Multicilin Redundancy in the Zebrafish MCC Programme as a Result of Evolutionary Divergence

Cilia play a crucial role in left-right symmetry and fetus development. Cells with dozens of cilia are termed as multiciliated cells (MCCs). Improper MCC differentiation may result in impeded fluid flow, a cause of airway infections, primary ciliary dyskinesia, situs inversus, and retinal degradation, classified under a broad term RGMC (reduced generation of motile cilia). In humans, multicilin (MCI) is inhibited by geminin (GMNN), and overexpression resulted in ectopic MCC differentiation. We sought to explore whether mci functions similarly in zebrafish. By cloning and transforming mci into E. coli, then extracting it and transcribing it, we injected them into 1-cell stage zebrafish gmnn knockout embryos. We then stained these embryos for cilia, basal bodies, and nuclei, before checking for the presence of ectopic cilia under confocal microscopy. Overexpression of mci did not result in complete ectopic MCC differentiation, with isolated ectopic cilia in various tissues. This means that gmnn may not be completely inhibiting mci in zebrafish, rather it is somewhat dispensable in the zebrafish MCC differentiation programme. This may be due to is evolutionary history, as its sequence diverges significantly from other mci homologues. These findings altogether may indicate that the zebrafish MCC programme diverges from humans and may lay the foundation for understanding causes of RGMC and how it may be treated.