A Rapid F0 CRISPR Screen in Zebrafish to Identify Regulator Genes of Neuronal Development in the Enteric Nervous System

Abstract

Background: The neural crest-derived enteric nervous system (ENS) provides the intrinsic innervation of the gut with diverse neuronal subtypes and glial cells. The ENS regulates all essential gut functions, such as motility, nutrient uptake, immune response, and microbiota colonization. Deficits in ENS neuron numbers and composition cause debilitating gut dysfunction. Yet, few studies have identified genes that control neuronal differentiation and the generation of the diverse neuronal subtypes in the ENS.

Methods: Utilizing existing CRISPR/Cas9 genome editing technology in zebrafish, we have developed a rapid and scalable screening approach for identifying genes that regulate ENS neurogenesis.

Key results: As a proof-of-concept, F0 guide RNA-injected larvae (F0 crispants) targeting the known ENS regulator genes sox10, ret, or phox2bb phenocopied known ENS phenotypes with high efficiency. We evaluated 10 transcription factor candidate genes as regulators of ENS neurogenesis and function. F0 crispants for five of the tested genes have fewer ENS neurons. Secondary assays in F0 crispants for a subset of the genes that had fewer neurons reveal no effect on enteric progenitor cell migration but differential changes in gut motility.

Conclusions: Our multistep, yet straightforward CRISPR screening approach in zebrafish tests the genetic basis of ENS developmental and disease gene functions that will facilitate the high-throughput evaluation of candidate genes from transcriptomic, genome-wide association, or other ENS-omics studies. Such in vivo ENS F0 crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.

Keywords: CRISPR/Cas9; ENS neurons; ENS neuropathies; enteric progenitor cells; intestinal transit.

FIGURE 1

F0 crispants targeting ret, sox10, or phox2bb recapitulate known ENS phenotypes. (A–E) F0 crispants targeting ret phenocopy the lack of phox2bb:GFP ⁺ ENS cells (green) in stable ret mutant larvae (C) in comparison to wildtype‐level ENS cells in control larvae (A). Compare ENS phenotypes between (C) and (D), arrows point to remaining ENS neurons in the anterior gut. (E) A small portion of ret F0 crispants show ENS cell reduction (B) Schematic of zebrafish larvae, the gray boxed area indicates the area shown in A‐M. Line indicates length used for quantification of anterior neuron numbers in ret crispants in (F). (F) Significantly fewer GFP⁺ cells are present within 200 um of anterior gut in ret F0 crispants compared to control injected larvae (p < 0.0001, 2 experiments, % shown as mean ± SEM, each data point equals one larva). (G) % of ENS phenotypes in ret F0 crispants (2 experiments, % shown as mean ± SEM). (H–J) F0 crispants targeting sox10 (I) phenocopy the lack of GFP⁺ ENS cells in stable sox10 mutant larvae (H). A small portion of F0 crispants targeting sox10 show ENS cell reduction (J). (K) % of ENS phenotypes in control slc24a5, sox10, and phox2bb F0 crispants (≥ 2 experiments, % shown as mean ± SEM). (L, M) The majority of F0 crispants lack GFP⁺ ENS cells (L). A small portion have reduction in ENS cells (M). (A, C–E, H–J, L–M) Whole‐mount side views at 6 days post fertilization (dpf). Asterisks: autofluorescence in gut epithelium; dashed line: Gut outline. Scale bar = 100 μm.

FIGURE 2

F0 CRISPR screen identifies new regulators of ENS neurogenesis. The screening pipeline is designed to be performed within 10 days. (A) Schematic of zebrafish larvae, the boxed area indicates the area shown in B–G‴. (B) Uninjected control and (C) slc24a5 F0 crispants show wildtype levels of GFP⁺ ENS neurons (green) at 6 dpf. (D, F, G) F0 crispants at 6 dpf with fewer GFP⁺ cells targeting jarid2a + jarid2b (pool 1), mycn + foxj2 + foxj3 (pool3), dlx1a + foxn3 + phox2a (pool 4), but not (E) homeza + homezb (pool 2). F0 crispants, which target each gene individually from the three pools with ENS phenotypes, show fewer GFP⁺ cells for jarid2a (D′), mycn (F′), foxj3 (F‴), dlx1a (G′) and phox2a (G‴), but not jarid2b (D″), foxj2 (F″), and foxn3 (G″). (H) Average percentage of F0 crispants with and without reduced ENS phenotypes for each pool and each individual gene (≥ 2 experiments, % shown as mean ± SEM). The dashed line shows the percent phenotypes for the slc24a5 control F0 crispants. Asterisks show a consistent and significant difference using an unpaired t‐test (p < 0.05) to slc24a5 F0 crispant controls. (I) Average percentage of F0 crispants with and without reduced ENS phenotypes for second screen using two additional gRNAs for the individual genes with a phenotype (≥ 2 experiments, % shown as mean ± SEM). The dashed line shows the percent phenotypes for the slc24a5 control F0 crispants. Asterisks show a consistent and significant difference to slc24a5 F0 crispant controls using an unpaired t‐test (p < 0.05). (J) F0 crispants for each gene show a high percentage of indels in PCR amplicons of the target area (≥ 2 experiments, % shown as mean ± SEM, each data point equals one larva). (K) F0 crispants for each gene show a high percentage of indels in PCR amplicons of the target area (2 experiments, % shown as mean ± SEM, each data point equals one larva). Asterisks: Autofluorescence in gut epithelium; dashed line: Gut outline. Scale bar = 100 μm.

FIGURE 3

Secondary assays analyzing enteric progenitor cell migration and intestinal transit in F0 crispants. (A, top) slc24a5 F0 crispant control and embryo schematic (A, bottom) show the bilateral streams of GFP⁺ enteric progenitor cells (EPCs, green) migrating posteriorly to populate the gut. The gray box outlines the region of the embryo (A, top) and scale bar denotes the distance measured between the EPC migration front and the end of the gut (migration progress). (B) Quantification of migration progress in F0 crispants for jarid2a, dlx1a, and mycn compared to F0 slc24a5 crispant controls showed no significant (ns) difference (two experiments, each data point equals one embryo). (C) Experimental set‐up for the intestinal transit assay. Phenotypically sorted larvae are fed fluorescently labeled food at 6 and 7dpf. On day 7, 4 h post‐feeding, larvae that have eaten are selected (“feeders”) and placed in a new dish without food. Sixteen hours later, clearance of labeled food from the gut is determined. In sox10 mutants (E) and jarid2a F0 crispants (F) fluorescently labeled food (green) is still visible in the guts compared to the cleared guts of controls (D). (G) Quantification of percent larvae with cleared guts (≥ 2 experiments, % shown as mean ± SEM, each data point equals one experiment, each experiment ≥ 6 larvae). sox10 mutants and jarid2a F0 crispants have significantly lower percentages of larvae with cleared guts compared to controls. Asterisks show a significant difference to controls using an unpaired t‐test (p < 0.05) or no significant (ns) difference. dlx1a and mycn F0 crispants trend towards fewer larvae with cleared guts. Whole‐mount side views of 66 h post fertilization (hpf) embryo (A) and 8 days post fertilization (dpf) larvae (D–F) in the area indicated in the schematics. Scale bar = 100 μm.

FIGURE 4

Working model for functions of candidate genes with reduced ENS neurons. The working model combines our F0 screen results with known roles in neuronal development. Based on this, we propose which steps and stages of ENS neuronal development (migration or differentiation) and ENS function the different genes regulate.