Extended Data Fig. 4: Cluster heterogeneity and distinguishing features.
From: Unique functional responses differentially map onto genetic subtypes of dopamine neurons
(a) Quantification of stability (via normalized Jaccard similarity index) of all 15 clusters from n = 100 iterations of stability calculations (simulated randomly down-sampled datasets, see Methods). Lower stability measurements imply the possibility of further subdivisions within the cluster, or additional subpopulations that may have been split across adjacent clusters. Center represents median, upper and lower box bounds represent 75th and 25th percentiles respectively, whiskers represent maxima and minima excluding outliers (data points more than 1.5 times the IQR outside the box bounds). (b) Mapping clusters across progressively higher resolutions reveals potential subdivisions either within clusters (for example, the splitting of Cluster 8 into two stable clusters) or across adjacent clusters (for example a novel cluster emerging at the intersection of clusters 3 and 10 as resolution increases). Four clusters with lower stability, as shown in panel A, are colored to highlight the potential source of their instabilities. (c) Scatter plots comparing the average expression for all genes across two clusters. Several examples of distinguishing genes with notably enriched expression patterns are highlighted. Top: Clusters 4 (Anxa1+/Aldh1a1+) vs. 1 (Anxa1-/Aldh1a1+). Bottom: Clusters 11 (Calb1+ SNc) vs. 9 (Vglut2+ SNc/SNL). Transcriptomic similarity of cluster pairs can be approximated by the correlation coefficient of their average gene expressions. (d) In situ hybridization images from the Allen Mouse Brain Atlas of ventral tier marker genes. Note that Hs6st3, which is highly enriched in our Anxa1+ cluster, appears limited to ventral-most SNc and highly resembles the expression of Anxa1 (black arrows). Images available from Allen Mouse Brain Atlas, mouse.brain-map.org (e) Additional ISH images showing expression of two marker genes that distinguish Cluster 9 (Vglut2+) from Cluster 11 (Calb1+), further corroborating the distinct identities of these populations. DAT expression is shown for reference to highlight the localization of these markers to SN pars lateralis, matching the previously described location of Vglut2+ SN DA neurons and thus supporting Cluster 9 as the Vglut2+ neurons investigated in the GCaMP activity recordings in this study. Source images available from Allen Mouse Brain Atlas, mouse.brain-map.org.
