bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Deficiency of intellectual disability-related Brpf1 reduced inhibitory

2 neurotransmission and Map2k7 expression in GABAergic interneurons

3 Jingli Cao1, Weiwei Xian1, Maierdan Palihati1, Yu Zhu1, Guoxiang Wang2, Yunli Xie2,

4 Guomin Zhou1,3#, Linya You1#

5

6 1Department of Human Anatomy & Histoembryology, School of Basic Medical

7 Sciences, Fudan University, Shanghai 200032, China

8 2Institutes of Brain Sciences, Fudan University, Shanghai 200032, China

9 3Key Laboratory of Medical Imaging Computing and Computer Assisted Intervention

10 of Shanghai, Shanghai 200032, China

11

12 #Correspondences to: [email protected]; [email protected].

13

14 Conflict of interest: The authors declare no conflicts of interest.

15

16 Running Title: Brpf1 loss reduced neurotransmission

17

18 Keywords: intellectual disability; Brpf1; MGE-derived GABAergic interneurons;

19 differentiation; dendritic morphology, electrophysiology; in vivo migration;

20 mRNA-Seq

21

22 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

23 Abstract

24 Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1

25 was specifically expressed in medial ganglionic eminence (MGE), a developmental

26 niche of GABAergic interneurons, and patients with BRPF1 mutations were mentally

27 retarded. To test its role in development and function of MGE-derived GABAergic

28 interneurons, we performed immunofluorescence staining, whole-cell patch-clamp,

29 MGE transplantation and mRNA-Seq to understand its effect on neuronal

30 differentiation, dendritic morphology, electrophysiology, migration and gene

31 regulation, using mouse MGE-derived GABAergic interneurons infected with

32 AAV-shBrpf1. We found a decreasing trend on parvalbumin+ interneuron

33 differentiation. Moreover, increased firing threshold, decreased number of evoked

34 APs, and a reduced amplitude of mIPSCs were observed before any significant

35 change of MAP2+ dendritic morphology and in vivo migration appeared. Finally,

36 mRNA-Seq analysis revealed that related to neurodevelopment and synaptic

37 transmission such as Map2k7 were dysregulated. Our results demonstrated a key role

38 of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic

39 interneurons.

40

41 Introduction

42 Intellectual disability (ID) usually refers to a series of neurodevelopmental

43 disorders that are obviously impaired in intelligence and adaptive function due to

44 brain hypoplasia or organic damage[1, 2], affecting approximately 2-3% of the total bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

45 population[3], with a prevalence of up to 2% in children or adolescents[4, 5].

46 Intellectual disability not only causes a heavy burden on patients and families, but

47 also brings a series of problems in public health, society and education. At present,

48 studies have shown that there are around 1396 genes related to ID[6], and

49 bromodomain and PHD finger-containing 1 (BRPF1) is one of them. In the

50 last few years, a total of 40 clinical cases of mental retardation caused by de novo or

51 inherited BRPF1 mutations have been reported[7-11] . Yan et al. reported that 10

52 patients with BRPF1 mutations had symptoms such as developmental delay, language

53 expression disorder and mental retardation[7]. Another team found 6 patients with

54 BRPF1 mutations whose clinical manifestations were growth retardation, sagging

55 eyelid and cerebellar malformations[8]. Recent studies have further confirmed that

56 patients with BRPF1 mutations often exhibited clinical symptoms such as ID, general

57 developmental delay, gross motor delay, facial and ocular deformities (such as

58 blepharoptosis and ptosis)[9-11].

59 BRPF1 is a unique epigenetic regulatory factor, which contains two PHD fingers,

60 one bromodomain and one PWWP domain. BRPF1 can recognize different epigenetic

61 markers and activate three histone acetyltransferases MOZ (also known as KAT6A),

62 MORF (also known as KAT6B) and HBO1[12-14]. Our previous work indicated that

63 mouse Brpf1 plays a key role in early embryo development[15], forebrain

64 development[16, 17], and embryonic hematopoietic stem cell maintenance[18].

65 Global knockout mice were lethal before E9.5 with defects such as closure,

66 angiogenesis, and placental development[15]. Forebrain-specific loss of Brpf1 led to bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

67 early postnatal lethality and growth retardation[17], and it has also been found that

68 loss of Brpf1 can regulate neuronal migration, cell cycle progression and

69 transcriptional control, resulting in abnormal hippocampal morphogenesis[16]. Other

70 group reported that heterozygous loss of Brpf1 led to reduced dendritic complexity in

71 both hippocampal granular cells and cortical pyramidal neurons[19].

72 ID is closely related to the imbalance of inhibitory/excitatory neural circuits,

73 especially the imbalance of inhibitory circuits[20-22]. GABAergic interneurons are

74 the major cellular units of the inhibitory neural circuit[23, 24], and are mainly

75 produced in the medial and caudal ganglionic eminence (MGE and CGE) and preoptic

76 area (PoA) of the telencephalon[25, 26]. After differentiation, GABAergic

77 interneurons migrate tangentially to the neocortex, with some of them to the cortical

78 marginal zone[27]. MGE-derived GABAergic interneurons are mainly differentiated

79 into somatostatin (SST)+ and parvalbumin (PV)+ interneurons[26, 28], and growth

80 number reaches a peak at E13.5[27]. GABAergic interneurons stay on the MGE for a

81 period of time, then mostly tangentially migrate at E15.5-E16.5. Our previous work

82 showed that mouse Brpf1 is specifically expressed in MGE[29]. Relatedly, the haploid

83 mutation of Brd1 (also called Brpf2) significantly reduced the number of PV+

84 interneurons in the anterior cingulate cortex of mouse[30].We hypothesized that Brpf1

85 regulates MGE-derived GABAergic interneurons and affects inhibitory neural circuits,

86 which will eventually lead to abnormal learning, memory and cognitive ability.

