ngf Nppc , Npy Npy WT.MGE.HPC Mgat4c + - - type WT.MGE.HPC | G utp noainbsdo akrepeso A, expression marker on based annotation subtype MGE SST.4 PVALB.2 expression + validationfrom other studies : SST.2 SST.3 Putative nomenclature based on marker marker onbased nomenclature Putative GABAergic long GABAergic Fast TH.1

Rbp4 Syt2 Tac1 Pvalb WT.MGE.CX

Crh Nr4a2 Snhg11 O O O Bistratified

Zbtb20 - Axo PVALB .1 spiking /basket spiking bistratified - - - Septum projecting Septum LM / LM / LM / Basket - Bistratified Neurogliaform axonic WT.MGE.CX Martinotti Martinotti Martinotti ; SST.5 SST.6 Ivy this versionpostedJune12,2020. - - chandelier range projection - like PVALB TH.1 - - - WT.MGE.HPC family family family Bistratified - PVALB.1 type SST.5 SST.6 Basket WT.MGE.HPC B, asic s Ta 2016 C

S subclusters, SST Id2 Cacna2d1 Lamp5 Hapln1 NGFC.1 WT.MGE.CX X X X X et al., Paul et al.,et Paul NGFC.2 2017

Hapln1 Epha4 Opn3 Cnr1 X X X X X NGFC.1 WT.MGE.HPC WT.MGE.CX Harris et al.,2018 The copyrightholderforthispreprint X X X X X X X X X X NGFC.2 NGFC Axo PVALB.2 - axonic C, al.,2018 asic s Ta - X X X X X X type

GCsubclusters, NGFC Gabra5 Egfr Ngf Reln et WT.MGE.CX WT.MGE.HPC Hodgeet al.,2019 X X X X X X Axo PVALB.2 Yao et al., al., et Yao WT.MGE.HPC - axonic 2020 X X X X X X Table bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 31 of 46 USC 105 and is also made available for use under a CC0 license.

8 Ai Aii HPC.Grin1fl/fl HPC.Grin1wt/wt

6 2 X 1000X 3 4

1 2 Cells / mm Cells

0 0 P30 P60 P210 P30 P30 Tomato+ PVALB SST

20 Bi Bii CX.Grin1fl/fl CX.Grin1wt/wt 15

X 1000X 10 0.044 0.027 3 10 8 0.001 0.001 6 5 4 Cells / mm Cells 2

0 0 P30 P60 P210 P30 P30 PVALB SST Tomato+

Figure 13: Figure2, Supp.1 | MGE subtype abundances upon Grin1 -ablation A, Boxplots in- dicating the cell counts of hippocampal MGE-derived interneurons expressing (Ai) Ai14/tdTomato, (Aii) PV, SST immunostaining from Grin1 wt/wtand Grin1 fl/fl B, Boxplots indicating the cell counts of corti- cal MGE-derived interneurons expressing (Bi) Ai14/tdTomato, and (Bii) PV, SST immunostaining from Grin1 wt/wtand Grin1 fl/fl. t-test for statistical analysis.

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CX.Grin1wt/wt CX.Grin1fl/fl

Ai B WT.MGE.CX NULL.MGE.CX WT.MGE.HPC NULL.MGE.HPC #cells SST.all 4024 946 1532 1108 PVALB.all 1265 286 401 340 NGFC.all 287 100 1032 743 All.MGE 5576 1332 2965 2191

WT.MGE.CX NULL.MGE.CX WT.MGE.HPC NULL.MGE.HPC Aii #cells SST.1 310 201 26 25 SST.2 992 186 262 256 SST SST.3 667 107 399 420 PVALB SST.4 764 90 143 149 NGFC SST.5 93 39 87 63 SST.6 853 151 505 103 wt/wt fl/fl TH.1 345 172 110 92 HPC.Grin1 HPC.Grin1 SST.all 4024 946 1532 1108

#cells PVALB.1 1112 144 294 226 PVALB.2 115 78 106 107 PVALB.3 38 64 1 7 PVALB.all 1265 286 401 340

#cells NGFC.1 187 85 471 606 NGFC.2 100 15 561 137 NGFC.all 287 100 1032 743 SST PVALB NGFC

C

CX.Grin1wt/wt CX.Grin1fl/fl HPC.Grin1wt/wt HPC.Grin1fl/fl

Figure 14: Figure2, Supp.2 | scRNAseq differential recoveries of MGEsubtypes Ai, Number of cells recovered across cardinal subtypes SST, PV and NGFC. Aii, Number of cells recov- ered within the subtypes of PV / SST/ NGFC. B, UMAP representation colored by cardinal MGE-derived interneuron subtypes SST, PVALB and NGFC, highlighting the differential enrichments of cells C, Rep- resentative UMAP plots indicating the granularity among PV/SST/NGFC subtypes between both brain regions and both genotypes.

