Making sense of carbonic anhydrase function in zebrafish using antisense morpholinos.

Understanding gene function in vertebrate development requires tools that allow precise and timely manipulation of gene expression. Zebrafish (Danio rerio), with its transparent embryos and rapid development, offers an ideal model to study vertebrate biology. This review explores how morpholino oligonucleotides (MOs), a widely used tool for transient gene knockdown, have been employed to investigate the roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs) in zebrafish. CAs are metalloenzymes, while CARPs are inactive isozymes that play critical roles in pH regulation, ion transport, CO₂ metabolism, and protein interactions influencing diverse biological functions. Many of the MO knockdown studies presented here have been extensively conducted in our laboratory over the past decade, revealing novel roles for CAs in neural development, reproduction, and swim bladder formation. These studies also confirm roles previously reported in humans, such as pigmentation, acid-base homeostasis, neural development, and motor coordination. We discuss technical aspects of MO design, delivery, and validation, and address common challenges such as off-target effects, transient gene silencing, and the necessity of rescue experiments. In addition, the review includes a comparative analysis of MOs versus CRISPR/Cas9-based genome editing, underscoring their respective advantages and limitations for functional genomics. In conclusion, this review provides not only a methodological guide but also biological insights into CA function in zebrafish, highlighting how antisense technology continues to inform vertebrate development and disease modeling. The lessons learned here may inform the study of other gene families and support translational research in carbonic anhydrase-related human disorders.