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.

Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons.

Autonomous functions of the gastrointestinal tract require the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the range of enteric neuron diversity and how it emerges during development remain largely unknown.We here make a novel molecular definition of 12 enteric neuron classes (ENCs) within the myenteric plexus of the mouse small intestine. We identify communication features and provide histochemical markers for discrete motor, sensory, and interneurons together with genetic tools for class-specific targeting. Transcriptome analysis of embryonic ENS reveals a largely post-mitotic principle of diversification, where only ENC1 or ENC8 phenotypic traits arise through a binary neurogenic trajectory, and other identities form through subsequent differentiation. We propose generic and class-specific transcriptional regulators and functionally connect the transcription factor Pbx3 to one post-mitotic identity conversion.Our results offers a conceptual and molecular resource for dissecting ENS circuits, and predicting key regulators for the directed differentiation of distinct enteric neuron classes.

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis.

The enteric nervous system (ENS) is organized into neural circuits within the gastrointestinal wall where it controls the peristaltic movements, secretion, and blood flow. Although proper gut function relies on the complex neuronal composition of the ENS, little is known about the transcriptional networks that regulate the diversification into different classes of enteric neurons and glia during development. Here we redefine the role of Ascl1 (Mash1), one of the few regulatory transcription factors described during ENS development. We show that enteric glia and all enteric neuronal subtypes appear to be derived from Ascl1-expressing progenitor cells. In the gut of Ascl1 Ϫ/Ϫ mutant mice, neurogenesis is delayed and reduced, and posterior gliogenesis impaired. The ratio of neurons expressing Calbindin, TH, and VIP is selectively decreased while, for instance, 5-HT ϩ neurons, which previously were believed to be Ascl1-dependent, are formed in normal numbers. Essentially the same differentiation defects are observed in Ascl1KINgn2 transgenic mutants, where the proneural activity of Ngn2 replaces Ascl1, demonstrating that Ascl1 is required for the acquisition of specific enteric neuronal subtype features independent of its role in neurogenesis. In this study, we provide novel insights into the expression and function of Ascl1 in the differentiation process of specific neuronal subtypes during ENS development.