The landscape of regulatory genes in brain-wide neuronal phenotypes of a vertebrate brain

  1. Hui Zhang
  2. Haifang Wang
  3. Xiaoyu Shen
  4. Xinling Jia
  5. Shuguang Yu
  6. Xiaoying Qiu
  7. Yufan Wang
  8. Jiulin Du  Is a corresponding author
  9. Jun Yan  Is a corresponding author
  10. Jie He  Is a corresponding author
  1. Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, China
  4. School of Future Technology, University of Chinese Academy of Sciences, China
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Molecular classification of whole-brain cells in larval zebrafish.

(A) The t-distributed stochastic neighbor embedding (t-SNE) plot of 45,746 single-cell transcriptomes pooled from whole brains (n = 4) and four different individual brain regions (n = 2 each). The …

Figure 1—source data 1

Bioinformatics processing of raw reads of single-cell samples.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig1-data1-v3.xlsx
Figure 1—source data 2

The annotation of 68 clusters of whole-brain sample.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig1-data2-v3.xlsx
Figure 1—source data 3

The regional origins and neurotransmitter-type annotation of each whole-brain cluster with well-known markers.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig1-data3-v3.xlsx
Figure 1—source data 4

Top 20 marker genes of whole-brain larval zebrafish 68 clusters.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig1-data4-v3.xlsx
Figure 1—source data 5

Marker genes of major six cell type in whole brain.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig1-data5-v3.xlsx
Figure 1—figure supplement 1
Molecular classification of whole-brain cells in larval zebrafish brain.

(A) The schematic showing each samples of whole brain and different brain regions. (B) t-Distributed stochastic neighbor embedding (t-SNE) plot of pooled single-cell transcriptome data from …

Figure 1—figure supplement 2
Molecular classification of whole-brain cells in larval zebrafish brain.

(A) t-Distributed stochastic neighbor embedding (t-SNE) plots showing the expression (in red) of specific markers (eomesa, foxg1a, dlx5a, pitx2 as markers for the forebrain; tal1, en2a as markers …

Figure 2 with 1 supplement
Molecular classification of neuromodulator-type neurons.

(A) The schematic showing the procedure of collecting single-cell transcriptomes of neuromodulator neurons with fluorescence-activated cell sorting (FACS). Using Tg (ETvmat2:GFP) fishline, we could …

Figure 2—source data 1

The annotation of neuromodulator-type neuronal types with well-known markers.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Molecular classification of neuromodulator neurons.

(A) The JaccardRainCloundPlot showing the stability of vmat2+ neuromodulator clusters in Figure 2B. Red line indicated the 0.6 cutoff of Jaccard index to evaluate the cluster stability. Red …

Figure 3 with 4 supplements
The transcription factor (TF) regulatory landscape in whole-brain neuronal clusters.

(A) Schematic showing the strategies to assess the cluster similarity based on effector gene and TF profiles. We focused on clusters of whole-brain glutamatergic/GABAergic neurons and neuromodulator …

Figure 3—figure supplement 1
Hierarchical clustering analysis of whole-brain glutamatergic/GABAegric neurotransmitter-type neurons based on effector gene and transcription factor (TF) profiles.

(A) Hierarchical clustering of 39 glutamatergic/GABAegric neurotransmitter-type neurons based on 1099 effector gene profiles in highly variable genes. Red boxes were highlighted to indicate the …

Figure 3—figure supplement 2
The transcription factor (TF) regulatory landscape in whole-brain glutamatergic/GABAergic neuronal clusters.

(A) Schematic showing the sister clusters identification based on hierarchical clustering of 39 whole-brain glutamatergic/GABAergic neurotransmitter neuronal clusters (IIa: glutamatergic neurons and …

Figure 3—figure supplement 3
Similar pair clusters of neuromodulator-type neurons.

(A-B) Hierarchical clustering of neuromodulator-type neurons based on 1783 effector genes or 319 transcription factors (TFs) in highly variable genes. The tree plots visualizing the matching nodes …

Figure 3—figure supplement 4
Divergent pattern of glutamatergic/GABAergic neurotransmitter-type neuronal clusters.

