Diversification of molecularly defined myenteric neuron classes revealed by single cell RNA-sequencing

Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of enteric neuron types and how they emerge during development remain largely unknown. Here, we define a molecular taxonomy of twelve enteric neuron classes within the myenteric plexus of the mouse small intestine using single cell RNA-sequencing. We present cell-cell communication features, histochemical markers for motor, sensory, and interneurons together with transgenic tools for class-specific targeting. Transcriptome analysis of embryonic ENS uncovers a novel principle of neuronal diversification, where two neuron classes arise through a binary neurogenic branching, and all other identities emerge through subsequent post-mitotic differentiation. We identify generic and class-specific transcriptional regulators and functionally connect Pbx3 to a post-mitotic fate transition. Our results offer a conceptual and molecular resource for dissecting ENS circuits, and predicting key regulators for directed differentiation of distinct enteric neuron classes.