A multi-omics dataset of the developing zebrafish intestine: longitudinal transcriptomic and microbiome profiles in wild-type and IgZ knockout models

Technical Summary: A Multi-omics Dataset of the Developing Zebrafish Intestine

Problem Statement
The interaction between the host immune system and the gut microbiota is fundamental to mucosal immune homeostasis. In mammals, Immunoglobulin A (IgA) is a critical regulator of intestinal immunity and microbial composition. Teleost fish possess Immunoglobulin Z (IgZ, or IgT), a functional equivalent of IgA, which is enriched in mucosal tissues and mediates immune responses against pathogens. While the structural and functional similarities between IgZ and IgA are established, the specific developmental role of IgZ in regulating gut immunity and microbiota homeostasis remains unclear. Specifically, there is a lack of data regarding how sustained IgZ deficiency impacts intestinal immune regulation and microbial community structure across different developmental stages. Previous studies in mammals (Selective IgA Deficiency) and teleosts (IgT depletion) suggest that the absence of these immunoglobulins leads to barrier dysfunction, inflammation, and microbial dysbiosis, but the long-term kinetic effects of IgZ deficiency during zebrafish development have not been systematically characterized.

Methodology
This study employed a multi-omics approach to investigate the interplay between IgZ-mediated immunity and the gut microbiome in zebrafish.

• Model Generation: A stable zebrafish IgZ knockout (IgZ⁻/⁻) model was generated using CRISPR/Cas9 technology targeting a specific exon of the igz gene to induce premature translation termination. Homozygous mutants were established through heterozygous breeding.
• Experimental Design: The study utilized wild-type (WT) and IgZ⁻/⁻ zebrafish at three distinct developmental time points: 1, 2, and 3 months post-hatching. For each time point and genotype, five biological replicates were used for both transcriptomic and microbiome analyses.
• Sample Collection: Intestinal tissues and mucus were collected following euthanasia and blood clearance. Samples were processed for protein analysis, RNA extraction, and DNA extraction.
• Omics Profiling:
  • Microbiome: The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced on an Illumina NextSeq2000 platform. Data processing involved quality control (fastp), merging (FLASH), denoising (DADA2), and taxonomic classification against the SILVA database.
  • Transcriptome: Total RNA from intestinal tissues was sequenced using the NovaSeq X Plus platform (PE150). Data analysis included adapter trimming, alignment to the reference genome (STAR), and differential expression analysis (DESeq2).
• Validation: The knockout efficiency was confirmed via Western blotting and qRT-PCR, showing a significant deficiency in IgZ protein in serum and mucus. Transcriptome reliability was validated by comparing RNA-seq results with qRT-PCR for 20 randomly selected differentially expressed genes (correlation coefficient R = 0.96).

Key Contributions and Results
The primary contribution of this work is the generation and public release of an integrated multi-omics dataset comprising longitudinal transcriptomic and microbiome profiles of WT and IgZ⁻/⁻ zebrafish.

• Data Generation: The study produced high-quality sequencing data, identifying a total of 1,397 amplicon sequence variants (ASVs) from the microbiome and generating approximately 1.28 billion clean reads for the transcriptome.
• Knockout Validation: Technical validation confirmed that the IgZ⁻/⁻ model successfully lacked IgZ protein in the serum and intestinal mucus while maintaining stable transcriptional levels of other immunoglobulins (IgM and IgD) in various tissues.
• Data Quality: Sequencing metrics demonstrated high reliability. For 16S data, Q20 values exceeded 98%, and average sequence lengths were consistent with the V3–V4 region. For RNA-seq, Q30 scores exceeded 96%, with mapping rates between 88.62% and 93.78%. Principal Component Analysis (PCA) and PCoA plots confirmed clear separation between experimental groups and tight clustering of biological replicates, indicating high reproducibility.
• Phenotypic Assessment: The dataset includes assessments of IgZ secretion status, intestinal pathology, and inflammatory responses, providing a comprehensive view of the consequences of IgZ deficiency.

Significance
The paper claims that this dataset provides critical insights into the interplay between gut immunity and the microbiota in zebrafish following IgZ deficiency across different developmental stages. By integrating longitudinal microbiome and transcriptome data, the study addresses the gap in understanding how host immune homeostasis is maintained in the absence of IgZ during development. The authors posit that this resource will facilitate further research into the mechanisms of mucosal immunity and microbial regulation in teleosts, serving as a foundation for understanding the evolutionary conservation of IgA/IgZ functions in vertebrate gut health. The data is publicly available via the Sequence Read Archive (SRA) under BioProject accession numbers PRJNA1419456 (16S) and PRJNA1419499 (transcriptome).