(A) A schematic for divergent pattern (left) and a summary table (right) showing that neuronal clusters as sister cluster pairs within transcription factor (TF)-based hierarchy were divergent at …

Figure 4 with 1 supplement
Combinatorial transcription factors (TFs) in marking tectal morphological subclasses.

(A) Left: the schematic showing the procedure of collecting single-cell transcriptomes of tectal glutamatergic neurons with fluorescence-activated cell sorting (FACS) using Tg (vglut2a:loxp-DsRed-lox…

Figure 4—source data 1

The annotation of transcription factors (TFs) expression in each tectal glutamatergic clusters.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig4-data1-v3.xlsx
Figure 4—source data 2

Gene labeled morphological analysis of tectal glutamatergic neurons.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig4-data2-v3.xlsx
Figure 4—figure supplement 1
Combinatorial transcription factors (TFs) in marking tectal morphological subclasses.

(A) Canonical correlation analysis (CCA) of cells from two independent experiments of Tg (vglut2a:loxp-DsRed-loxp-gfp), two rounds overlapping indicated data reproducibility. Two rounds of data were …

Figure 5 with 1 supplement
The post-transcriptional regulatory landscape in neurons with different terminal features but similar transcription factor (TF) profiles.

(A) Dot plot showing Gene Ontology (GO) analysis of differentially expressed genes between each pair clusters with divergent pattern. Red highlighted the category of post-transcriptional regulators. …

Figure 5—source data 1

Differentially gene expression between sister pair clusters with matched, convergent, and divergent patterns.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig5-data1-v3.xlsx
Figure 5—source data 2

The p-value of differential gene expression between sister pair clusters.

https://cdn.elifesciences.org/articles/68224/elife-68224-fig5-data2-v3.xlsx
Figure 5—source data 3

Annotation the function of RNA-binding proteins (RBPs).

https://cdn.elifesciences.org/articles/68224/elife-68224-fig5-data3-v3.xlsx
Figure 5—source data 4

The binding sites of pattern specific RNA-binding proteins (RBPs).

https://cdn.elifesciences.org/articles/68224/elife-68224-fig5-data4-v3.xlsx
Figure 5—source data 5

The combinatorial usage of RNA-binding proteins (RBPs).

https://cdn.elifesciences.org/articles/68224/elife-68224-fig5-data5-v3.xlsx
Figure 5—figure supplement 1
The post-transcriptional regulatory landscape in neurons with different terminal features but similar transcription factor (TF) profiles.

(A) Dot graph showing the Gene Ontology (GO) analysis of differentially expressed genes between neuronal clusters with convergent pattern. Red highlighted the category of post-transcriptional …

Organization of transcriptional and post-transcriptional regulators in the specification of brain-wide neuronal clusters.

