Skip to main content
. 2020 Apr 21;9:e54074. doi: 10.7554/eLife.54074

Figure 8. Identification of neuropeptide expressing cells.

(A) Heatmap of the mean log-normalized expression (0–4), by cluster (bottom), of neuropeptide genes (right). Only clusters with at least one neuropeptide gene expressing in excess of mean log-normalized value of 10 are shown. Only neuropeptide genes that were expressed in at least one cell at a level >10 transcripts per cell are shown. Arrowhead highlights cluster 84. Dendrograms represent hierarchical clustering. (B) t-SNE plot highlighting neuropeptide expressing cluster 84. (C) Schematic of neuropeptide processing steps highlighting enzymes (in red) identified as enriched markers for cluster 84. Propeptides are cleaved by the pro-hormone convertase Amontillado (Amon); the carboxypeptidase Silver (Svr) then removes the C-terminal cleavage sequence. C-terminal amidation occurs through the combined actions of Peptidyl-α-hydroxyglycine-α-amidating lyase 2 (Pal2) and Peptidylglycin-α-hydroxylating monooxygenase (Phm) (reviewed in Pauls et al., 2014). (D, E). Expression of neuropeptide processing enzymes (D) and multiple neuropeptide genes (E) in t-SNE cluster 84. Intensity of red is proportional to the log-normalized expression level.

Figure 8—source data 1. Neuropeptide expression levels.
Table of neuropeptide expressed genes, their maximum observed log-normalized expression, the rank position of this expression, the percentile of this expression, and their associated receptors. n.d. = no data available regarding associated receptors.

Figure 8.

Figure 8—figure supplement 1. Neuropeptide gene expression levels.

Figure 8—figure supplement 1.

(A) Violin plot showing the number of transcripts (UMI) per cell in the whole VNC data set (all clusters) compared to neuropeptide-expressing cluster 84. Data represents expression between 1,200–10,000 UMI (dashed lines). (B) Box plots representing number of UMI per cell of all detected neuropeptide genes across the whole VNC data set. (C) Ranked mean number of UMI per cell of the top 10 expressing cells for each GOI. Neuropeptide genes are highlighted in red, many of which have the highest number of UMI/cell in the genome. (D) Ranked mean percent of UMI per cell (GOI UMI/total UMI) of the top 10 proportion of UMI cells for each GOI. Neuropeptide genes are highlighted in red, many of which have the highest proportion of transcripts per cell in the genome.
Figure 8—figure supplement 2. Analysis of VNC data removing upper limit on transcript number (UMI) per cell.

Figure 8—figure supplement 2.

(A) Re-analyzed t-SNE plot of VNC data without the 10,000 UMI high cut-off. Neuropeptide (NP) enriched cluster and Proctolin expressing motor neuron (Proc+ MN) cluster are highlighted. (B) t-SNE of the number of UMI. Color is scaled from 10,000 to 20,000 UMI. Most cells with greater than 10,000 UMI are in the NP cluster or the Proc+ MN cluster. (C) Violin plot showing the number of UMI per cell without 10,000 UMI filtering cut-off in the whole VNC data set (all clusters) compared to the NP cluster. Dashed lines represent the low cut-off of 1,200 UMI and the now removed cut-off of 10,000 UMI. (D) Box plots representing number of UMI per cell of all expressed neuropeptide genes across the whole VNC data set without the 10,000 UMI filtering cut-off. Multiple NP genes exceed the expression level of 10,000 UMI. (E) t-SNE plots of the NP cluster showing the spatial distribution of the number of expressed genes (nGene), number of transcripts (nUMI) and proportion of transcripts that are mitochondrial (prop.mito). (F, G) Expression of neuropeptide processing enzymes (F) and multiple neuropeptide genes (G) in t-SNE of NP cluster. Intensity of red is proportional to the log-normalized expression level.
Figure 8—figure supplement 3. Orcokinin expression analysis.

Figure 8—figure supplement 3.

Violin plot of raw UMI Orcokinin expression by cluster.