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. 2021 Feb 16;10:e60005. doi: 10.7554/eLife.60005

Figure 5. Differentiating enteric neurons captured during key transitional stage of subtype diversification within 68–70 hpf sox10:GFP+ larval cells.

(A) tSNE plot reveals five distinct sub-clusters following the subset analysis and re-clustering of Clusters 5 and 12 from the 68–70 hpf data set. (B) Dot plot depicts expression levels of enteric neuron markers across resulting Sub-clusters. Each marker was expressed at low levels in Sub-cluster 1 and were found to be expressed at higher levels within Sub-cluster 3. (C) tSNE feature plots further depict the expression of enteric neuron markers by illustrating the levels and localization of expression within the Sub-cluster architecture. Feature plots supplement dot plot and demonstrate the prominent expression of enteric neuron markers within Sub-cluster 3, which appears to emanate from Sub-cluster 1. (D,E) Violin and feature plots reveal expression levels of acetylcholine-associated and excitatory neuron markers reported to distinguish enteric IPANs. These markers were found in a discrete pocket of cells forming the distal-most region of Sub-cluster 3 (red arrowhead). Violin data points depicted in each Sub-cluster represent single cells expressing each gene shown. (F) Graphical model summarizes expression patterns observed in 68–70 hpf data set and HCR validation. Common enteric neuroblast capable of diverging into subsequent lineages, IPAN, inhibitory neuron, and interneuron through lineage restricted gene expression. pbx3b promotes assumption of IPAN role through loss of nos1 and vipb and begins expressing calb2a, ache, and slc18a3a. (G) Whole mount HCR analysis reveals co-expression of IPAN marker genes, pbx3b and calb2a, and inhibitory neurochemical marker genes, vipb and nos1 (white arrowheads), within the foregut (dashed white line) at 68 hpf. Vesicular acetylcholine transferase, slc18a3a, was not observed in tandem with pbx3b but was co-expressed with calb2a, vipb, and nos1 (yellow arrowheads). Scale bar: 50 μM. (H) Feature plots reveal expression of opioid receptor genes, oprl1 and oprd1b, within the differentiated enteric neuron Sub-cluster 3. (I–N) Whole mount HCR analysis validates expression of oprl1 in combination with vipb and phox2bb (yellow arrowheads) in enteric neurons localized to the foregut region of a 68 hpf embryo. Scale bar: 10 μM.

Figure 5—source data 1. List of marker genes per Sub-cluster, following subset and re-clustering of enteric Clusters 5 and 12 at 68–70 hpf.
Table reporting the Seurat output for genes for each Sub-cluster (0–4), including p-values (<0.01), average log-fold change (.25), adjusted p-values (1.0), pct.1 summarizing proportion of cells expressing the individual gene in the Sub-cluster, pct.2 showing the proportion of cells expressing the individual gene in all other Sub-clusters in the sub-data set.
Figure 5—source data 2. List of enriched pathways within enteric neuron Sub-cluster 3 and genes present in specific opioid proenkephalin pathway identified following PANTHER Overrepresentation Test.
Source Data table contains two sheets. Enriched Gene Pathways sheet lists the enriched pathways identified following the use of the statistical test, Fisher’s exact test that compares the number of genes in a given pathway within the reference Danio rerio list to the number of genes in a given pathway within the enteric neuron Sub-cluster 3 list. This analysis produced 43 statistically overrepresented pathways. Column B: Number of genes within the given pathway present with Danio rerio reference list of 25888 genes. Column C: Number of genes within the given pathway present within Sub-cluster 3 gene list. Column D: Number of pathway genes expected to be present within the Sub-cluster 3 gene list based on the percentage of pathway genes present in the Danio rerio reference list. Column E: Denotes that more pathway genes were present in Sub-cluster 3 gene list than expected. Column F: Fold enrichment of Sub-cluster 3 pathway genes comparative to reference list. Column G: p-values calculated following Fisher’s exact test comparing expected number of pathway genes to number of genes pathway genes in Sub-cluster 3. Column H: p-value following Benjamini-Hochberg false discovery rate (FDR) correction. Opioid proenkephalin pathway sheet lists genes associated with this pathway that are present within the enteric neuron Sub-cluster 3 gene list, notably, opioid receptor oprd1b.

Figure 5.

Figure 5—figure supplement 1. Enteric neuron subtype diversification gene expression patterns seen in enteric neuron Sub-clusters related to Figure 5D,E Panel of tSNE feature plots magnified and cropped to focus on progressively differentiating enteric neurons (highlighted by etv1 expression).

Figure 5—figure supplement 1.

Subtype diversification and IPAN emergence depicted via combinatorial gene expression (etv1, ntng1a, pbx3b, slc18a3a, calb2a, and ache) localized to the distal tip of Sub-cluster 3. Inhibitory neuron markers, nos1, vip, and galanin (galn) were present within the pocket of diverging enteric subtypes.
Figure 5—figure supplement 2. Enteric and sympathetic neuron markers distinguished among common autonomic neuron precursors.

Figure 5—figure supplement 2.

UMAP analysis of Sub-clusters depicts enteric and sympathetic neurons delineating from a common pool of autonomic neurons. (A) UMAP plot generated following re-clustering of Clusters 5 and 12 from the 68–70 hpf data set. (B) Dot plot depicts expression levels of general autonomic neuron makers (ascl1a, hand2, phox2a, phox2bb), sympathetic neuron markers (th, dbh, lmo1, insm1a) and enteric neuron markers (vipb, etv1, nos1, gfra1a) within Sub-clusters 0–4. (C) Dendrogram denotes similarity of Sub-clusters based on average gene expression of each cell within the Sub-clusters, which reveals transcriptomic distinction of enteric neuron Sub-cluster 3. (D–F). Feature plots highlight the expression of autonomic, sympathetic, and enteric neuron gene markers within UMAP Sub-clusters.