Additional file 1 of A cell atlas of the larval Aedes aegypti ventral nerve cord

Additional file 1: Fig. S1. Quality control measurements for larval Aedes VNC dataset. (A) Histogram showing the log10 distribution of cells expressing the indicated number of UMIs. Red line represents the nUMI cutoff, with cells below 1000 UMI excluded from further analysis. (B) Histogram showing the log10 distribution of cells with the indicated number of expressed genes (nGene). Red line represents the nGene cutoff; cells with fewer than 500 expressed genes were excluded from further analysis. (C) Histogram showing the distribution of cells with indicated proportion of mitochondrial transcripts. Red line represents the mitochondrial proportion cutoff (18%), above which cells were eliminated from further analysis. (D) Histogram showing the distribution of cells with the indicated proportion of transcripts from ribosomal genes. Cells with ribosomal gene proportions less than 5% or greater than 40% were eliminated from further analysis. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S2. Identification of putative glial cells in the larval Aedes VNC. (A) Cell atlas from initial clustering, which contains 25 distinct cell clusters. Putative glial cells, identified based on marker gene expression, are indicated. (B) Cells from this putative glial cluster were isolated and reclustered to reveal putative glial subtypes. Feature plots depict expression of glial marker genes including the pan-glial marker repo, cortex glia marker wrapper, surface glia marker gemini and four astrocyte glial markers wunen-2 (wun2), Excitatory amino acid transporter 1 (Eaat1), Gat and glutamine sythetase [57]. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S3. Distribution of neuronal marker gene expression in the Aedes larval VNC cell atlas. Feature plots depict expression of nSyb, Syt, and CadN which are present in all clusters. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S4. Neurotransmitter marker gene expression in the larval Aedes VNC. (A-D) Feature plots showing expression of neurotransmitter marker genes ChAT, VGAT, Vmat, and Dat. (E) Histogram showing number and percentage of cells expressing the indicated neurotransmitter marker genes. (F) Venn diagram showing the number of cells expressing the cholinergic marker genes VAChT and ChAT. Among the 329 ChAT+ VAChT- cells, 240 expressed an additional FAN marker gene (VGlut, VGAT and/or Gad1). (G) Maximum projections of confocal stacks depicting anti-ChAT immunostaining of the abdominal ventral ganglion, which was additionally labeled with the nucleic acid stain Hoechst. ChAT immunoreactivity is distributed throughout the neuropil but largely absent from the cortical cell layers. (H) Maximum projections of confocal stacks depicting anti-VGlut immunostaining of the abdominal ventral ganglion, which was additionally labeled with the nucleic acid stain Hoechst. Punctate VGlut immunoreactivity was present throughout the ganglion. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S5. Identifying subsets of monoaminergic neurons. Neurons that highly expressed monoaminergic marker genes were isolated from the VNC cell atlas and re-clustered to identify monoaminergic subsets. (A-D) Feature plots depict expression of the monoaminergic marker genes Tbh, SerT, Dat, and TH. Note that these genes are expressed in mutually exclusive sub-populations. (E) Seurat UMAP plot depicting monoaminergic neuron subtypes assigned on the basis of marker gene expression. (F-H) Feature plots depict expression of FAN markers VAChT (F), Gad1 (G), and VGlut (H) in monoaminergic neurons. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S6. Identification of transcriptional markers for serotonergic neurons. Neurons that expressed the serotonergic marker SerT were isolated and re-analyzed. Feature plots depict expression of SerT (A) and two transcription factor genes en (B) and Hox-C3a (C) whose expression is highly correlated with SerT (D-E). Expression correlation plots additionally depict Gad1 expression colored according to a lookup table. Note that cells with the highest levels of SerT, en, and Hox-C3a exhibit low expression of Gad1. (F) Maximum projections of confocal stacks depict representative images of the abdominal VNC labeled with antibodies to serotonin (5-HT) and en as well as the nucleic acid stain Hoechst. Cells co-expressing 5-HT and en are indicated with yellow arrowheads. