Extended Data Fig. 2: Characterization of cortical projection neuron classes and layer selectivity across mouse lines.

a, Brain-wide projection patterns were visually inspected for every experiment and manually classified into one of six categories on the basis of projections to ipsilateral and contralateral cortex, striatum, thalamus, and midbrain, pons or medulla structures as described for IT, PT, and CT classes. b–d, Unsupervised hierarchical clustering (using Euclidean distance and average linkage) of projection weights validates and reveals major classes of cortical projection neurons. b, Each column of the heat map shows one of the 1,081 injection experiments. Colours in the ‘manual PN’ class are coded as in c for projection class. Rows show selected major brain regions that distinguish known classes of projection neurons. Values in each cell are the fractions of total brain projection volume in the given region. The dendrogram was split into nine clusters, with two subclusters identified post-hoc for cluster 5. The numbers of experiments per cluster were: 1, n = 24; 2, n = 4; 3, n = 204; 4, n = 158; 5a, n = 148; 5b, n = 230; 6, n = 174; 7, n = 12; 8, n = 16; 9, n = 111. The numbers of experiments per projection class were: CT, n = 119; IT, n = 342; IT PT, n = 158; IT PT CT, n = 189; local, n = 100; PT, n = 173. c, The relative frequency of experiments from manually assigned projection classes within each cluster is shown. There was significant enrichment of 1, or 2 related, classes in each cluster (dots; Fisher’s exact t-test, P < 0.01). d, Maximum intensity projections of GFP-labelled axons across the brain from one example per cluster. e, Characterization of layer selectivity in wild-type mice and 14 Cre lines derived from injection experiments. Number of experiments per line is listed in Supplementary Table 1. For every injection and line, we assessed layer selectivity on the basis of the manually annotated injection sites. Polygons were drawn around every injection site so that, after registration to the CCF, injection volume in each layer could be informatically derived. A layer-selectivity index was calculated for each experiment (the fraction of the total injection volume contained in each layer, scaled by the relative volume of each layer in the injection source region, because layer volumes differ by area). Plots show individual data points and the average layer selectivity index ± 95% confidence intervals (in black) for the set of 15 mouse lines. Red lines in each Cre graph show average values from C57BL/6J experiments. Red lines in the C57BL/6J graph are averages from the Emx1-IRES-Cre experiments, which also labels cells across all layers. There is a bias towards L5 neuron infection in both C57BL/6J and Emx1-IRES-Cre mice, highlighting the importance of using layer-selective Cre lines for better coverage of cortical outputs.