Cilia motility studies in zebrafish embryos

Abstract

Motile ciliary dysfunctions cause specific Ciliopathies that affect mainly the respiratory tract, fertilization and left-right body establishment. The embryonic organ where left-right decisions are first taken is called the organizer, a ciliated organ where a leftward cilia driven fluid-flow is generated. The organizer is named node in the mouse and Kupffer’s vesicle (KV) in zebrafish. The correct left-right axis formation is highly dependent on signaling pathways downstream of such directional fluid-flow. Motile cilia need to be coordinated and Ciliary Beat Frequency (CBF) is characteristic of different types of cilia depending on their function. Using zebrafish as a model, our group has been studying cilia length regulation and motility in wild-type and deltaD-/- mutant embryos. Recently, we showed that Notch signalling was directly involved in the control of cilia length in the KV cells given that the deltaD-/- mutant present shorter KV cilia. The goal of this project was to characterize the CBF of deltaD-/- KV cilia vs. wild-type cilia and reveal how potential differences in CBF impact on KV fluid flow, using spectral analysis associated with highspeed videomicroscopy. By decomposing and comparing the obtained CBF with Fast Fourier Transform, we identified two major populations of motile cilia in wild-type as well as in deltaD-/- mutant embryos. However, we found the CBF populations had differential relative contributions and different distributions between wild-type and mutant embryos. Furthermore, by measuring the velocity of native particles we studied the KV fluid-flow and concluded that the dispersion of the flow velocity was much wider in the deltaD-/- mutants. On the other hand, based on a gene expression study of motility genes downstream of DeltaD, we concluded that motility related genes (dnah7, rsph3 and foxj1a) were deregulated in the mutants. During this project we generated data that led to new hypotheses that will allow us to test the causality between the described correlations.