Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

Abstract

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR), which is caused by defective ENS development. Zebrafish have proven to be fruitful in the identification of novel genes involved in ENS development and HSCR pathology. However, the composition and specification of enteric neurons and glial subtypes of the larval zebrafish at a single cell level, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified both vagal neural crest progenitors and Schwann cell precursors, as well as four clusters of early differentiated neurons. Interestingly, since we took an unbiased approach where we sequenced total intestines, an elavl3+/phox2bb- population of neurons and the presence of cx43+/phox2bb- enteric glia were identified in larval zebrafish. These populations have not been described before. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, which happens via a notch-responsive state. Together, our data revealed previously unrecognized ENS populations and serve as a resource to gain new insights on ENS development and specification, proving that the zebrafish is a valuable model organism in the quest towards understanding and treating congenital enteric neuropathies.

Human enteric glia diversity in health and disease: new avenues for the treatment of Hirschsprung disease

“…To investigate this, we made use of the available zebrafish HSCR model, ret hu2846/hu2846 (herein referred to as ret-/-), and performed scRNA-seq on 5dpf zebrafish intestines presenting with a phenotype reminiscent of total colonic aganglionosis (52) (Fig 6A). The scRNA-seq dataset obtained, was compared to the one we have previously reported, for wild-type 5dpf zebrafish intestines using the same approach (50). As expected, an overall dramatic loss of ENS cells in the ret-/-zebrafish was observed (Fig 6B).…”

“…To further explore the therapeutic potential of Schwann-like enteric glia, the zebrafish was used as a model organism. Previous studies have shown that in the intestine of both larvae (5 days postfertilization (dpf)) and adult zebrafish, enteric glia with neurogenic potential are present (50,51). In addition, SCPs have also been reported at 5dpf and were shown to play a key role in enteric neurogenesis during larvae development, as well as in case of injury (46,47).…”

“…scRNA sequencing analysis was performed using Seurat V3 (69). For analysis, scRNA-seq datasets obtained for the intestines of 5dpf wildtype (50) and ret-/-zebrafish, were integrated. Both datasets were down-sampled to 9200 cells to obtain an equal number of cells (nFeature_RNA > 100 & nFeature_RNA <4200 & percent.mito < 0.05), and the Seurat pipeline was used for normalization, integration and downstream analysis.…”

“…We used 50 dimensions with a resolution of 0.8 for the clustering, and UMAP for the processing of the individual datasets and also for the post-processing of the merged dataset. This led to 49 clusters of which four expressed neuronal and/or enteric progenitor markers (50). These four clusters were selected for a subset analysis, using 20 dimensions with a resolution of 0.5 for the clustering and UMAP.…”

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system

“…Recently, integration of single-cell RNA sequencing (scRNA-seq) into the zebrafish model for exploration of ENS development has yielded invaluable insights into what genes are expressed during ENS developmental phases (Howard et al, 2021;Kuil et al, 2023). Concurrently, the successful adoption of CRISPR technology for gene disruption in zebrafish, known for its high efficiency in analyzing phenotypes directly in the injected generation (F0), also known as "crispants", facilitates swift assessment of candidate genes (Gui et al, 2017;Hwang et al, 2013).…”