Single-Cell RNA-Seq of Mouse Olfactory Bulb Reveals Cellular Heterogeneity and Activity-Dependent Molecular Census of Adult-Born Neurons
題目：小鼠嗅球的單細(xì)胞rna-seq揭示了細(xì)胞的異質(zhì)性和成體神經(jīng)元活性依賴的分子普查
作者及單位：
Burak Tepe, Matthew C. Hill, Brandon T. Pekarek, Patrick J. Hunt, Thomas J. Martin, James F. Martin, Benjamin R. Arenkiel
1 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
發(fā)表刊物及時(shí)間：
December 4, 2018, Cell Reports Vol. 25, Issue 10, p2689–2703.e3
Highlights
- ?Single-cell sequencing reveals cellular heterogeneity in the mouse olfactory bulb
- ?Differential gene expression uncovers selective markers for cell types
- ?Pseudotemporal ordering of adult-born neurons reveals developmentally governed genes
- ?Olfactory experience changes the cellular composition of olfactory bulb circuits
亮點(diǎn)：
?單細(xì)胞測序揭示了小鼠嗅球的細(xì)胞異質(zhì)性
?差異基因表達(dá)揭示細(xì)胞類型的選擇性標(biāo)記
?從出生到成體的神經(jīng)元的偽時(shí)間排序揭示了發(fā)育上控制的基因
?嗅覺經(jīng)歷改變了嗅球回路的細(xì)胞組成
Summary
Cellular heterogeneity within the mammalian brain poses a challenge toward understanding its complex functions. Within the olfactory bulb, odor information is processed by subtypes of inhibitory interneurons whose heterogeneity and functionality are influenced by ongoing adult neurogenesis. To investigate this cellular heterogeneity and better understand adult-born neuron development, we utilized single-cell RNA sequencing and computational modeling to reveal diverse and transcriptionally distinct neuronal and nonneuronal cell types. We also analyzed molecular changes during adult-born interneuron maturation and uncovered developmental programs within their gene expression profiles. Finally, we identified that distinct neuronal subtypes are differentially affected by sensory experience. Together, these data provide a transcriptome-based foundation for investigating subtype-specific neuronal function in the olfactory bulb (OB), charting the molecular profiles that arise during the maturation and integration of adult-born neurons and how they dynamically change in an activity-dependent manner.
摘要
哺乳動(dòng)物腦細(xì)胞的異質(zhì)性對于理解其復(fù)雜功能來說一直是一種 挑戰(zhàn)， 在嗅球中扼褪， 氣味信息由抑制性中間神經(jīng)元的亞型處理砂碉， 其異質(zhì) 性和功能被不斷形成的成體神經(jīng)發(fā)生所影響五辽。 為了研究其細(xì)胞異質(zhì)性以及 更好的理解 adult-born 神經(jīng)元發(fā)育過程眯牧， 我們利用單細(xì)胞 RNA 測序 和計(jì)算模型來揭示不同的和轉(zhuǎn)錄不同的神經(jīng)元和非神經(jīng)元細(xì)胞類型蹋岩。 我們還分析了 adult-born 中間神經(jīng)元成熟期間的分子變化以及在其基 因表達(dá)譜中發(fā)現(xiàn)的發(fā)育程序。 最后炸站， 我們發(fā)現(xiàn)不同的神經(jīng)元亞型受感 覺經(jīng)驗(yàn)的不同影響星澳。 總之疚顷， 這些數(shù)據(jù)提供了基于轉(zhuǎn)錄組的基礎(chǔ)旱易， 用于 研究嗅球（OB） 中的亞型特異性神經(jīng)元功能， 繪制成人出生神經(jīng)元 的成熟和整合期間出現(xiàn)的分子譜以及它們?nèi)绾我曰顒?dòng)依賴性方式動(dòng) 態(tài)變化腿堤。
圖表選析
