Homology comparison of Ace homologues in different species. (A) The secondary structures of Ace homologues in D. rerio, H. sapiens, M. musculus, and X. tropicalis. This diagram was generated using SMART online software (http://smart.embl-heidelberg.de/, accessed on 1 December 2023). Red squares, signal peptides; blue rectangles, transmembrane regions; M2, metallopeptidase 2 domain. The similarity index was derived by DNAMAN software (9.0.1.116). (B) The three-dimensional structures of D. rerio ACE, H. sapiens ACE, M. musculus ACE, and X. tropicalis ACE. This diagram was generated using the Alphafold online server (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb accessed on 5 December 2023). AlphaFold produces a per-residue model confidence score (pLDDT, predicted local distance difference test) between 0 and 100. Some regions below 50 pLDDT may be unstructured in isolation. (C) A phylogenetic tree of Ace homologues of various species was constructed with IQ-tree using the maximum likelihood method. Hs: H. sapiens (NP_000780), Mm: M. musculus (NP_997507), Gg: G. gallus (NP_001161204), La: L. agilis (XP_033024785), Xt: X. tropicalis (NP_001116882), Cc: C. carcharias (XP_041029546), Dr: D. rerio (XP_694336), Pn: P. nyererei (XP_005721704), Lo: L. oculatus (XP_015217378), Pm: P. marinus (XP_032821231, XP_032807619), Ci: C. intestinalis (XP_026693950), Sk: S. kowalevskii (XP_002741143), and Ta: T. adhaerens (XP_002111333). Each node was bootstrapped with 1000 replications to estimate its reliability. (D) Syntenic analysis among zebrafish ace genomic neighborhoods and those of humans, frogs, and flameback cichlids. Different colors indicate different genes. Orthologs of these genes in other species are shown in corresponding colors.

Homology comparison of Ace homologues in different species. (A) The secondary structures of Ace homologues in D. rerio, H. sapiens, M. musculus, and X. tropicalis. This diagram was generated using SMART online software (http://smart.embl-heidelberg.de/, accessed on 1 December 2023). Red squares, signal peptides; blue rectangles, transmembrane regions; M2, metallopeptidase 2 domain. The similarity index was derived by DNAMAN software (9.0.1.116). (B) The three-dimensional structures of D. rerio ACE, H. sapiens ACE, M. musculus ACE, and X. tropicalis ACE. This diagram was generated using the Alphafold online server (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb accessed on 5 December 2023). AlphaFold produces a per-residue model confidence score (pLDDT, predicted local distance difference test) between 0 and 100. Some regions below 50 pLDDT may be unstructured in isolation. (C) A phylogenetic tree of Ace homologues of various species was constructed with IQ-tree using the maximum likelihood method. Hs: H. sapiens (NP_000780), Mm: M. musculus (NP_997507), Gg: G. gallus (NP_001161204), La: L. agilis (XP_033024785), Xt: X. tropicalis (NP_001116882), Cc: C. carcharias (XP_041029546), Dr: D. rerio (XP_694336), Pn: P. nyererei (XP_005721704), Lo: L. oculatus (XP_015217378), Pm: P. marinus (XP_032821231, XP_032807619), Ci: C. intestinalis (XP_026693950), Sk: S. kowalevskii (XP_002741143), and Ta: T. adhaerens (XP_002111333). Each node was bootstrapped with 1000 replications to estimate its reliability. (D) Syntenic analysis among zebrafish ace genomic neighborhoods and those of humans, frogs, and flameback cichlids. Different colors indicate different genes. Orthologs of these genes in other species are shown in corresponding colors.

Expression patterns of ace mRNA in zebrafish. (A) Transcriptional expression patterns of ace mRNA in different tissues (heart, liver, spleen, gill, kidney, muscle, intestine, eye, brain, skin, testis, and ovary) were detected by RT-PCR. β-actin was used as an internal control. (B,C) Relative transcriptional expression of ace in different tissues and different developmental stages as measured by RT-qPCR. hpf: hours post-fertilization. β-actin was used as an internal control. (D) Spatio-temporal expression patterns of ace in embryos detected by WISH. “a–d” represents the results of anti-sense probe hybridization, while “e–h” represents the results of sense probe hybridization (negative control). dpf: days post-fertilization. (E) The intestinal region of the larvae at 5 dpf after WISH was frozen and sectioned (8 µm). The red line indicates the position of the frozen section.