807 of hippocampal neurogliaform cells, Nature Communications 8 (1) (2017) 152–152. doi:10.1038/s41467- 808 017-00218-y. 809 URL https://dx.doi.org/10.1038/s41467-017-00218-y 810 [10] J. H. Cornford, M. S. Mercier, M. Leite, V. Magloire, M. Häusser, D. M. Kullmann, Dendritic 811 NMDA receptors in parvalbumin neurons enable strong and stable neuronal assemblies (2019). 812 doi:10.7554/elife.49872. 813 URL https://dx.doi.org/10.7554/elife.49872 814 [11] N. V. D. M. García, R. Priya, S. N. Tuncdemir, G. Fishell, T. Karayannis, Sensory inputs control

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Grin1fl/fl B i PVALB.Pthlh.2 vs PVALB.Pthlh.3

A MGE.CX.Grin1fl/fl MGE.HPC.Grin1fl/fl A i Aii Aiii CX HPC FIGURE 2 Prox1 fl/fl Prox1 fl/fl TH.1 NGFC.1 NGFC.2 PVALB.1 PVALB.2 PVALB.3 A MGE.CX.Grin1 MGE.HPC.Grin1 PVALB.3 SST.1 156 14 TH.1 SST.1 SST.2 SST.3 SST.4 SST.5 SST.6 CX HPCSST.2 43 FIGURE9 2 Prox1 Prox1 Chodl Kcnip2 Kcnip2Hapln1 Hapln1SST.1 156 SST14 .3 258 408 SST.2 43 SST9 .4 101 520 Tcap TcapId2 Id2 Elfn1 PVALB.3 PVALB.3SST.3 258 408SST.5 219 244 SST.4 101 PROX1.1520 30 27 Tac1 Cacna2d1Tac1 Cacna2d1 PVALB.3Npas1 PVALB.3 SST.5 219 244TH.1 139 60 Lamp5 Lamp5PROX1.1 30 PVALB27 .1 3a 255 Cdh7 Pthlh Pthlh SST.2 SST.2 TH.1 139 PVALB60 .2 75 110 Sema3c Sema3c Myh8 C1ql1 C1ql1 PVALB.1 3a PVALB255 .3 68 7 Bii Grin1fl/fl Grin1wt/wt SST.2 SST.2 PVALB.2 75 NGFC110 .1 93 685 Nfix Unc5b Unc5bTrpc5 Trpc5 PVALB.1 PVALB.1 PVALB.3 68 7 PVALB.2 PVALB.2 NGFC.1 93 685 Grin1PVALB.3fl/fl Grin1wt/wt NGFC.1 Etv1 Gabra5Etv1 Gabra5 NGFC.1 NGFC.2 Reln SST.1 SST.1 B D Norm. Tac1 Reln SST.2 SST.2Reln Pthlh Pthlh Pvalb C1ql1 Pvalb C1ql1 expression

fl/fl wt/wt Lamp5 Hapln1 Lamp5 Th Crabp1 Crabp1Ngf Ngf Grin1 Grin1 SST.3 SST.3Hapln1

