Quantification of Enteric Neuron Maturation

To determine the proportion of terminally differentiated enteric neurons, 5 dpf F2 zebrafish larvae (tfap2b^+/-, phox2bb:GFP) treated with PTU were immunostained with antiHuC/HuD, a mature neuronal marker. Larvae were incubated on ice for 30 min before being fixed in 4% PFA and washed in 1x phosphate buffer solution/0.25% Triton X-100 for 1 h, at room temperature. Whole mount antibody staining was performed according to previous reports (Uyttebroek et al., 2010 (link)). Anti HuC/HuD (1:100, A-21271, Invitrogen, Waltham, Massachusetts, United States) was used as primary antibody and Cy3 Mouse IgG (1:500, Thermo Fisher Scientific, Waltham, Massachusetts, United States) as secondary antibody. Larvae were imaged under the confocal microscope (Leica SP5 AOBS, Leica Camera, Wetzlar, Germany). The number of phox2bb:GFP⁺ and HuC/HuD⁺ cells were counted using Fiji ImageJ software. The proportion of differentiated enteric neurons was determined by calculating the ratio of HuC/HuD⁺ cells to phox2bb:GFP⁺ cells. All larvae were genotyped at the end of the experiment.