Subcellular localization of Ace in HEK293T cells. Recombinant plasmids pcDNA3.1/V5/Ace/eGFP and pcDNA3.1/V5/eGFP (control) were transiently transfected into HET293T cells. DAPI stained the nucleus. Red arrows point to the cell membrane and white arrowheads point to the endoplasmic reticulum.

Knockout of ace and morphological examination of ace^−/− mutant larvae. (A) The generation of ace mutations in zebrafish through CRISPR/Cas9 technology. (a). The targeted exon 8 of ace containing the knockout site. Exons are indicated by boxes, blue box means coding region of exon, while white box means non-coding region of exon, introns are represented by fold lines. The red letter in the wild type (WT) means the position some changes will happen, while the dotted line and blue letter corresponded to red letters means deletion and transition happened in the mutant (MT), respectively. (b,c). In comparison to the wild type, the mutants had a deletion of 7 nucleotides in exon 8, leading to skipped mutation (red letters), and premature termination of translation at the 468th amino acid. The asterisk in the figure indicates the position of protein translation termination. (d). To assess the impact of ace knockout, RT-PCR was performed to analyze ace expression in both ace^−/− and wild-type samples (B) Expression patterns of fabp2 in ace^−/− mutants and wild-type larvae at 5 dpf, as detected by WISH. (C) The morphology of the intestinal epithelium in ace^−/− mutants and wild-type larvae at 5 dpf, as shown by HE staining.

RNA-seq analysis of ace^−/− mutants and wild type at 5 dpf. (A,B) GO and KEGG analysis of the pathways through the enrichment of DEGs between ace^−/− mutants and wild type. The red box signifies the peptide enzyme or immune-related signaling pathways. (C) The table shows some DEGs with significant changes, including genes highly expressed in intestinal epithelium and some immune genes. (D) Single-cell profile of ACE in fxr^−/− zebrafish larvae at 6 dpf.

Inflammatory response evaluation for ace^−/− mutants and wild-type larvae at 5 dpf. (A,B) qRT-PCR analysis of selected inflammation-related genes. (C) AB-PAS staining sections label mucus in the intestine of both ace^−/− mutants and wild-type larvae at 5 dpf (upper panel). The lower panels provide enlarged photographs of the intestinal bulb and mid- and posterior intestine regions. The red arrowhead in the figure indicates the staining signal. (D) Cell statistics for Alcian blue staining in the digestive tract of ace^−/− mutants and wild-type larvae at 5 dpf. Data are presented as the mean ± SD of 10 fish. ** p < 0.01, *** p < 0.001. The experiments were independently repeated three times.

The IBD model was successfully constructed using DSS. (A) A diagram of IBD model development. (B) Changes in DSS-treated and untreated wild-type larvae were examined by qRT-PCR for several pro-inflammatory factors: ilb, lect2l, cxcl8b, mmp9, and mpx. Data represent the mean ± SD. * p < 0.05, ** p < 0.01. Three independent biological replicates were performed. (C) Line chart of survival rates comparing ace^−/− mutants and wild-type larvae treated as the DSS dose (% (w/v)) increased from 0% to 1% (n = 50). The significance of differences is annotated above the nodes in the line chart. * p < 0.05, ** p < 0.01. (D) HE staining shows a more severely disorganized intestinal epithelium in ace^−/− mutants compared with the wild type at 6 dpf following DSS treatment.

Histopathological assessment comparing ace^−/− mutant and wild-type larvae treated with DSS. (A) AB-PAS staining sections highlighting mucus in the intestine of DSS-treated and untreated ace^−/− mutants and wild-type larvae (upper panel). The lower panels show enlarged photographs of the intestinal bulb and the mid- and posterior intestine regions. At the bottom, the control group exhibits AB-PAS staining sections that did not undergo DSS treatment. The red arrow in the figure indicates the staining signal. (B) (a). Quantification of Alcian blue-stained cells in the digestive tract of ace^−/− mutants and wild-type larvae at 6 dpf. Data represent the mean ± SD of 10 fish. (b–f). The expression of several pro-inflammatory factors in ace^−/− mutants and wild-type larvae examined by qRT-PCR. Data are presented as the mean ± SD. Different lowercase letters indicate significant differences (p < 0.05) within the group; different uppercase letters indicate significant differences (p < 0.05) between the groups after DSS treatment; different Latin letters indicate significant differences (p < 0.05) between the groups before DSS treatment.