.1 SST.4 SST.4 SST.1 SST.1 D SST.5 SST.5 0 3 B SST.2 SST.2 Norm. Tac1 SST.6 Norm.expressionSST.6 Expression % Expression% cells SST.3 SST.3 TH.1 TH.1 .1 Prox1 PVALB.1 PVALB.2 TH.1 SST.1 SST.2 SST.3 SST.4 SST.5 NGFC SST.4 PVALB.3 SST.4 PVALB.1 0PVALB.1 3 0 50 110000 SST.5 SST.5 0 3 PVALB.2 PVALB.2 SST.6 SST.6 NGFC.1 NGFC.1% cells TH.1 TH.1 NGFC.2 NGFC.2 PVALB.1 PVALB.2 Prox1 TH.1 SST.1 SST.2 SST.3 SST.4 SST.5 NGFC Gad1 Figure 15:PVALB.3 Figure2, Supp.3 | Select marker gene expressionPVALB.1 acrossPVALB.1 the0 subtypes50 100 of merged wt/wt fl/flPVALB.2 PVALB.2 MGE-derived interneurons from Grin1 and Grin1 NGFC.1A, ViolinNGFC.1 plot showing the distribution Gad1 Gad2 of expression levels of well-known representative cell-type-enrichedNGFC.2 markerNGFC.2 genes across the MGE subtypes. B , −log10 False Discovery Rate (FDR) versus log2 fold change (FC) between Pthlh-PVALB.2 and Pthlh- Lhx6 i Gad2 PVALB.3 at a fold change ≥0.5 and FDR <10e-3. Bii, Dot plots representing the normalized expressions Nkx2.1 of NGFC marker genes mis expressed in Pthlhfl/fl -PVALB.3 upon Grin1 -ablation. Lhx6 Grin1 Sst Nkx2.1 fl/fl 815 the integration of neurogliaformGrin1 interneurons into cortical circuits, Nature Neuroscience 18 (3) (2015) Th Sst 816 393–401. doi:10.1038/nn.3946. Pvalb 817 URL https://dx.doi.org/10.1038/nn.3946 Th 818 [12] J. A. Matta, K. A. Pelkey, M. T. Craig, R. Chittajallu, B. W. Jeffries, C. J. McBain, Developmental Lamp5 819 origin dictates interneuron AMPA and NMDA receptor subunit composition and plasticity, Nature Pvalb 820 Neuroscience 16 (8) (2013) 1032–1041. doi:10.1038/nn.3459. Prox1 821 URL https://dx.doi.org/10.1038/nn.3459 Lamp5 822 [13] R. Luján, R. Shigemoto, G. López-Bendito, Glutamate and GABA receptor signalling in the developing Htr3a Prox1 823 brain, Neuroscience 130 (3) (2005) 567–580. doi:10.1016/j.neuroscience.2004.09.042. Vip 824 URL https://dx.doi.org/10.1016/j.neuroscience.2004.09.042 Htr3a 825 [14] J. B. Manent, Glutamate Acting on AMPA But Not NMDA Receptors Modulates the Migration of Hip- 826 pocampal Interneurons, Journal of Neuroscience 26 (22) (2006) 5901–5909. doi:10.1523/jneurosci.1033- Vip 827 06.2006. 828 URL https://dx.doi.org/10.1523/jneurosci.1033-06.2006 829 [15] J. M. Soria, Receptor-activated Calcium Signals in Tangentially Migrating Cortical Cells, Cerebral 830 Cortex 12 (8) (2002) 831–839. doi:10.1093/cercor/12.8.831. 831 URL https://dx.doi.org/10.1093/cercor/12.8.831 832 [16] E. Hanson, M. Armbruster, L. A. Lau, M. E. Sommer, Z.-J. Klaft, S. A. Swanger, S. F. Traynelis, S. J. 833 Moss, F. Noubary, J. Chadchankar, C. G. Dulla, Tonic Activation of GluN2C/GluN2D-Containing 834 NMDA Receptors by Ambient Glutamate Facilitates Cortical Interneuron Maturation, The Journal 835 of Neuroscience 39 (19) (2019) 3611–3626. doi:10.1523/jneurosci.1392-18.2019. 836 URL https://dx.doi.org/10.1523/jneurosci.1392-18.2019

Mahadevan et al, 2020 NMDAR-mediated transcriptional control of gene expression in the specification of interneuron subtype identity Mahadevan (which wasnotcertifiedbypeerreview)istheauthor/funder.ThisarticleaUSGovernmentwork.Itsubjecttocopyrightunder17 bioRxiv preprint et os yaia ern n non-neurons. and neurons pyramidal rons, A Grin1 16: Figure al, i , 2020 pi-ilnpo rmbt eoye niaigteepeso of expression the indicating genotypes both from plot Split-violin -ablation doi: B iue,Sp. xrsino oto ee nteMEsbye usqetto subsequent subtypes MGE the in genes control of Expression | Supp.4 Figure2, A https://doi.org/10.1101/2020.06.10.144295 Cortical and hippocampal hippocampal and Cortical USC 105andisalsomadeavailableforuseunderaCC0license.

Non-neurons Grin1

GABA.MGE.All expression GLUT.Pyramidal B, Progenitors ersnaieUA lt fcria G akr genes. markers MGE cardinal of plots UMAP Representative ; this versionpostedJune12,2020. HPC. HPC. CX. CX. Nkx2.1:Ai14: Nkx2.1:Ai14: Grin1 Grin1 Grin1 Grin1 fl/fl wt/ fl/fl wt/ wt wt expression Grin1 Grin1 nteMEdrvdinterneu- MGE-derived the in in The copyrightholderforthispreprint NMDAR-mediated fl/fl fl/fl the specification - Mutant of transcriptional interneuron subtype control Page 34 of identity gene of 46 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17Page 35 of 46 USC 105 and is also made available for use under a CC0 license.