87 To test this, we applied mouse MGE-derived GABAergic interneurons and

88 AAV-shBrpf1 to examine the effect of Brpf1 deficiency on the differentiation, bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

89 dendritic morphology, electrophysiological properties, migration, and gene expression

90 of MGE-derived GABAergic interneurons by immunofluorescence staining,

91 whole-cell patch-clamp, MGE cell transplantation and mRNA-seq. This study will

92 help us understand the key role of Brpf1 on the morphology and function of

93 GABAergic interneurons, and provide us with new insights into the pathogenesis of

94 ID caused by BRPF1 mutations.

95

96 Results

97 Brpf1 deficiency led to a mild decreased trend on the differentiation of PV+

98 interneurons

99 To study the effect of Brpf1 knockdown on the differentiation of MGE-derived

100 GABAergic interneurons, we dissected mouse MGEs from E14.5 embryos for

101 primary culture of GABAergic interneurons. Neurons were infected with

102 AAV-scramble-GFP or AAV-shBrpf1-GFP at DIV3, and immunolabeling was

103 performed at DIV14-15. We confirmed a knockdown efficiency of approximately 40%

104 by RT-qPCR (Figure 1D). Then the proportion of GABA+GFP+, PV+GFP+ and

105 SST+GFP+ neurons in GFP+ neurons was calculated (Figure 1). The results showed

106 that the ratio of GABA+GFP+ neurons in GFP+ neurons reached almost 100%,

107 indicating that the majority of the cultured MGE-derived neurons were GABAergic

108 interneurons (Figure 1A and 1E). GABAergic interneurons are mainly differentiated

109 into SST and PV interneurons[26, 28]. Thus, we examined the ratio of PV+ GFP+ or

110 SST+GFP+ in total GFP+ neurons (Figure 1B-1C, 1F-1G), and found little effect on bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

111 SST+ neuronal differentiation but a mild decreased trend on PV+ neuronal

112 differentiation upon Brpf1 knockdown.

113 Brpf1 deficiency had little effect on the dendritic morphology of MGE-derived

114 GABAergic interneurons

115 The dendrites of neurons are essential for neuronal functions. Studies have shown

116 that the complexity of neurites is closely related to some development-related

117 diseases[31-33]. To study the effect of Brpf1 knockdown on the dendritic morphology

118 of MGE-derived GABAergic interneurons, we dissociated E14.5 MGEs and infected

119 primary cultured neurons with AAV-scramble-GFP or AAV-shBrpf1-GFP.

120 Immunostaining was performed at DIV 14-15 with anti-MAP2 antibody (a specific

121 marker for dendrites) and the quantification was calculated from 3 independent

122 experiments, totally 33 and 37 neurons in the AAV-Scramble and AAV-shBrpf1 group

123 were analyzed, respectively, using Sholl analysis[34]. The results showed that there

124 was no significant difference in the number of intersections and total length of

125 dendrites between AAV-scramble and AAV-shBrpf1 groups, indicating that Brpf1

126 knockdown had little effect on the dendritic morphology of MGE-derived GABAergic

127 interneurons.

128 Brpf1 deficiency attenuated inhibitory neurotransmission by decreasing mIPSC

129 amplitude and increasing firing threshold

130 Patients with ID usually have reduced learning and memory abilities, delay of

131 which is closely related to synaptic dysfunction[35-37]. Synaptic dysfunction is

132 usually determined by the characteristics of cell membranes and synaptic bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

133 transmission between neurons. To assess the functional consequences of Brpf1

134 knockdown on synaptic transmission, we performed whole-cell patch clamp to

135 record the electrophysiological properties of MGE-derived GABAergic interneurons

136 at DIV 15 after AAV-scramble or –shBrpf1 infection at DIV3 (Figure 3). For

137 inhibitory neurotransmission, we measured miniature inhibitory postsynaptic

138 current (mIPSC). Each neuron was recorded for 6 minutes and the signal of the later

139 3 minutes was selected for statistical analysis (Figure 3A, a short fragment of the

140 signal was shown). The amplitude but not the frequency of mIPSC decreased

141 significantly (Figure 3B-3C). For membrane characteristics, we measured resting

142 membrane potential (RMP), action potential (AP), and evoked AP. RMP had little

143 change (Figure 3D). Due to few or no spontaneous APs after many trials, we

144 recorded evoked APs. The AAV-shBrpf1 group required a larger incident current to

145 induce the evoked APs, with a significant increase in the firing threshold (Figure

146 3E), although the maximum frequency of evoked APs was not affected (Figure 3F).

