Morphological pseudotime ordering and fate mapping reveal diversification of cerebellar inhibitory interneurons

Understanding how diverse neurons are assembled into circuits requires a framework for describing cell types and their developmental trajectories. Here we combine genetic fate-mapping, pseudotemporal profiling of morphogenesis, and dual morphology and RNA labeling to resolve the diversification of mouse cerebellar inhibitory interneurons. Molecular layer interneurons (MLIs) derive from a common progenitor population but comprise diverse dendritic-, somatic-, and axon initial segment-targeting interneurons. Using quantitative morphology from 79 mature MLIs, we identify two discrete morphological types and presence of extensive within-class heterogeneity. Pseudotime trajectory inference using 732 developmental morphologies indicate the emergence of distinct MLI types during migration, before reaching their final positions. By comparing MLI identities from morphological and transcriptomic signatures, we demonstrate the dissociation between these modalities and that subtype divergence can be resolved from axonal morphogenesis prior to marker gene expression. Our study illustrates the utility of applying single-cell methods to quantify morphology for defining neuronal diversification.