Single-Cell Sequencing Establishes a Molecular Census of Olfactory Bulb Cells To elucidate the overall cellular heterogeneity and activity dependent changes in olfactory bulb composition, we profiled the transcriptomes of 51,246 single cells collected from the olfactory bulbs of wild-type adult mice (Figure 1A). Mice were naive, olfactory deprived through naris occlusion, or olfactory enriched through training on an olfactory-discrimination learning paradigm. To block olfactory sensory input, we performed unilateral naris occlusion, with the occluded side serving as the sensory-deprived sample and the open side as the control (Najbauer and Leon, 1995, Quast et al., 2017, Yamaguchi and Mori, 2005). Mice were trained to discriminate various odorants using an olfactory-cued learning paradigm (Liu et al., 2017, Liu et al., 2018). This form of olfactory training exposed mice to several different odorants while also actively engaging the olfactory system to facilitate olfactory-discrimination learning. Cells from naive, olfactory-deprived, and enriched mice were clustered together after single-cell sequencing based on similarities in their transcriptional profiles using an unsupervised principal-component analysis (Macosko et al., 2015) and visualized using t-distributed stochastic neighbor embedding (t-SNE) (Van der Maaten, 2014, Van Der Maaten and Hinton, 2008) (Figure 1B). We identified 38 distinct cellular clusters, each composed of cells from different olfactory experience paradigms, indicating that experimental conditions did not bias the identity of the clusters (Figure S1). Using the expression patterns of cluster-enriched genes, we next assigned identities to each cluster. In total, we observed 16 neuronal (Syt1+/Tubb3+), three astrocytic (Gfap+), five olfactory ensheathing cell-based (Sox10+), six hematopoietic (all Aif1+; three Siglich+ microglia, one CD52+ macrophage, one CD74+ monocyte, and one Hba-a1+ red blood cell), four blood-vessel-based (two Slco1c1+ endothelial and two Pdgfrb+ mural), one oligodendrocyte-precursor-based (Olig2+), one myelinating-oligodendrocyte-based (Mag+), and two mesenchymal clusters (Figures 1B and 1D). Together, these data reveal the overall transcriptional heterogeneity of the cell types that comprise the mammalian olfactory bulb and identify molecular markers to further investigate the diversity and function of olfactory bulb cell types. Transcriptome-Based Clustering of Neurons Identifies Markers of Neuronal Subtypes To assign specific identities to neuronal subtypes within the olfactory bulb, we filtered and subclustered neurons identified from initial clustering (Figure 1B, Neuron01–Neuron16). Neurons were initially identified utilizing gene enrichment data (Figure 1C; Table S1) by selective expression of known neuronal markers such as Syt1 and Tubb3.