147 More specifically, when the evoked APs were induced against a serial step of

148 increasing currents, there was a significant decrease in the number of spikes at

149 current 130pA, 170pA and 190pA (Figure 3G). All the results indicated that Brpf1

150 knockdown attenuated inhibitory neurotransmission of MGE-derived GABAergic

151 interneurons.

152 Brpf1 deficiency had little effect on GABAergic interneuron migration in vivo

153 Neurons after birth migrate to specific locations and form a network with

154 surrounding cells to perform their specific functions. In order to study the localization bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

155 and migration ability of MGE-derived GABAergic interneurons in the cortex upon

156 Brpf1 knockdown, we adopted a transplantation assay[38, 39]. Briefly, E13.5 MGE

157 cells were infected with AAV-scramble-GFP or AAV-shBrpf1-GFP, then injected into

158 the cortex of P1 wildtype mice by micro-syringe (Figure 4A). The transplanted cells

159 were allowed to develop in vivo for 35 days (a time point at which mature

160 interneuronal markers were expressed) and the proportion of GFP+ cells in the cortex

161 was analyzed (Figure 4B-4D). First, we analyzed the deep (V and VI) and superficial

162 (I-IV) layers of the cortex, and found the proportion of GFP+ cells showed no

163 significant difference upon Brpf1 knockdown (Figure 4C). Upon further dividing

164 cortex layers into layer I, II-IV, V and VI, the ratio of GFP+ cells in each layer still had

165 little change (Figure 4D). Thus, the mild knockdown of Brpf1 did not affect the

166 neuronal migration and cortical localization of MGE-derived GABAergic

167 interneurons.

168 Brpf1 deficiency led to dysregulated MAPK pathway via Map2k7 in MGE-derived

169 GABAergic interneurons

170 To study the molecular mechanism of Brpf1 knockdown leading to changes in

171 electrophysiological properties of MGE-derived GABAergic interneurons, three

172 batches of RNA from AAV-scramble and AAV-shBrpf1 groups were extracted for

173 mRNA-Seq. The number of fragments per kilobase fragment (FPKM) of the

174 transcript per thousand fragments was used to calculate the expression of the known

175 gene. DESeq2 software was used to screen the differentially expressed genes (DEGs)

176 between different sample groups. DEGs with p < 0.05 and log2 (fold change) >=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

177 were considered to be up-regulated genes. Similarly, DEGs with p < 0.05 and log2

178 (fold change) <= -1 were considered to be down-regulated genes. 24 and 22 genes

179 were upregulated and downregulated, respectively, upon Brpf1 knockdown (Figure

180 5A and Table S1).

181 GO and KEGG enrichment analysis of these genes revealed that most of the 24

182 up-regulated genes were involved in gene negative transcription and inflammatory

183 response (Figure 5B and Table S1). Among them, Tceal7, Mecom, Vdr, Lrch4 were

184 involved in negative regulation of transcription, and Hrh1, Mecom, Stab1 in

185 inflammatory response. Vdr and Hrh1 were verified by RT-qPCR (Figure 5C).

186 However, for the 22 downregulated genes, no pathways were significantly enriched.

187 To further explore the underlying molecular mechanism, we selected most

188 neuron-related genes among the 46 DEGs (24 plus 22) and validated by RT-qPCR

189 (Figure 5C). For downregulated DEGs, SHROOM4 was implicated in neural

190 development, as its mutations were associated with human X-linked mental

191 retardation[40]; ion transport related genes such as Asic3[41], Ak4[42], and Slpr5[43],

192 can promote the release of cations to the outside of the cell, and thus a greater current

193 stimulation is required to induce an AP; Map2k7 is a key gene that activates MAPK

194 signaling pathway[44], Morf (one of the three KATs Brpf1 binds) can also specifically

195 regulate this pathway[45]. Moreover, Map2k7 heterozygous mice showed insufficient

196 prefrontal cortex-dependent working memory[44, 46], consistent with the learning

197 and memory impairment in Brpf1 mutant mice[19]; Nlrp6 is part of Nlrp6

198 inflammasome and plays a crucial role in innate immunity and inflammation. Nlrp6 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

199 could induce apoptosis to regulate the survival of neurons[47].Interestingly, study

200 found that the space exploration ability and associative learning of Nlrp6-/- male mice

201 were weakened[48]. We validated that Map2k7 and Nlrp6 were significantly

202 decreased upon Brpf1 knockdown (Figure 5C). Of note, Both Map2k7 heterozygous

203 and tissue-specific knockout mice showed symptoms related to mental retardation,

204 such as cognitive dysfunction and motor dysfunction (see discussion for details) [44,

205 49], which is similar as that of Brpf1 knockout. For upregulated DEGs,

206 overexpression of Rnf170 inhibited IP3 receptor[50], which plays a key role in cell

207 signaling; overexpression of transporter genes such as Vdr[51], Kctd20[52], affects

208 the assembly of some cation channels protein, and eventually leads to changes in

209 membrane ion permeability.