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S7. Classification of dopaminergic neurons according to TH levels and GABA expression. (A) Histogram showing the distribution of dopaminergic cells with indicated intensities of anti-TH fluorescence (CTCF, corrected total cell fluorescence). Cells were classified according to breaks in the distribution: low TH cells had TH levels below 15,000 CTCF; high TH cells had TH levels greater than 50,000 CTCF; medium TH cells had intermediate TH levels (between 15,000 and 50,000 CTCF). (B) Histogram depicts the average number of low-, medium-, and high-expressing TH cells in each segment of the larval VNC. N = 6 animals, 251 neurons. (C-E) Relationship between TH and GABA expression. (C) Histogram showing the number of GABA-positive and GABA-negative low-, medium-, and high-expressing TH cells. Most TH+, GABA+ double-positive cells have low anti-TH levels. N = 3 animals, 129 neurons. (D) High TH-expressing cells (n = 60) were isolated from the global VNC dataset and reclustered. The feature plot depicts expression of the GABAergic neuron marker gene VGAT in dopaminergic neurons. (E) TH and Gad1 expression levels are inversely related in dopaminergic neurons. Scatter plot depicts expression of TH (x-axis), Gad1 (y-axis), and VGAT (shading) in 106 dopaminergic neurons. High-TH-expressing cells express low levels of Gad1 and VGAT1, and vice-versa. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S8. Expression patterns of neuropeptides and peptide processing enzymes. Dot plots depict expression levels of (A) neuropeptide genes and (B) neuropeptide processing enzyme genes in each cell cluster of the VNC cell atlas. Dot color indicates mean expression level across the cluster and dot diameter indicates the proportion of cells in the cluster with nonzero expression of the gene. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S9. Transcriptional markers of VNC cell clusters. Dot plots depict expression of transcription factors identified as marker genes for the indicated cell clusters, grouped according to transmitter identity: aminergic and peptidergic (A), cholinergic (B), GABAergic (C), and glutamatergic neurons (D). Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S10. Co-expression of neuropeptide genes in neurosecretory cluster 19. (A) Histogram showing the number and proportion of cluster 19 cells that express the following neuropeptide genes: DH31, Mip, burs, CCAP, pburs, and ASTCC. (B) Histogram showing the number and proportion of cluster 19 cells that co-express multiple neuropeptides in the same cell. Half of cluster 19 cells express 6 neuropeptide genes simultaneously. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S11. Expression pattern of the transcription factor gene ct. (A) Feature plot showing ct expression across the VNC cell atlas. (B) Maximum projections of confocal stacks depicting anti-Ct immunoreactivity in a representative segment of the abdominal VNC additionally labeled with the nuclear dye Hoechst. As with ct mRNA, Ct protein is broadly expressed at varying levels in the VNC. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S12. Expression patterns of neurotransmitter and neuropeptide receptor genes. Dot plots depict expression levels of (A) FAN receptor genes, (B) monoamine receptor genes, and (C) neuropeptide receptor genes in each cell cluster of the Aedes VNC cell atlas. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S13. Expression patterns of Hox transcription factors. (A) Schematic diagram indicating putative expression patterns of Hox genes mapped alongside a composite image of the larval VNC and segmental nerves visualized in a fillet preparation of a brp-T2A-QF2w, QUAS-mcd8GFP larva. (B) Maximum intensity projections depict representative results from antibody staining of thoracic (T2) and abdominal (A4) segments of the larval VNC with antibodies to Antp and Ubx/Abd-A (antibodies recognize both Ubx and Abd-A). (C) Feature plots depicting Hox gene expression across the larval VNC cell atlas. (D-E) Histograms depict (D) the overall number of cells and (E) the proportion of cells from each cluster which express the indicated Hox genes. Colors are used to indicate neuronal subtypes. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S14. Marker gene expression in neural progenitors and immature neurons. Dot plot depicts expression levels of pan-neuronal and FAN marker genes in cluster 10 sub-clusters. Putative neuroblasts (sub-cluster 4) exhibit limited expression of neuronal marker genes, sub-cluster 0 expresses neuronal markers but no FAN markers, and sub-clusters 1–3 expressed neuronal markers and an individual FAN marker. