Single-cell transcriptome profiling of the Ciona larval brain - PubMed (original) (raw)

Single-cell transcriptome profiling of the Ciona larval brain

Sarthak Sharma et al. Dev Biol. 2019.

Abstract

The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.

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Figures

Figure 1.

Figure 1.. scRNAseq of sorted Fascin>tagRFP+ cells

A) Ciona robusta larva electroporated with Fascin>tagRFP, labeling brain cells. Scale bar = 25 μm. B) tSNE plot showing all 2607 cells that passed quality control (see Materials and methods for details), clustered by Seurat into 10 clusters (1–10). Clusters were assigned putative identities based on gene expression data publicly available on ANISEED. Number of cells in each cluster is indicated in parentheses. Clusters containing mostly neural cells are indicated in bold italic. C) Comparison of gene expression profiles of Clusters 1–10 to previously published lists of genes enriched in Ciona brain (Hamada et al. 2011) and nervous system (Mochizuki et al. 2003). Colored circles indicate the fraction of genes from each previously published study that is also enriched in the corresponding cluster from the present study. Numbers in parentheses indicate total number of genes in each fraction (see Supplemental Table 2 for details).

Figure 2.

Figure 2.. Marker genes reveal cell identities of major brain regions

A) Clusters 3, 5, and 9, corresponding to the majority of the Ciona larval brain, highlighted in the original tSNE plot (see Figure 1). Other clusters and outlier cells are shaded in gray. B) Gene expression of neuronal markers Synaptotagmin 1 (Syt1), Celf3/4/5 (also known as Etr-1), Rlbp1 (also known as Cralbp) plotted on tSNE plots of clustered cells. High expression of neuronal markers in these cells delineates Cluster 3, comprised of neurons and photoreceptors mostly in the dorsolateral regions of the brain. C) Cluster 5 cells show expression of brain vesicle wall (ependymal) cell markers Aquaporin 8 (Aqp8), Zeb, Gonadotropin-releasing hormone.b (Gnrh.b, previously named GnRH2) and gene KH.C10.493. D) Cluster 9 is distinctly marked by expression of Ptf1a, GTP cyclohydrolase 1 (GCH1) Phosphodiesterase 9a (Pde9a) and Guanylate cyclase-related (Gucy-r). According to Razy-Krajka et al. (2015), these cells, comprising the ventral-most part of the brain, represent the tunicate homologue of a proto-hypothalamo-retinal region in the olfactorian ancestor that gave rise to both hypothalamus and retinal neurons in vertebrates. In all gene expression maps, red color indicates high expression, yellow indicates low expression. Gene expression violin plots (right) are color coded according to clusters in Figure 1.

Figure 3.

Figure 3.. Re-clustering of putative neural cells

tSNE plot showing re-clustering of 644 cells from original clusters 3, 5, 8 and 9, which were deemed to be primarily neural in composition. Cluster I was manually separated from Cluster B, and Cluster J was manually separated from Cluster C. Putative identities are based on known gene expression patterns available on ANISEED (see Results for details). Number of cells in each cluster is in parentheses. BV: brain vesicle, PHR: proto-hypothalamus/retina, H/T: head/tail.

Figure 4.

Figure 4.. Comparison of gene expression profiling of coronet cells by scRNAseq and bulk RNAseq

A) Expression of coronet cell marker Tyrosine hydroxylase (TH) mapped onto Cluster E cells on tSNE plot. B) Magnified view of brain of Ciona larva electroporated with Fascin>H2B:mCherry (pink nuclei) and TH>Unc-76::eGFP (green), revealing coronet cells of the ventral brain/proto-hypothalamo-retinal region. C) Comparison of differential gene expression analyses based on bulk RNAseq (enriched/depleted in sorted TH+ cells relative to whole brain; see Supplemental Table 4) and scRNAseq (enriched/depleted in Cluster E, Supplemental Table 2, sheet 5), indicating a strong correlation (Pearson’s correlation coefficient, r = 0.749) between the two methods. Only genes that were differentially expressed in Cluster E were analyzed (n = 942 total genes. See Supplemental Table 5). “Gap” in scRNAseq values is due to a default average log(FC) cutoff of +/−0.25. For perspective, little correlation was found when comparing values for genes differentially expressed in other neurons, like Cluster B (r = −0.327) or Cluster I (r = −0.271).

Figure 5.

Figure 5.. Epidermal and palp neurons

A) Gene expression of Thymosin beta-related mapped onto the tSNE plot. Dotted outline indicates region magnified in inset at far right, highlighting clusters H, J, and C, excluding certain outlier cells. Top center inset: gene expression violin plots. B) In situ hybridization of Thymosin beta-related in a late tailbud embryo, showing expression in developing epidermal neurons of the head and tail. C) Expression of palp neuron marker Crystallin beta/gamma marks small 12-cell cluster (Cluster J). Top right inset: gene expression violin plot color coded as in (A). D) Larva electroporated with Crystallin beta/gamma>Unc-76::eGFP (green), revealing palp neurons (arrowheads). Expression is also strong in the otolith pigment cell (asterisk), which does not express Fascin and was therefore not found in our dataset. E) Expression of another known marker of palp neurons, Islet, also maps to Cluster J. Top right inset: gene expression violin plot. F) Mid-tailbud embryo electroporated with Islet[palp+notochord]>Unc-76::eGFP, labeling developing palp neurons (arrowheads), notochord, and bipolar tail neurons. This particular Islet driver, corresponding to the first intron and the proximal promoter region, was identified in Wagner et al. (2014).

Figure 6.

Figure 6.. Major subdivision of brain neurons into putative GABAergic and cholinergic neurons

A) tSNE plot of neural-specific cells from Figure 3. Inset: enlarged view of Clusters B and I, corresponding to dorsolateral brain neurons, with some outliers omitted. B) Expression of GABA decarboxylase (GAD) superimposed on clusters B and I, showing enrichment in Cluster B and depletion in Cluster I. Gene expression violin plot in inset, color coded as in (A). C) Larva electroporated with GAD>tagRFP, revealing GAD + neurons of the brain, likely corresponding to Cluster B. D) Expression of Slc32a1 (also known as Vesicular GABA transporter, VGAT) in a subset of _GAD_-expressing neurons. E) Larva co-electroporated with Fascin>H2B::mCherry (pink nuclei) and Slc32a1>Unc-76::eGFP (green), showing VGAT + brain neurons. All nuclei counterstained with DAPI (blue). F) Expression of KH.C1.498 operon, consisting of Slc18a (also known as Vesicular acetylcholine transporter, VAChT) and Choline acetyltransferase (Chat) plotted onto clusters B and I, showing enrichment in Cluster I. G) Larva electroporated with Fascin>H2B::mCherry (pink nuclei) and Slc18a>Unc-76::eGFP (green), showing cholinergic neurons of the brain. All nuclei counterstained with DAPI (blue). H) Expression of Slc1a2 encoding a Glutamate transporter and Grin, encoding an NMDA-type ionotropic glutamate receptor, labels primarily Cluster I, suggesting that cholinergic neurons are the principal targets of glutamatergic neurotransmission in the Ciona larval brain. I) Expression plots of various genes showing enrichment in either Cluster B (top row) or Cluster I (bottom row).

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