Fig. 3: Gene regulatory network analysis identifies regulons essential for SGN diversification.

a Dotplot showing the developmental progression of regulons associated with the different cell states and transitions along the diversification tree shown in Fig. 2a; the color bars on the left indicate cell states as in Fig. 2a. b Plot showing developmental progression of Neurod1(+) after reducing the threshold score. c Plots showing developmental (pseudotime) progression of two regimes of activity of Neurod1(+) along the differentiation tree. A first regime is active at the beginning of the unspecialized population state, is associated with neurogenesis targets and decreases progressively towards the first bifurcation (representing fate choice between I_c and I_a/I_b/II); a second regime is progressively active in cells as they reach the first bifurcation and continues to be active in the I_c trajectory. d Gene regulatory network representation of Neurod1(+) showing its two regimes of activity associated with either neurogenesis (left) or I_c targets (right). e Genetic strategy for conditional deletion of Neurod1 from postmitotic SGNs. f–j Neuronal diversification phenotype in cochlea of Neurod1^cKO mice at E16.5, P0 and P3. At P3 (f, g) and P0 (h), I_a-, I_b- and I_c-SGNs are CR⁺, CR⁺/Lypd1⁺ and Lypd1⁺/CR^- (arrowheads in f), respectively. At E16.5 (i, j), SGNs are immunostained for βIII-tubulin and only emerging I_c-SGNs express Lypd1 (see Fig. 1). Quantifications of labelling are shown in g, h and j. Data in a represent max normalized fitted AUC scores. Data in c represent mean of minmax normalized gene expression from I_c targets and neurogenesis targets, shown as single cell data points as well as gam fit (line) over pseudotime trajectory. Data in g, h and j are presented as mean ± SEM; circles represent values from individual animals (1 cochlea per animal analyzed, minimum of 5 sections per cochlea, basal and mid-basal regions; n = 2–6 animals per genotype, per stage); t-test, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. Genes referenced through the text are highlighted in red in a and d. Scale bars: 20 µm. Ctrl. control.