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S15. Global pseudotime analysis of VNC development. (A) Pseudo-time trajectory analysis of the entire cell atlas, anchored at cluster 10. Cells are colored according to their pseudo-time score along the trajectory, with darker colors marking more immature neurons and lighter colors indicating less immature neurons. (B) UMAP plot highlighting cluster 6 and cluster 10, both of which are comprised principally of immature neurons. (C) Box plots showing pseudotime scores for each cluster. Clusters are grouped according to their principle FAN and ordered according to pseudotime scores. Genotype: brp-T2A-QF2w / +; QUAS-mcd8GFP / +. Fig. S16. Identification of mitotic cells in the larval VNC. (A) Maximum intensity projection of confocal stack depicting anti-PH3 immunoreactivity in thoracic ventral ganglion additionally labeled with anti-Lamin antibodies to facilitate cell counting. (B) Quantification of PH3+ cells in thoracic and abdominal segments at day 3 and day 5 of larval development. *P < 0.05, Kruskal-Wallis test with a post-hoc Dunn’s test. Ganglia number (N) for each sample: 3-day thorax (18), 3-day abdomen (37), 5-day thorax (29), 5-day abdomen (75). Genotype: wild type. Fig. S17. Positive control for efficacy of TUNEL labeling. Larval tissue was fixed, permeabilized, and pre-incubated with the endonuclease DNaseI for 10 min to induce DNA fragmentation prior to TUNEL labeling. Images depict TUNEL labeling of a larval ganglion (A) additionally labeled with Hoechst (B). Table S1. Antibodies used in this study. The antigen, host, source and working dilution of all antibodies used in this study are indicated. Many of the primary antibodies were raised against Drosophila antigens and cross-reactivity has not been extensively documented, therefore we include antibodies that showed no specific cross-reactivity even though the antibody target was detectably expressed by RNAseq. Table S2. Putative glial cluster marker genes were identified by applying the ‘FindAllMarkers’ function as described in the Materials and Methods to cluster 12 from our preliminary clustering analysis (Fig. S2). Avg_log2FC represents the enrichment (FC, fold change) of the marker gene in cluster 12 compared to global mean expression values for that marker. Pct.1 and pct.2 represent the percent of cells in cluster 12 and percent of cells in all other clusters, respectively, that express that marker. Table S3. Proportion of cells in each cluster expressing neurotransmitter marker genes. For each cluster, the proportion of cells expressing VAChT and/or ChAT (Cholinergic), GAT (GABAergic), VGlut and/or Gad1 (Glutamatergic), and Vmat (Monoaminergic) is shown. Clusters with a predominant (expressed in > 70% of cells) transmitter fate are indicated with highlights color-coded by transmitter type. Table S4. Proportion of cells in each cluster expressing marker genes for 0, 1, or multiple neurotransmitters. For each cluster, the proportion of cells expressing 0–4 different types of neurotransmitter marker genes is shown [VAChT and/or ChAT (Cholinergic), GAT (GABAergic), VGlut and/or Gad1 (Glutamatergic), and Vmat (Monoaminergic)]. Highlights mark clusters containing > 20% of cells with no detectable neurotransmitter marker gene expression. Table S5. Genes exhibiting the most highly correlated expression to SerT. Correlation in Gene-gene expression values was calculated using Markov Affinity-based Graph Imputation of Cells (MAGIC) algorithls, as described in the Materials and Methods. The output of this analysis (Magic_dismay_SerT_cor) represents the expression correlation (positive or negative; negative represents an inverse gene expression relationship) between each gene and SerT. Genes are rank-ordered according to the absolute value of the expression correlation (abs_cor). Table S6. Aedes aegypti neuropeptide genes. The name, Aedes aegypti accession numbers, and coding gene (CG) number for Drosophila orthologues are shown for each Aedes aegypti neuropeptide gene identified. NA, not available. Table S7. VNC Cell Atlas cluster markers. Markers for each of the 35 VNC cell clusters were identified using the ‘FindAllMarkers’ function as described in the Materials and Methods. Avg_log2FC represents the enrichment (FC, fold change) of the marker gene in that particular cluster compared to global mean expression values for that marker. Pct.1 and pct.2 represent the percent of cells in that particular cluster and percent of cells in all other clusters, respectively, that express that marker. Table S8. Cluster 10 sub-cluster marker genes. Immature neurons from cluster 10 were subsetted from the filtered dataset and markers for ‘FindAllMarkers’ function. Avg_log2FC represents the enrichment (FC, fold change) of the marker gene in that particular subcluster compared to mean expression values for that marker across cluster 10. Pct.1 and pct.2 represent the percent of cells in that particular subcluster and percent of cells across cluster 10, respectively, that express that marker.