210

211 Discussion

212 Accumulating clinical studies has reported a total of 40 cases of patients with

213 BRPF1 monoallelic mutations, with symptoms such as mental retardation,

214 developmental delay and epilepsy[7-11]. Impaired GABA neurotransmission is

215 associated with mental disorders such as epilepsy and mental retardation. Our

216 previous work has found that Brpf1 global knockout is embryonically lethal[15], and

217 Brpf1 forebrain-specific knockout showed hypoplasia in , corpus

218 callosum and cortex[16, 17]. Moreover, we found that Brpf1 is specifically expressed

219 in MGE at E14.5[29], a niche where most GABAergic interneurons were born. To

220 study monoallelic loss effect of Brpf1, we mildly knocked down Brpf1 in bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

221 MGE-derived GABAergic interneurons with the knockdown efficiency of about 40%

222 (Figure 1D). Interestingly, this study found that Brpf1 mild knockdown led to changes

223 in electrophysiology (Figure 3) and PV+ neuronal differentiation (Figure 1) before any

224 influence showed in dendritic morphology (Figure 2) and neuronal migration (Figure

225 4). More specifically, Brpf1 mild knockdown can sensitively cause attenuated

226 inhibitory neurotransmission by decreasing mIPSC amplitude and increasing firing

227 threshold of evoked APs (Figure 3). The underlying mechanism may involve

228 dysregulated MAPK pathway via Map2k7, as indicated by mRNA-Seq (Figure 5).

229 We found that the p value of the comparison of PV+ neurons is 0.056, suggesting a

230 decreased trend of MGE-derived GABAergic interneurons to differentiate into PV+

231 interneurons upon Brpf1 knockdown. Consistently, Brd1 (also known as Brpf2)

232 heterozygous mice also displayed a decrease in the number of PV+ interneurons[30,

233 53]. Moz and Morf (two of the three enzymes Brpf1 binds) were involved in

234 regulating [54-56]. Morf mutant mice showed reduced ability of neural

235 stem cells/progenitor cells differentiating into neurons, astrocytes, and

236 oligodendrocytes, but not limited to specific neuronal types[54]. In our study, we

237 observed a decreased trend of PV+ interneuronal differentiation upon mild Brpf1

238 reduction. Further confirmation with a more efficient knockdown/knockout in vivo

239 will be needed to study Brpf1’s effect on PV+ interneuronal differentiation.

240 Dysregulation of excitatory/inhibitory neuronal circuits was closely related to

241 various neurodevelopmental disorders[57-59] such as Parkinson, intellectual disability,

242 epilepsy, schizophrenia, etc. Our study found that insufficient Brpf1 led to abnormal bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

243 synaptic transmission in MGE-derived GABAergic interneurons with reduced

244 amplitude of mIPSC and increased firing threshold of evoked AP. Consistently,

245 previous studies showed that Brpf1 forebrain-specific (by Emx1-Cre) heterozygotes

246 had increased firing threshold of AP, a downward trend of spike number, reduced

247 mEPSC frequency and amplitude in hippocampus[19]. In addition, Brd1+/- pyramidal

248 neurons showed decreased frequency of spontaneous IPSCs and mIPSCs[60]. These

249 results indicated that membrane properties and functional maturation of MGE-derived

250 GABAergic interneurons were sensitive to Brpf1 dose even with mild knockdown.

251 Interestingly, the mild knockdown dose of Brpf1 in our study only led to significant

252 changes in electrophysiology properties before any major influence showed in

253 dendritic morphology and neuronal migration. Thus we performed mRNA-Seq

254 analysis to mainly explain the change in electrophysiology. The analysis revealed that

255 Map2k7 and Nlrp6 genes were significantly downregulated. Relatedly, MOZ

256 knockdown led to a decrease in JNK(a key component of MAPK signaling pathway)

257 phosphorylation[56], and patients with JNK mutations were detected with ID[61].

258 MORF disruption leads to a Noonan syndrome-like mainly via

259 hyperactivated MAPK signaling in human and mice[45]. Map2k7 also plays an

260 important role in the regulation of MAPK signaling pathway[44]. In addition, Map2k7

261 heterozygous mice exhibited cognitive dysfunction[44] and neuron-specific Map2k7

262 knockout exhibited motor dysfunction such as muscle weakness and abnormal

263 walking[49]. For Nlrp6, Nlrp6-/- mice showed symptoms related to mental

264 retardation[48]. The mechanism of how Brpf1 regulates Map2k7 expression merits bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

265 further investigation.

266 This study helps us understand the key roles of Brpf1 in differentiation, dendritic

267 morphology, electrophysiological characteristics, migration and gene expression of

268 MGE-derived GABAergic interneurons from a neurobiological level, and provides us

269 with new insights into the pathogenesis of ID related to BRPF1 mutations.

270

271 References

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437

438 Materials and Methods

439 Animals

440 C57BL/6 mice were used in this study and were purchased from Shanghai Slac

441 Laboratory Animal CO., LTD. The day when vaginal plug is detected was considered

442 to be embryonic day 0.5 (E0.5). All experimental animals were kept in an animal

443 facility at Fudan University and all experiments were conducted in accordance with

444 guidelines approved by Fudan University.

445 Primary Neuron Culture, Immunofluorescence, and Quantification

446 Primary GABAergic interneurons were prepared from E14.5 wild-type pregnant

447 mice. MGEs were isolated from the embryonic brains in ice-cold HBSS

448 (Thermo,14185052) with 1% HEPES (Thermo, 15630106) and the MGEs were

449 transferred to a 15 ml centrifuge tube for digestion with 3ml HBSS/HEPES containing

450 100 μg / ml DNase (Sigma, D4527) and 0.05% trypsin (Sigma, T8658-1vl).