單細(xì)胞測序建立了嗅球細(xì)胞的分子普查 為了闡明嗅球組成的整體細(xì)胞異質(zhì)性和活性依賴性變化阀坏，我們分 析了從野生型成年小鼠的嗅球中收集的 51,246 個(gè)單細(xì)胞的轉(zhuǎn)錄 組（圖 1A）。通過鼻竇阻塞或通過嗅覺辨別學(xué)習(xí)范例的訓(xùn)練豐富 嗅覺笆檀，小鼠是缺乏嗅覺經(jīng)歷的忌堂； 通過單側(cè)鼻孔閉塞導(dǎo)致嗅覺剝奪； 或者通過嗅覺識別學(xué)習(xí)模型的訓(xùn)練酗洒，豐富了嗅覺士修。為了阻斷嗅覺 感官的輸入，我們進(jìn)行了單側(cè)鼻孔閉塞樱衷，閉塞側(cè)作為感覺缺失的 樣本棋嘲，開放側(cè)作為對照(Najbauer and Leon, 1995, Quast et al., 2017, Yamaguchi and Mori, 2005)。 訓(xùn)練小鼠區(qū)分各種氣味使用嗅覺暗 示的學(xué)習(xí)模型(Liu et al., 2017, Liu et al., 2018)矩桂。這種形式的嗅覺 訓(xùn)練使小鼠暴露于幾種不同的氣味沸移，同時(shí)還積極地參與嗅覺系統(tǒng) 以促進(jìn)嗅覺辨別學(xué)習(xí)。 來自嗅覺無經(jīng)驗(yàn)，嗅覺剝奪和豐富嗅覺經(jīng) 驗(yàn)小鼠的細(xì)胞在單細(xì)胞測序后基于其轉(zhuǎn)錄譜中的相似性雹锣，使用無 監(jiān)督的主成分分析聚集在一起(Macosko et al., 2015)网沾，并使用 t 分 布隨機(jī)鄰域嵌入（t-SNE）可視化(Van der Maaten, 2014, Van Der Maaten and Hinton, 2008)（圖 1B）。 我們鑒定了 38 個(gè)不同的細(xì)胞 簇蕊爵，每個(gè)由來自不同嗅覺經(jīng)驗(yàn)?zāi)Ｐ偷募?xì)胞組成辉哥，表明實(shí)驗(yàn)條件對 聚類的識別沒有偏倚（圖 S1）。接下來在辆，我們使用簇富集的基因 的表達(dá)模式证薇，為每個(gè)群集分配了身份。 總共匆篓，我們觀察到 16 個(gè) 神經(jīng)元（Syt1 + / Tubb3 +）浑度， 3 個(gè)星形細(xì)胞（Gfap +）， 5 個(gè)嗅鞘細(xì) 胞（Sox10 +）鸦概， 6 個(gè)造血（所有都是 Aif1 + ; 3 個(gè) Siglich+ 小膠質(zhì) 細(xì)胞箩张， 1 個(gè) CD52+ 巨噬細(xì)胞，一個(gè) CD74+ 單核細(xì)胞和一個(gè) Hbaa1+ 紅細(xì)胞）窗市，四個(gè)基于血管的（兩個(gè) Slco1c1+ 血管內(nèi)皮和兩個(gè) Pdgfrb+ 外壁）先慷，一個(gè)基于少突膠質(zhì)細(xì)胞前體（Olig2 +），一個(gè)基 于髓鞘-少突膠質(zhì)細(xì)胞（Mag +）和兩個(gè)間充質(zhì)簇（圖 1B 和 1D）咨察。 總之论熙，這些數(shù)據(jù)揭示了構(gòu)成哺乳動(dòng)物嗅球的細(xì)胞類型的整體轉(zhuǎn)錄 異質(zhì)性，并鑒定分子標(biāo)記以進(jìn)一步研究嗅球細(xì)胞類型的多樣性和 功能摄狱。 基于轉(zhuǎn)錄組的神經(jīng)元聚類識別神經(jīng)元亞型的標(biāo)記 為了將特定身份分配給嗅球內(nèi)的神經(jīng)元亞型脓诡，我們過濾并從初始 聚類中鑒定出亞群集神經(jīng)元（圖 1B， Neuron01-Neuron16）媒役。最初 通過選擇性表達(dá)已知的神經(jīng)元標(biāo)志物如 Syt1 和 Tubb3祝谚，利用基 因富集數(shù)據(jù)（圖 1C ; 表 S1）鑒定神經(jīng)元。
Figure 1Single-Cell Transcriptome Analysis Delineates Mouse Olfactory Bulb Cellular Heterogeneity
圖1. 單細(xì)胞轉(zhuǎn)錄組測序揭示小鼠嗅 球細(xì)胞異質(zhì)性
(A) Schematic view of the experimental workflow.
(B) Cellular composition of the olfactory bulb was visualized using t-distributed stochastic neighbor embedding (t-SNE). Individual single-cell transcriptomes were colored according to cluster identity in (B)–(D).
(C) Dendrogram depicting hierarchical relationships between distinct cell populations. Heatmap illustrating the genes most highly enriched in each cluster, with each column representing a gene and each row representing average expression level of that gene in each cluster.
(D) Graph showing number of cells per cluster, number of unique molecular identifiers (UMIs) per cluster (mean ± SEM; scale is in thousands), and number of genes detected per cluster (mean ± SEM; scale is in thousands). Violin plots show expression of cell-type-specific marker genes for each cluster.