A 500 i UP DOWN 400

300

200

100

0

SST.CX NGFC.CXSST.HPC PVALB.CX NGFC.HPC PVALB.HPC

Bi Bii Biii adj P

10 Ci Cii Ciii Log -

Log2 fold change

Figure 17: (Figure3, Supp.1) | Differential gene expression in the MGE subtypes subsequent to Grin1 -ablation Ai, Split-violin plot from both genotypes indicating the expression of Grin1 in the MGE- derived interneurons, pyramidal neurons and non-neurons. A ii , Bar plot denoting the number of genes up/downregulated in the cortical and hippocampal MGE clusters. Volcano plot representing the −log10 False Discovery Rate (FDR) versus log2 fold change (FC) between B, hippocampal and C,cortical MGE cardinal clusters upon Grin1 -loss, at a fold change ≥0.2 and FDR <10e-6.

837 [17] W. Kelsch, Z. Li, S. Wieland, O. Senkov, A. Herb, C. Gongrich, H. Monyer, GluN2B-Containing 838 NMDA Receptors Promote Glutamate Synapse Development in Hippocampal Interneurons, Journal 839 of Neuroscience 34 (48) (2014) 16022–16030. doi:10.1523/jneurosci.1210-14.2014. 840 URL https://dx.doi.org/10.1523/jneurosci.1210-14.2014 841 [18] G. Akgül, C. J. McBain, AMPA receptor deletion in developing MGE-derived hippocampal interneu- 842 rons causes a redistribution of excitatory synapses and attenuates postnatal network oscillatory ac- 843 tivity, Scientific Reports 10 (1) (2020) 1333–1333. doi:10.1038/s41598-020-58068-6. 844 URL https://dx.doi.org/10.1038/s41598-020-58068-6

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SST.CX SST.HP A A C i PV.CX SST.CX PV.HPC SST.HPC ii 142 214 145 (28%) (43%) (29%) 80 106 140 (12%) 130 123 (19%) 64 (20%) (19%) (22%) (11%) PV.CX PV.HP 69 154 C 70 (10%) 75 39 (27%) 35 (10%) (11%) (7%) (6%) 169 189 234 (29%) (32%) (40%) 122 52 (18%) (9%) NGFC.CX NGFC.HPC

NGFC.CX NGFC.HPC 208 130 150 (43%) (27%) (31%)

B 6.0e-09 Neuroactive ligand-receptor interaction (39/348) 9.9e-11 Nicotine addiction (15/40) 8.8e-09 CAMP signaling pathway (29/210) 7.8e-07 Ca2+ signaling pathway (24/188) 2.6e-10 Long-term potentiation (18/66) 4.1e-10 Gap junction (20/86) 5.3e-07 Neuroactive peptide secretion (15/74) 1.8e-07 Growth hormone secretion (16/78) 2.0e-08 Growth factor secretion (18/86) 2.0e-11 Circadian entrainment (23/98) 3.1e-14 Glutamatergic synapse (28/113) 3.3e-11 Dopaminergic synapse (26/131) 2.0e-08 Amphetamine addiction (16/67) 8.6e-08 Oxytocin signaling pathway (23/153) 2.5e-10 Cholinergic synapse (23/112) 4.5e-12 Morphine addiction (23/91) 5.7e-17 GABAergic synapse (28/89) 4.3e-13 Retrograde endocannabinoid signaling (30/146) 3.0e-07 MAPK signaling pathway (32/294) C 1.5e-07 Synaptic vesicle cycle (16/77)

50 SST.CX PVALB.CX NGFC.CX SST.HPC PVALB.HPC NGFC.HPC 40

30

20

# of of genes # 10

0 Membrane Gene Synaptic Axon Kinase, Cytoskeleton, Ca2+ Neuropeptide, Vesicle Energetics Second excitability expression organizers Guidance, Phosphatase modifiers homeostasis GPCR release (35) Messengers (78) (67) (61) migration (49) (49) (47) signaling (36) (33) (53) (46)