451 Centrifuge tube was transferred to a 37℃ water bath for 15min and gently shaked

452 every 3-5 minutes. 5 ml Neurobasal medium (Gibco, 211030049) supplemented with

453 10% FBS (Gibco, 10091148), 2% B27 supplement (Gibco, 17504044), and 1%

454 GlutaMAX-1(Gibco, 35050061) was added to stop the digestion. The mixed liquid

455 was centrifuged at 800 rpm for 5 minutes and the supernatant was removed.

456 Neurobasal medium supplemented with 10% FBS, 2% B27 supplement, and 1%

457 GlutaMAX-1 was added again. The neurons were then dissociated by pipetting, and bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

458 the suspension was centrifuged at 1000 rpm for 5 minutes. Cells were then

459 resuspended, plated at a density of 2-5 × 104 cells / well on coverslips in a 24-well

460 plate pre-coated with poly-L-lysine (Sigma, P9155) and cultured in a Neurobasal

461 medium supplemented with 2% B27 supplement, and 1% GlutaMAX-1.

462 Neurons were infected with AAV-scramble-GFP or AAV-shBrpf1-GFP virus at

463 DIV3. The shBrpf1 sequence was shown in Table 1. After DIV10, GFP-expressing

464 neurons could be gradually observed under a fluorescent microscope. At DIV14-15,

465 immunofluorescent staining was performed to quantify the number of GABAergic

466 interneurons that differentiated into PV+ or SST+ interneurons. Briefly, neurons were

467 fixed in 4% paraformaldehyde (Biosharp, BL539A) in PBS (Hyclone, SH30243.01)

468 for 20 minutes, permeabilized with 0.1% Triton X-100 (Sigma, X-100) in PBS for 1

469 hour, and blocked with 10% goat serum (Proteintech, b900780) in PBS for 1 hour at

470 room temperature. Neurons were then incubated with primary antibodies at 4 ℃

471 overnight and second antibodies for 1 hour. The primary antibodies used were mouse

472 anti-MAP2 (Proteintech, 67015-1-ig, 1:1000), rabbit anti-GFP (Proteintech,

473 50430-2-AP, 1:500), mouse anti-GABA (Sigma, A0310, 1:200), rabbit anti-PV

474 (Proteintech, 26521-1-ap, 1:500), rabbit anti-SST (Proteintech, 17512-1-ap, 1:500),

475 and mouse anti-GFP (Proteintech, 66002-1, 1:500). Neurons were finally incubated

476 with DAPI (Sigma, D9542) at room temperature for 10 minutes and mounted with

477 antifade mounting medium.

478 To study the effect of Brpf1 knockdown on the differentiation of GABAergic

479 interneurons, we used ImageJ Fiji software to manually count the number of GABA+ bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

480 GFP+, SST+GFP+, PV+GFP+ neurons and total number of GFP+ neurons, the

481 proportion of co-labeled neurons in GFP+ neurons was then calculated.

482 For analysis of dendritic morphology, coverslips were observed under an inverted

483 fluorescence microscope (Olympus) and confocal microscope (leica SP8). The images

484 were analyzed using ImageJ Fiji. To calculate the branches and the total length of

485 dendrites, Sholl analysis was applied. Simple Neurite Tracer plug-in was used to track

486 the path of the neuron, and the radius was increased to cover the entire path of the

487 neurons with the cell body as the center and a fixed value of 10um. The Sholl analysis

488 plug-in was used to calculate the number of intersections between the ring and the

489 neurite. The total length of dendrites was also displayed when tracing the path of

490 neurites.

491 Electrophysiological Recordings and analysis

492 For whole-cell patch-clamp recording, neurons were infected with a reduced titer of

493 AAV-scramble-GFP or AAV-shBrpf1-GFP virus at DIV3 to achieve sparse labeling

494 and the recordings were performed at DIV15. Neurons expressing GFP under

495 high-resolution fluorescent microscope were chosen for electrophysiological

496 recordings. Briefly, coverslips covered with neurons were transferred to a holding

497 chamber attached to the microscope stage, containing normal-temperature artificial

498 cerebrospinal fluid (ACSF). Electrodes with electrode solution were used to patch

499 neurons under the fluorescent microscope. To characterize the intrinsic membrane

500 properties of neurons, current clamp recordings were performed and hyperpolarized

501 and depolarized current steps were injected in 20 pA increments. The following bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

502 parameters were measured to characterize the neuronal membrane characteristics: the

503 resting membrane potential was recorded immediately after the neuronal membrane

504 was ruptured, and the number of APs induced after different current injections was

505 measured. The AP current threshold is defined as the first rectangular current injection

506 that causes a spike. Offline analysis was performed using Clampfit software. To study

507 the synaptic transmission between neurons, mIPSC was recorded. 1 μM tetrodotoxin,

508 10 μM NBQX and 50 μM D-AP5 were added to ACSF. The same method to patch the

509 neurons was applied and the data was recorded for 6 minutes. The amplitude and

510 frequency were analyzed by mini-analysis software.

