Fig. 2

Examination on the inflammatory bowel disease (IBD)-like phenotype in ctla-4-/- zebrafish. (A) Generation of a homozygous Cytotoxic T lymphocyte antigen-4 (ctla-4)-deficient (ctla-4-/-) zebrafish line through CRISPR/Cas9-based knockout of ctla-4 gene on chromosome 9. A 14 bp deletion mutation in exon 2 results in a premature stop at codon 82, which is predicted to produce a defective Ctla-4 protein containing 81 amino acids. (B) Genotyping of the deficiency of ctla-4 gene by Sanger sequencing. (C) Knockout efficiency of Ctla-4 selectively examined in spleen and gut tissues of ctla-4-/- zebrafish by Western blot analysis. Gapdh serves as a loading control. (D) Normal gross appearance of adult wild-type (WT) and ctla-4-/- zebrafish. (E) Body weight statistics of WT and ctla-4-/- zebrafish (n=30). (F) The change of intestine length in WT and ctla-4-/- zebrafish. (G) The change of splenic size in WT and ctla-4-/- zebrafish. (H) Representative H&E staining analysis of histopathological changes and quantitation of histology scores in the anterior, mid, and posterior intestines from WT and ctla-4-/- zebrafish. Red arrows denote mucosal inflammatory cell infiltration, and black arrow indicates transmural inflammatory cell infiltration. (I) Alcian Blue and Periodic Acid-Schiff (AB-PAS) staining was used to analyze the mucin components and the number of goblet cells in anterior intestine from WT and ctla-4-/- zebrafish (n=5). (J) Quantitation analysis of goblet cells of each villus in the foregut of WT and ctla-4-/- zebrafish (n=8). (K) Observation of cell junctions between intestinal epithelial cells in posterior intestines from WT and ctla-4-/- zebrafish under TEM (Hitachi Model H-7650). White triangles indicate tight junctions, black triangles indicate adhesion junctions, and red triangles indicate desmosomes. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed through an unpaired Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).