Dynamic expression of the zebrafish (Danio rerio) proteome across early larval development

Zebrafish is one of the most important model organisms across diverse disciplines from developmental biology to environmental science and engineering, and toxicology, substantially contributing to reductions in the use of mammals in disciplines that have previously relied on rodent models. The use of immature zebrafish in particular have arguably contributed to the largest reduction in animal usage, at least during toxicology experimentation, since these ages are not counted as laboratory animals unless they have reached the free-feeding stage (> 5 dpf for zebrafish reared at 28 °C). Despite its use as a developmental and animal alternative model, baseline biomolecule expression dynamics are poorly understood throughout the zebrafish lifecycle. While the adult (> 3 months) and embryonic proteome (2-120 hpf) of this species have been previously characterized, variations in proteome expression across early life stages is limited. Here, we examined proteome expression patterns in larval zebrafish at 4-, 7- and 10 dpf. We specifically identified 11,344 proteins (5 % false discovery rate) using bottom-up shotgun proteomics, with many proteins unique to experimental time points and minimal overlap among age groups (∼ 5-12 %). Longitudinal examination of differentially expressed proteins indicates coordinated expression of the muscular system and contractile proteins with extracellular matrix structure aligning with growth, muscle development, and movement observations in larval fish. Considering the use of early life stage zebrafish as a model organism and an animal alternative vertebrate method, differential expression and enrichment of evolutionarily conserved processes, especially associated with developmental periods of metabolic stress, highlight potential experimental confounders requiring further investigation.