A transgenic zebrafish forin vivovisualization of cilia

Abstract

Cilia are organelles for cellular signalling and motility. Mutations affecting ciliary function are also associated with cilia-related disorders (ciliopathies). The identification of cilia markers is critical for studying their function at the cellular level. Due to the lack of a conserved, short ciliary localization motif, the full-length ARL13b or 5HT 6 proteins are normally used for cilia labelling. Overexpression of these genes, however, can affect the function of cilia, leading to artefacts in cilia studies. Here, we show that Nephrocystin-3 (Nphp3) is highly conserved among vertebrates and demonstrate that the N-terminal truncated peptide of zebrafish Nphp3 can be used as a gratuitous cilia-specific marker. To visualize the dynamics of cilia in vivo , we generated a stable transgenic zebrafish Tg ( β-actin : nphp3N-mCherry ) sx1001 . The cilia in multiple cell types are efficiently labelled by the encoded fusion protein from embryonic stages to adulthood, without any developmental and physiological defects. We show that the line allows live imaging of ciliary dynamics and trafficking of cilia proteins, such as Kif7 and Smo, key regulators of the Hedgehog signalling pathway. Thus, we have generated an effective new tool for in vivo cilia studies that will help shed further light on the roles of these important organelles.

The role of cilia during organogenesis in zebrafish

“…It exhibits an 87% similarity with the human genome, with more than 82% of human pathogenic genes having homologues in zebrafish [55]. Zebrafish is a major model for live imaging in vertebrates, owing to its in vitro development and transparent embryos [9,[56][57][58]. In terms of genetic manipulation, forward genetics techniques are commonly employed in zebrafish to screen mutants through radiation, chemical reagents and DNA insertion, enabling genetic research in embryonic development and organ formation [59].…”

Structures and functions of cilia during vertebrate embryo development

“…In addition to developmental defects, dysregulation of cilia assembly/disassembly has been implicated in cancer (Cao et al, 2021 ; L. Wang & Dynlacht, 2018 ). While many studies of cilia dynamics have been done using tractable in vitro systems, tools developed for in vivo imaging studies (examples include [Ford et al, 2018 ; H. Zhang, Huang, et al, 2022 ]) provide opportunities to better understand the molecular cues and mechanisms that regulate the dynamic building and deconstruction of cilia in vertebrate embryos.…”