(A) 實(shí)驗(yàn)流程的示意圖 (B) 用 t-SNE 可視化嗅球的細(xì)胞組成酣衷。 根據(jù)（B） –（D） 中的簇 同一性對每個(gè)單細(xì)胞轉(zhuǎn)錄本進(jìn)行著色交惯。 (C) 描述不同細(xì)胞群體之間的層次關(guān)系的樹狀圖。 熱圖顯示了每 個(gè)簇中富集程度高的基因穿仪， (D) 每一列代表一個(gè)基因席爽， 每一行代表該基因在每個(gè)簇中的平均 表達(dá)水平。 (E) 顯示每個(gè)簇的細(xì)胞數(shù)啊片、 每個(gè)簇的獨(dú)特分子標(biāo)識符(UMI)的數(shù) 目(平均±SEM只锻； 標(biāo)度為千)的圖表， 以及每簇檢測到的基因（平均 ±SEM钠龙； 規(guī)模以千計(jì)） 炬藤。 小提琴圖顯示了每個(gè)簇的細(xì)胞類型特異性標(biāo) 記基因的表達(dá)御铃。
Figure 3Pseudo-Timeline Analysis Reveals Transcriptional Changes during Maturation and Integration along Distinct Developmental Axes
(A) Schematic sagittal view of the mouse olfactory system. Inset: summary diagram of olfactory bulb adult-neurogenesis illustrating broad morphological and developmental changes throughout maturation of adult-born neurons. LV, lateral ventricle; RMS, rostral migratory stream. 小鼠嗅覺系統(tǒng) 的示意圖矢狀位圖。內(nèi)嵌：嗅球成體神經(jīng)發(fā)生簡圖沈矿，說明成年出生神經(jīng)元在成熟過程中的廣泛形態(tài)和發(fā) 育變化上真。左室，側(cè)腦室羹膳；均方根睡互，吻移流 2018/12/25 Untitled Document 7/16
(B) (Top left) Monocle2 pseudotime trajectory of adult-born neurons. Cells are colored by pseudotime score, with dark colors representing immature cell stages and light colors representing mature cell stages. (Top right) Monocle2 pseudotime trajectory of adult-born neurons with cells colored by cluster identity according to Figure 2. (Bottom) Adult-born neuron cluster density plot projected to the x axis of the bifurcating Monocle2 pseudotime trajectory, indicating which arm of the timeline each cell type is located. (左上角)成年神經(jīng)元的單時(shí)間假時(shí)間軌跡。細(xì)胞用假時(shí)間記分染色陵像， 深色代表不成熟的細(xì)胞階段就珠，淺色代表成熟的細(xì)胞期。(右上)按圖2所示醒颖，帶有團(tuán)簇同一性的細(xì)胞的成年 出生神經(jīng)元的單時(shí)間假時(shí)間軌跡妻怎。(底部)成年神經(jīng)元團(tuán)簇密度圖投射到分叉單時(shí)間軌跡的x軸，指示每種 細(xì)胞類型的時(shí)間線的哪個(gè)臂泞歉。
(C) (Top) Axis A of Monocle2 pseudotime trajectory colored according to cluster identity. (Bottom) Axis A pseudotime trajectory colored by pseudotime score, with the dark color representing an immature cell stage and the light color representing a mature cell stage. (頂)軸a為單點(diǎn)2偽時(shí)軌跡逼侦， 按簇同一性著色。(下)軸用假時(shí)間分?jǐn)?shù)著色的偽時(shí)間軌跡腰耙，暗顏色代表不成熟的細(xì)胞階段榛丢，淺色代表成 熟的細(xì)胞期。
(D) Axis A: 4 distinct groups of pseudotime-dependent genes with dynamic expression patterns plotted across pseudotime as heatmaps, with blue indicating low levels and red indicating high levels of expression. (Middle) Gene expression trends for each gene (black) with the trend line highlighted in red. (Right) Top 6 enriched gene ontology (GO) terms for each temporal cluster. 軸a：4組具有動(dòng) 態(tài)表達(dá)模式的假時(shí)相關(guān)基因作為熱圖挺庞，藍(lán)色表示低水平晰赞，紅色表示高表達(dá)水平。(中)每個(gè)基因(黑色)的 基因表達(dá)趨勢选侨，趨勢線以紅色突出顯示掖鱼。(右)前6個(gè)豐富基因本體論(GO)術(shù)語為每個(gè)時(shí)間簇。
(E) Differential expression patterns of one example gene from each group of genes along developmental axis A. 一例基因與每組基因沿發(fā)育軸a的差異表達(dá)模式侵俗。