(A) Graphic summary describes the general organization of transcription factors (TFs) and post-transcriptional regulators in the specification of neuronal clusters at the whole-brain level. Effector …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiers Additional information
Strain, strain background (Danio rerio)Wild typeDr WilliamAB
Strain, strain background (Danio rerio)gad1b:EGFPWang et al., 2020ZDB-TGCONSTRCT-210507–9Tg (gad1b:EGFP)
Strain, strain background (Danio rerio)vglut2a:loxp-DsRed:loxp-GFPSatou et al., 2012ZDB-FISH-150901–9050Tg (vglut2a:loxp-DsRed:loxp-GFP)
Strain, strain background (Danio rerio)glyT2:GFPMcLean et al., 2007ZDB-ALT-070514–1Tg (glyT2:GFP)
Strain, strain background (Danio rerio)vmat2:GFPWen et al., 2008ZDB-PUB-080102–11Tg (ETvmat2:GFP)
Strain, strain background (Danio rerio)elavl3: H2B-GCaMP6sFreeman et al., 2014ZDB-TGCONSTRCT-190827–1Tg (elavl3: H2B-GCaMP6s)
Recombinant DNA reagentpTol2-10xUAS:loxp-stop-loxp-tdtomatoThis paperWe made this plasmid by ligated three PCR fragments: loxp-stop-loxp, tdTomatocaax and 10× uas backbone
Recombinant DNA reagentvglut2a:creThis paperBAC plasmid use BAC (CH211-111D5)
Recombinant DNA reagentzic1:gal4FFThis paperBAC plasmid use BAC (CH211-95F4)
Recombinant DNA reagentbhlhe22:gal4FFThis paperBAC plasmid use BAC (CH211-277b21)
Recombinant DNA reagenten2b:gal4FFThis paperBAC plasmid use BAC (DKEY-265A7)
Recombinant DNA reagentfoxb1a:gal4FFThis paperBAC plasmid use BAC (CH211-2C17R)
Recombinant DNA reagentzbtb18:gal4FFThis paperBAC plasmid use BAC (CH211-221N23)
Recombinant DNA reagentirx1a:gal4FFThis paperBAC plasmid use BAC (CH73-211K12)
Commercial assay or kitSingle Cell 3' Library and Gel Bead kit v2 Chip kit10× Genomics120237scRNA-seq
Commercial assay or kitdsDNA High Sensitivity Assay KitAATIDNF-474–0500scRNA-seq
Commercial assay or kitClonExpressMultiS One Step Cloning KitVazymeCat#C112-01/02Prepare recombinant plasmid
Chemical compound, drugpapainWorthington Biochemical CorporationLS003126Prepare papain solution to dissociation cells
Chemical compound, drugDNase ISigmaCat#DN25Prepare papain solution to dissociate cells
Chemical compound, drugL-cysteineSigmaCat#C6852Prepare papain solution to dissociate cells
Chemical compound, drugDMEM/F12InvitrogenCat#11330032Prepare papain solution to dissociate cells
Chemical compound, drug45% glucoseGibcoCat#04196545SBPrepare wash buffer during dissociation
Chemical compound, drugHEPESSigmaCat#H4034Prepare wash buffer during dissociation
Chemical compound, drugFBSGibcoCat#10270106Prepare wash buffer during dissociation
Chemical compound, drugDPBSInvitrogenCat#14190–144Prepare wash buffer during dissociation
Chemical compound, drugMS222SigmaCat#A5040Anaesthesia
Chemical compound, drugLow melting agaroseSigmaCat#A0701Embedded fish
Software, algorithmR 3.5.1R-projecthttps://www.r-project.org/Data analysis
Software, algorithmCell Ranger Single Cell Software Suite (v2.1.0)10× Genomicshttps://support.10xgenomics.comscRNA-seq data analysis
Software, algorithmSeurathttps://satijalab.org/seurat/http://satijalab.org/seurat/scRNA-seq data analysis
Software, algorithmbioDisthttps://www.bioconductor.orghttps://www.bioconductor.org/packages/release/bioc/html/bioDist.html
Software, algorithmscclustevalR-package, Jaccard indexhttps://github.com/crazyhottommy/scclusteval; Tang et al., 2020Jaccard index could be used to evaluate the robustness of clusters
Software, algorithmTreeDistR-package, calculate two tree similarityhttps://github.com/ms609/TreeDist; Smith, 2021Calculate tree distance
Software, algorithmFIJIPMID:22743772http://fiji.sc/Analysis image
Software, algorithmGraphPad PrismGraphPad Softwarehttps://www.graphpad.comData analysis
Software, algorithmFV10-ASW 4.0 ViewerOlympushttps://www.olympus-global.comAnalysis image
Software, algorithmclusterProfilerR-packagehttps://bioconductor.org/packages/release/bioc/html/clusterProfiler.htmlGO analysis
Software, algorithmNNLSR-package, non-negative least squares solver from Lawson and Hansonhttps://github.com/rdeits/NNLS.jl; Deits, 2021Compare correlation of clusters

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