Schematic representation of the GI tract in mammalians and zebrafish. (A) Schematic representation of the layers present in the GI tract of mammalians and zebrafish. Zebrafish lack the muscularis mucosa and submucosal plexus. In addition, the enteric neurons are not organized in ganglia, but rather as individual cells (Wallace and Pack, 2003). (B) Schematic representation of the mammalian (human) and the zebrafish digestive system. While the human GI tract is divided in stomach, duodenum, jejenum, ileum and colon, the GI tract of the zebrafish is traditionally subdivided in three major components, rostral intestinal bulb, mid intestine and posterior segment (Wang et al., 2010). A new division based on conserved transcriptional profiles has also been proposed, which is depicted by color alterations and labels in regular font (Lickwar et al., 2017). Note that boundaries between sections should not necessarily be considered discrete. (C) Schematic representation of ENS development in zebrafish showing that the NCCs depicted in green are first detected in the developing intestine around 32–36 h post-fertilization (hpf). NCCs migrate in two parallel chains caudally, between 36 and 66–72 hpf. Starting rostrally, the NCCs start to migrate laterally to form a network. At 4 days post-fertilization (dpf) the ENS network has been formed around the total length of the intestine. (D) Schematic representation of ENS development in mammals. Vagal NCCs colonize the foregut at embryonic day (E)7-E9.5 in mice and gestational week (GW) 4 in humans. From E10.5, NCCs migrate caudally in various multicellular strands.

The transparency of zebrafish larvae allows non-invasive visualization of enteric neurons (A), peristalsis (B), and gut transit (C). (A) Microscopy images of Tg(phox2bb:GFP) zebrafish larvae at 5 dpf. Scale bar represents 500 μm (top), 200 μm (middle) and 50 μm (bottom). (B) Still image of the zebrafish intestine (top) and spatiotemporal map (STMap) of gut flow (bottom). Scale bar represents 200 μm. (C) Microscopic images of Tg(phox2bb:GFP) zebrafish larvae fed with fluorescently labeled food, showing typical intestinal transit and describing the transitional zone. Graphs showing phenotypical scoring of larvae directly after food intake (top graph) and 16 h after food intake (bottom graph). Scale bar represents 200 μm.

Functional analysis of ret morphants and mutants. (A) Phenotypical variability observed in zebrafish, using MO targeting Ret expression and two different zebrafish ret mutant lines. Proposed phenotyping classifications are shown by microscopy images and illustrations. Scale bar represents 200 μm. (B) Microscopic images of Tg(−8.3phox2bb:keade) zebrafish injected with control or ret MO, show normal ENS colonization in controls and a total colonic HSCR phenotype in the ret MO injected fish. Peristaltic function is disrupted in zebrafish lacking enteric neurons, which is shown by the absence of bowel movement in the STMaps. Scale bar represents 200 μm. Note that the use of the terms hypoganglionosis and HSCR in zebrafish despite the lack of ganglia in this organism, refers to the reminiscent phenotype observed in mammals (humans).