511 RT-qPCR

512 All the following experiments were RNase-free. The total RNA was extracted by

513 the traditional Trizol (Thermo, 15596018) method(Groppe and Morse 1993),

514 dissolved in a suitable volume of RNase-free water, and the RNA concentration was

515 determined using a Nanodrop. The RNA was then reverse-transcribed using a reverse

516 transcription kit (Accurate Biotechnology Co., Ltd, AG11705). The cDNA solution

517 was subjected to RT-qPCR using TB Green Premix Ex Taq (Takara, rr420a) and prime

518 script rt master mix (Takara, rr036a). Gapdh was used as the internal control, and the

519 relative expression of the gene was calculated by 2-ΔΔCt method[62]. The primer

520 sequences were shown in Table 1.

521 mRNA sequencing and bioinformatic analyses

522 The original Sequencing data were analyzed using previously published

523 methods[63, 64].The total RNA was extracted using Trizol method and the RNA bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

524 integrity was determined using RNA Nano chip and Agilent 2100 bioanalyzer[65].

525 The samples were subjected to high-throughput sequencing through the paired-end

526 sequencing mode of the Illumina HiSeq sequencing platform. For each sample, the

527 pre-processing sequence was compared with the mouse genome sequence (release-98)

528 by STAR software after removing the linker sequence fragments and low-quality

529 fragments. For all samples, StringTie software was used to count the original

530 sequence counts of known genes, and the expression of known genes was calculated

531 using fragments per kilobase of transcript per million fragments mapped (FPKM).

532 The DESeq2 software was used to screen the differentially expressed genes between

533 different sample groups, and the differential expression range of 1 <= log2 (Fold

534 Change), log2 (Fold Change) <= -1 and P value < 0.05 was used to screen the

535 differentially expressed genes between the two groups. The function of differentially

536 expressed genes were analyzed by David[66, 67] website (https://david.ncifcrf.gov/),

537 including (GO) and kyoto encyclopedia of genes and genomes

538 (KEGG). The raw and processed data were deposited in Gene Expression Omnibus

539 database with GEO# 157903.

540 MGE cell transplantation and evaluation of migration behavior

541 MGEs from E13.5 wild-type embryonic brains were harvested and dissociated. The

542 cell suspensions were incubated with AAV-scramble-GFP or AAV-shBrpf1-GFP for 2

543 hours at 37°C, during which the liquid was gently mixed every half hour. Each group

544 had about 50,000 cells. The mixed liquid was centrifuged at 800 rpm for 5 minutes

545 and then the supernatant was removed. The centrifugation step was repeated 2 to 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

546 times. The pellet was resuspended in 10ul DMEM supplemented with 10% FBS. The

547 cell suspension was then injected into the neocortex of wild-type P1 host.

548 Transplanted neurons were permitted to migrate in vivo for 35 days before analysis.

549 On day 35, mice were anesthetized, perfused with PBS followed by 4% PFA

550 through the endocardium, and then the brain was carefully dissected. The brain was

551 incubated overnight in 4% PFA, dehydrated in 30% sucrose in PBS until the sample

552 sank to the bottom, embedded with optimum cutting temperature compound (O.C.T.,

553 Sakura,4583), and sectioned at a thickness of 20 μm. Rabbit anti-GFP (Proteintech,

554 1:500) and DAPI (1:1000) were used for immunofluorescence staining. The

555 proportion of GFP+ cells in each layer of the cortex was analyzed after that the stained

556 sections were photographed by confocal (leica SP8).

557 Statistical Analysis

558 Data were analyzed statistically using ImageJ Fiji and GraphPad Prism 8.0 software.

559 Differences between groups were tested by T test. The values of p <0.05 was

560 considered statistically significant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

561

562 Data and reagent availability

563 Gene expression data are available at GEO with the accession number:

564 GEO157903.

565

566 Acknowledgements

567 We thank members from Dr. Linya You’s lab for their support and help during the bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

568 experiment. This work was supported by the National Natural Science Foundation of

569 China (81771228) to Dr. Linya You and National Natural Science Foundation of

570 China (31571238) to Dr. Guomin Zhou.

571 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

572 Figure 1 Brpf1 knockdown led to a mild reduced trend on the differentiation of PV+

573 neurons from MGE-derived GABAergic interneurons

574 (A, B, C). Representative immunofluorescent images at DIV15, which were

575 co-labeled with GABA and GFP, PV and GFP, SST and GFP antibodies, respectively.

576 Scale bar, 100 µm. (D). Quantitative mRNA analysis showed that knockdown

577 efficiency of Brpf1 reached approximately 40% (AAV-scramble group, n = 5 batches;

578 AAV-shBrpf1 group, n = 5 batches; p<0.001). (E, F, G). The ratio of GABA+GFP+,

579 PV+GFP+, SST+GFP+ neurons in total GFP+ neurons upon Brpf1 knockdown was

580 quantified respectively. (E). n = 43 and 52 fields for AAV-scramble and -shBrpf1,

581 respectively, p=0.5875; (F). n = 55 and 50 fields for AAV-scramble and -shBrpf1,

582 respectively, p=0.0556; (G). n=49 and 45 fields for AAV-scramble and -shBrpf1,

583 respectively, p=0.7486). n was the number of fields, and the average cell count per

584 field was about 25 neurons.