(F) (Top) Axis B of Monocle2 pseudotime trajectory colored according to cluster identity. (Bottom) Axis B pseudotime trajectory colored by pseudotime score, with the dark color representing an immature cell stage and the light color representing a mature cell stage. (頂部)單點(diǎn)2偽時(shí)軌跡的軸b 按簇同一性著色锨用。(下)軸b偽時(shí)間軌跡用偽時(shí)間記分著色丰刊，暗色代表不成熟細(xì)胞期隘谣，淺色代表成熟細(xì)胞 期。
(G) Axis B: 4 distinct groups of pseudotime-dependent genes, with dynamic expression patterns plotted across pseudotime as heatmaps. Blue indicates low levels and red indicates high levels of expression. (Middle) Gene expression trends for each gene (black), with the trend line highlighted in red. (Right) Top 6 enriched gene ontology (GO) terms for each temporal cluster. 軸b：4組不同的假 時(shí)間依賴基因啄巧，其動(dòng)態(tài)表達(dá)模式以熱圖的形式繪制寻歧。藍(lán)色代表低層次，紅色代表高表達(dá)水平秩仆。(中)每個(gè) 基因(黑色)的基因表達(dá)趨勢码泛，趨勢線以紅色突出。(右)前6個(gè)豐富基因本體論(GO)術(shù)語為每個(gè)時(shí)間簇澄耍。
(H) Differential expression patterns of one example gene from each group of genes along developmental axis B. See also Tables S3 and S4. 一例基因與每組基因沿發(fā)育軸b的差異表達(dá)模式噪珊。 另見表S3和S4
Figure 5. Olfactory Activity Alters Adult-Born Interneuron Subtype Composition (A) Schematic view of olfactory bulb experimental procedures, which are detailed in methods. (B)Two-dimensionalt-SNE representation of 16, 302 adult-born interneurons colored according to experimental group. OC, narisoccluded; TR, olfactory trained; WT, wild-type. (C) t-SNE representation of 16,302 adult-born interneurons colored according to cluster identity. (D) Shifts in adult-born neuron cluster composition for each indicated experimental condition plotted across expression-based t-SNE (left and middle) and along the Monocle2 pseudotime trajectory (right). Pearson’s chi-square test residuals were calculated for the corresponding experimental group (left). Discrete values were determined from Pearson’s chi-square test (middle); if the p value was < 0.05, then clusters were assigned appropriate designation of increased or decreased (red or blue) based on their residual score. Clusters with no significant composition shift are highlighted in gray (no change, p value > 0.05).
圖 5.嗅覺活動(dòng)改變成人新生中間神經(jīng)元亞型組成 (A)嗅球?qū)嶒?yàn)過程詳細(xì)方法示意圖晌缘。 (B)根據(jù)實(shí)驗(yàn)組著色的 16， 302 例成人新生中間神經(jīng)元的二維 SNE 表示痢站。 OC磷箕，麻醉閉塞； TR阵难， 嗅覺訓(xùn)練岳枷； WT，野生型呜叫。 (C)依據(jù)聚類特征著色的 16空繁， 302 個(gè)成人新生中間神經(jīng)元的 t-SNE 表示。 (D)在基于表達(dá)的 t-SNE(左邊和中間)上和單峰 2偽時(shí)間軌跡(右)旁繪制的每個(gè)指示實(shí)驗(yàn)條件的 成人新生神經(jīng)元簇組成的變化朱庆。 計(jì)算相應(yīng)實(shí)驗(yàn)組(左邊)的皮爾遜(Pearson)卡方檢驗(yàn)殘值盛泡。 離 散值由 Pearson‘s 卡方檢驗(yàn)(中間)確定，如果 p 值<0.05娱颊，則根據(jù)其殘值給聚類分配適當(dāng)?shù)脑?減值(紅或藍(lán))饭于。無明顯組分變化的簇呈灰色(無變化， p 值>0.05)维蒙。
翻譯小組：
李碧琪掰吕、王俊豪、陳志榮颅痊、黃敬潼殖熟、陳凱星、黃子亮斑响、鄭凌伶
