The enteric nervous system, the vast mesh of neurons embedded in the walls of the gastrointestinal tract, is often called the “second brain.” It contains as many neurons as the spinal cord and operates largely autonomously, controlling digestion, nutrient absorption, and gut motility without input from the central nervous system. Yet how this complex circuit is built during development has remained surprisingly obscure.
A new study from researchers at the Université de Lyon, published in PNAS, provides the most detailed picture yet of enteric neurons as they first emerge and wire the gut. Using a combination of single-nucleus RNA sequencing at short time intervals, whole-gut 3D imaging, and cross-species comparisons, the team led by Valérie Castellani and Julien Falk has mapped the transcriptional and morphological dynamics of enteric neuron subtype diversification, and identified conserved guidance programs that drive axon pathfinding in chick, mouse, and human embryos.
Capturing a moving target
Enteric neurons arise from vagal neural crest cells that migrate into the developing gut and then differentiate into dozens of molecularly distinct subtypes. Capturing this process requires timing: the team performed single-nucleus transcriptomics at multiple closely spaced developmental stages, allowing them to reconstruct the precise sequence of transcriptional changes as progenitors committed to specific neuronal fates.
First author Maëlys André and colleagues found that emerging neurons express axon guidance gene programs that are fundamentally different from the migration programs used by their neural crest progenitors during the initial colonization of the gut. The guidance programs are also distinct between different emerging neuron subtypes, suggesting that each subtype is equipped with its own molecular navigation toolkit.
A rapidly growing network
Using whole-gut 3D imaging with selective plane illumination microscopy (SPIM) on cleared chick embryo tissue, the team visualized enteric axons as they grew. The images reveal highly dynamic axon network growth: a rapid increase in axon density and a diversification of spatial orientation as development proceeds. The axons do not simply follow the gut wall passively, they actively navigate, guided by molecular cues.
Conservation across species
The study’s most striking finding is the evolutionary conservation of these guidance programs. Cross-analysis with publicly available single-cell RNA sequencing data from mouse and human embryos revealed globally conserved enteric lineage trajectories. The same subtypes emerge in the same sequence, using the same guidance genes.
The team functionally validated two of these conserved genes, ISLR2 and DSCAM, in chick whole-gut cultures. When they manipulated DSCAM and ISLR2 signaling networks, enteric axon patterns were altered, confirming that these molecules actively direct the formation of enteric circuits.
Both genes have known links to human disease. DSCAM polymorphisms are associated with non-syndromic forms of Hirschsprung disease, a congenital condition in which the enteric nervous system fails to fully colonize the gut. ISLR2 mutations cause a neurodevelopmental disorder with gastrointestinal involvement. The new findings provide a developmental framework for understanding why these mutations produce their specific effects.
Why it matters
The enteric nervous system is increasingly recognized as a player in conditions far beyond classical gut disorders. Alzheimer’s, Parkinson’s, and autism spectrum conditions all involve gastrointestinal symptoms that precede or accompany neurological ones. The gut is also where amyloid pathology begins in some forms of neurodegenerative disease. Understanding how the enteric nervous system is built, which cells emerge when, how they find their targets, and which guidance molecules they use, provides the developmental baseline for understanding what goes wrong in disease.
The study also has direct clinical relevance for Hirschsprung disease, which affects approximately 1 in 5,000 newborns. Current treatment is surgical removal of the aganglionic gut segment, but the underlying developmental defect, failure of enteric neuron colonization, has been difficult to study in human tissue. The cross-species conservation validated in this study means that chick and mouse models can be used with greater confidence to develop cell-replacement or guidance-based therapies.
Limitations
The study was performed primarily in chick embryos, with cross-species analysis of existing mouse and human transcriptomic data. The functional validation (DSCAM and ISLR2 perturbations) was performed only in chick. Direct functional studies in human tissue or human organoids would be needed to confirm that the same guidance rules apply.
The time-resolved transcriptomic data captures gene expression at discrete developmental stages, the intervals between sampling points may miss rapid transitions or transient cell states.
Sources
1. André, M., Gury, R., Lepetit, M., Boismoreau, F., Bozon, M., Ganofsky, J., Heritier-Tellier, C., Plotton, I., Duclaux-Loras, R., Peretti, N., Marcy, G., Castellani, V., & Falk, J. (2026). Time-resolved morphological and transcriptomic characterization of early enteric neuron subtype emergence in chick. Proceedings of the National Academy of Sciences, 123(28), e2511442123. https://doi.org/10.1073/pnas.2511442123
2. Data available at NCBI GEO accession GSE282673 and GitLab: https://forge.univ-lyon1.fr/melis/Castellani_lab/andre_snrna_seq