585 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

586 Figure 2 Brpf1 knockdown had no significant effect on the dendritic morphology of

587 MGE-derived GABAergic interneurons

588 (A). Representative immunofluorescent images of MGE-derived GABAergic

589 interneurons immunostained with mouse anti-MAP2 and rabbit anti-GFP antibodies.

590 Scale bar, 75 µm. (B). The mean number of intersections between dendrites and

591 concentric circles with different radii (AAV-scramble group, n = 33 neurons;

592 AAV-shBrpf1 group, n = 37 neurons). (C) Comparison of mean total length of

593 neuronal dendrites (AAV-scramble group, n = 33 neurons; AAV-shBrpf1 group, n = 37

594 neurons; p=0.5651).

595 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

596 Figure 3 Brpf1 deficiency led to attenuated inhibitory neurotransmission of

597 MGE-derived GABAergic interneurons

598 (A) Representative traces of mIPSC in GABAergic interneurons at DIV 15 between

599 the AAV-scramble and AAV-shBrpf1 groups. 25pA and 100ms were shown. (B)

600 Statistical comparison of averaged mIPSC amplitude in GABAergic interneurons

601 (AAV-scramble group, n = 6 neurons; AAV-shBrpf1 group, n = 5 neurons; p=0.0495).

602 (C) Statistical comparison of averaged mIPSC frequency (AAV-scramble group, n = 6

603 neurons; AAV-shBrpf1 group, n = 5 neurons; p=0.2172). (D) Comparable resting

604 membrane potentials were observed (AAV-scramble group, n = 19 neurons;

605 AAV-shBrpf1 group, n = 22 neurons; p=0.9481). (E) The averaged firing threshold to

606 induce evoked APs were compared (AAV-scramble group, n = 19 neurons;

607 AAV-shBrpf1 group, n = 22 neurons; p=0.0193). (F) The maximum frequency of

608 evoked APs showed little change (AAV-scramble group, n = 19 neurons;

609 AAV-shBrpf1 group, n = 22 neurons; p=0.4430). (G) The number of evoked APs was

610 induced against depolarizing current steps of increasing amplitude starting from -70 to

611 210 pA. (AAV-scramble group, n = 19 neurons; AAV-shBrpf1 group, n = 22 neurons;

612 p= 0.03972 at 130pA; p= 0.0072 at 170pA; p= 0.0452 at 190pA).

613 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

614 Figure 4 Brpf1 deficiency had no effect on the neuronal migration of MGE-derived

615 GABAergic interneurons in vivo.

616 (A) Schematic diagram of MGE cell transplantation. In short, MGE neurons

617 dissociated from E13.5 embryonic brains were infected with GFP-carrying

618 adeno-associated virus (AAV-scramble-GFP or AAV-shBrpf1-GFP) and transplanted

619 into the P1 neocortex. The proportion of GFP+ neurons in each cortical layer were

620 analyzed after 35 days. (B) Representative images of GFP+ neurons in each layer of

621 cortex analyzed at day 35 after transplantation. Arrows indicated GFP+ neurons. Scale

622 bar: 50μm. (C) Quantitative analysis of the proportion of GFP+ neurons in deep (V

623 and VI) and superficial (I-IV) cortical layer, respectively (AAV-scramble, n=9 mice;

624 AAV-shBrpf1, n=9 mice). (D) Quantitative analysis of the ratio of GFP+ neurons in

625 layer I, II-IV, V, and VI, respectively (AAV-scramble, n=9 mice; AAV-shBrpf1, n=9

626 mice).

627 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

628 Figure 5 Brpf1 deficiency led to decreased Map2k7 expression in MGE-derived

629 GABAergic interneurons revealed by mRNA-Seq

630 (A). Volcano plot of DEGs from mRNA-Seq, log2(fold change) <= -1 or >= 1 was set

631 as significant downregulation or upregulation (AAV-scramble group, n = 3;

632 AAV-shBrpf1 group, n = 3). (B). GO-analysis of 24 upregulated and 22

633 downregulated genes revealed by mRNA-Seq. The number of genes involved in each

634 Go term was indicated. (C). Validation of selected DEGs from mRNA-Seq by

635 RT-qPCR. n indicated pairs of scramble and shBrpf1 groups validated by RT-qPCR. *,

636 p< 0.05, ***, p< 0.001. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table 1 Primer sequence

Primer name Primer sequence

shBrpf1 sense GGCTTACCGCTACTTGAACTT

antisense AAGTTCAAGTAGCGGTAAGCC

Gapdh F AGGTCGGTGTGAACGGATTTG

R TGTAGACCATGTAGTTGAGGTCA

mBrpf1 F ATGCAGCTGACCCCTTTCCT

R CACTGCTGCCCTGTAGAAGA

Rnf170 F AGAAGGAGTGAGCGACCAAGTC

R CTCGAAGCACTCTGACTAGCTC

Vdr F GCTCAAACGCTGCGTGGACATT

R GGATGGCGATAATGTGCTGTTGC

Hrh1 F AAGGTGACCTCTCCGACTGTCT

R TGGCTCAAGGTCTGGTCATTGG

Cldn19 F GCACTCGGGTTGGAGACAGTAA

R GGTAGCATACCAGGAGACAGCA

Pcdhgb1 F CTCAGGATCTCCTGTGCGATGA

R GGCTTGAGAGAAACGCCAGTCA

Sec1 F GGCGAATATGCCACGCTGTTTG

R CCTTTTGGCTGTGTCGCTATGC

Fgf2 F AAGCGGCTCTACTGCAAGAACG

R CCTTGATAGACACAACTCCTCTC bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Kctd20 F CCACTCAAGAGAAAGAAGGCAGC