Figure 18: (Figure3, Supp.2) | Molecular pathways differentially expressed in MGE subtypes Ai, Venn-diagrams indicating the percentages of DEGs common within MGE subtypes from cortex or hippocampus. A i,Venn-diagrams indicating the percentages of DEGs common within MGE subtypes from cortex and hippocampus. B, Hierarchical clustering tree summarizing the correlation among significant pathways enriched among the DEGs. Pathways with many shared genes are clustered together. Bigger dots indicate more significant P-values. C, Bar plot showing the classification of molecular functions of the DEGs, across the MGE subtypes. Total number of DEGs in the particular molecular class indicated in parentheses. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

CX HPC CX HPC CX HPC A B C Legends: Log2 normalized differential expression in MGE-Grin1fl/fl; FDR<0.01

SST PVALB NGFC second messengers second homeostasissignalingand 2+ kinase and phosphatase signaling phosphatase and kinase Misexpressed Subdued Ca Subdued Misexpressed

Figure 19: (Figure4, Supp.1) | Differential expression of intracellular signaling cascades across subtypes upon Grin1 -ablation Heatmap of log2 FC of significant DEGs in cortical and hippocam- pal MGE cardinal subtypes, showing a subset of A, genes regulating intracellular Ca2+ homeostasis and Ca2+ binding proteins; B, notable second messengers; and, C, Ca2+ dependent / activated kinases and phos- phatases.

845 [19] D. Bortone, F. Polleux, KCC2 Expression Promotes the Termination of Cortical Interneu- 846 ron Migration in a Voltage-Sensitive Calcium-Dependent Manner, Neuron 62 (1) (2009) 53–71. 847 doi:10.1016/j.neuron.2009.01.034. 848 URL https://dx.doi.org/10.1016/j.neuron.2009.01.034 849 [20] C. L. Magueresse, H. Monyer, GABAergic Interneurons Shape the Functional Maturation of the 850 Cortex, Neuron 77 (3) (2013) 388–405. doi:10.1016/j.neuron.2013.01.011. 851 URL https://dx.doi.org/10.1016/j.neuron.2013.01.011 852 [21] N. V. D. M. García, T. Karayannis, G. Fishell, Neuronal activity is required for the development of 853 specific cortical interneuron subtypes, Nature 472 (7343) (2011) 351–355. doi:10.1038/nature09865. 854 URL https://dx.doi.org/10.1038/nature09865 855 [22] M. Yozu, H. Tabata, N. König, K. Nakajima, Migratory Behavior of Presumptive Interneurons Is 856 Affected by AMPA Receptor Activation in Slice Cultures of Embryonic Mouse Neocortex, Develop- 857 mental Neuroscience 30 (1-3) (2008) 105–116. doi:10.1159/000109856. 858 URL https://dx.doi.org/10.1159/000109856 859 [23] O. Marín, Interneuron dysfunction in psychiatric disorders, Nat. Rev. Neurosci 13 (2012) 107–120. 860 [24] K. Nakazawa, K. Sapkota, The origin of NMDA receptor hypofunction in schizophrenia, Pharmacology 861 & Therapeutics 205 (2020) 107426–107426. doi:10.1016/j.pharmthera.2019.107426. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.10.144295; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Post-syn B Neurotransmitter A i Release machinery NGFC Pre-syn

S. lacunosum moleculare HPC Post-syn

S. radiatum PVALB CX Pre-syn * * * Sz Sz Sz Sz Sz Sz Sz Sz Sz Sz Sz Sz Sz PYR

S. pyramidale GABAergic synapse Glutamatergic Kcn / Scn families synapse CX HPC CX HPC SST Pre-syn CX HPC

Post-syn S. oriens Sz Sz* Sz

Sz As Sz Legends: Log2 normalized differential Sz * expression in MGE-Grin1fl/fl; FDR<0.01 Sz Sz Sz As Sz Sz As Sz SST Sz: Schizophrenia risk Sz PVALB As: Autism risk As Sz* NGFC : Both Sz and As * Sz * Sz Sz Sz

Misc. excitability As regulators Sz CX HPC As

Bii Postsynaptic / neurotransmitter receptor Sz Sz and ion channel Sz complexes Sz

Sz

Figure 20: (Figure6, Supp.1) | Aberrant NMDAR-signaling result in misexpression of regu- lators of membrane excitability that are high-risk Sz genes A, Schema representing the field of hippocampal pyramidal cell innervated by the interneurons. Heatmap of log2 FC of significant DEGs in cortical and hippocampal MGE cardinal subtypes, showing a subset of Bi, Neurotransmitter release ma- chinery. B ii, Postsynaptic GABA / Glutamate receptor complexes, Kcn-, Scn- ion channel complexes and other miscellaneous regulators of excitability.

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