R TTGCTGGCTGAGAGGCATAGTC

Rcvrn F CTTCTCGCTCTACGACGTAGAC

R TCTTCTCAGCCCGCTTTTCTGG

Shroom4 F TACAGGTTGCTGTCACGGTCCT

R TTGGCTTCCACTTGTCCACAGG

Map2k7 F TCAGGTGTGGAAGATGCGGTTC

R AAGGGCAGTCATGGCTCTTGAG

Asic3 F TATGTGGCTCGGAAGTGCGGAT

R CAGACACAAGTGTCCTTTCGCAG

Jph2 F CAACCAGGAGTCCAACATCGCC

R GCTCTCAGAGTTCTCCAGGATC

Prickle4 F AAGAAGGACCCGATCCAAGC

R AGGGTTTCCAGGGTGAGAGT

Ak4 F GAAGCAGTTGCTGCCAGGCTAA

R GCCAGATTCTGTTAGTCTCCGTC

S1pr5 F AGACTCCTCCAACAGCTTGCAG

R TAGAGCTGCGATCCAAGGTTGG

Nlrp6 F CTGGCGTCATTGTGGAACCTCT

R TCTCACTCAGCTCCACAGAGGT

bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table S1 Up- and down-regulated genes by mRNA-Seq Gene_ID Symbol Fold Change Overlap P value regulated ENSMUSG00000026857 Ntmt1 2.0299 0.0290 Up ENSMUSG00000041966 Dcaf17 2.0306 0.0072 Up ENSMUSG00000004031 Brinp2 2.1446 0.0031 Up ENSMUSG00000051550 Zfp579 2.1872 0.0082 Up ENSMUSG00000013878 Rnf170 2.3315 0.0090 Up ENSMUSG00000031163 Glod5 2.5318 0.0327 Up ENSMUSG00000079428 Tceal7 2.6004 0.0277 Up ENSMUSG00000006463 Zdhhc24 2.6018 0.0000 Up ENSMUSG00000093445 Lrch4 2.6850 0.0076 Up ENSMUSG00000078713 Tomm5 2.7962 0.0144 Up ENSMUSG00000005936 Kctd20 2.8236 0.0002 Up ENSMUSG00000025997 Ikzf2 2.9329 0.0016 Up ENSMUSG00000066058 Cldn19 3.0572 0.0402 Up ENSMUSG00000074570 Cass4 3.6390 0.0136 Up ENSMUSG00000049580 Tsku 3.9736 0.0000 Up ENSMUSG00000103037 Pcdhgb1 4.0694 0.0392 Up ENSMUSG00000027684 Mecom 4.1236 0.0118 Up ENSMUSG00000022479 Vdr 4.4849 0.0354 Up ENSMUSG00000042286 Stab1 4.6919 0.0427 Up ENSMUSG00000037225 Fgf2 4.7842 0.0495 Up ENSMUSG00000053004 Hrh1 6.5097 0.0001 Up ENSMUSG00000040364 Sec1 6.7241 0.0214 Up ENSMUSG00000062157 Ifnlr1 7.6755 0.0238 Up ENSMUSG00000098387 Pet117 28.4467 0.0010 Up ENSMUSG00000098754 Prn 0.1009 0.0118 Down ENSMUSG00000053158 Fes 0.1161 0.0341 Down ENSMUSG00000036832 Lpar3 0.1337 0.0305 Down ENSMUSG00000043017 Ptgir 0.1485 0.0413 Down ENSMUSG00000038276 Asic3 0.1553 0.0116 Down ENSMUSG00000071724 Smpd5 0.1589 0.0462 Down ENSMUSG00000050103 Agmo 0.1628 0.0468 Down ENSMUSG00000017817 Jph2 0.1703 0.0213 Down ENSMUSG00000047686 Zcchc5 0.1741 0.0467 Down ENSMUSG00000109061 Map2k7 0.1926 0.0013 Down ENSMUSG00000061311 Rag1 0.2006 0.0329 Down ENSMUSG00000096549 Prickle4 0.2132 0.0056 Down ENSMUSG00000028527 Ak4 0.2359 0.0154 Down ENSMUSG00000020907 Rcvrn 0.2986 0.0404 Down ENSMUSG00000033306 Lpp 0.3095 0.0244 Down ENSMUSG00000020088 Sar1a 0.3121 0.0000 Down ENSMUSG00000045087 S1pr5 0.3154 0.0116 Down bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426233; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ENSMUSG00000027401 Tgm3 0.3343 0.0208 Down ENSMUSG00000038745 Nlrp6 0.4239 0.0473 Down ENSMUSG00000004233 Wars2 0.4546 0.0478 Down ENSMUSG00000032384 Csnk1g1 0.4587 0.0034 Down ENSMUSG00000068270 Shroom4 0.4615 0.0284 Down