Abstract
Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes. Using CRISPR mutagenesis, we show that rsph9 is required for motility of presumptive 9 + 2 olfactory cilia and, unexpectedly, 9 + 0 neural cilia. rsph9 is also required for the structural integrity of 9 + 2 and 9 + 0 ciliary axonemes. rsph9 mutant larvae exhibit reduced initiation of the acoustic startle response consistent with hearing impairment, suggesting a novel role for Rsph9 in the kinocilia of the inner ear and/or lateral line neuromasts. These data identify novel roles for Rsph9 in 9 + 0 motile cilia and in sensory kinocilia, and establish a useful zebrafish PCD model.
Highlights
Respiratory infections, hearing impairment, infertility, situs inversus and hydrocephalus[16]
Since expression of radial spoke components has not been described in zebrafish, we isolated cDNA clones that encode Rsph[9] and Rsph4a and determined their expression patterns using whole mount in situ hybridization (WISH). rsph[9] was expressed in the ciliated structures, namely, Kupffer’s vesicle (KV) (Fig. 1A), pronephric ducts, otic placodes, and ventral spinal cord (Fig. 1B,C). rsph4a was expressed in a similar pattern (Fig. 1D). rsph[9] was expressed in the ventral midline of the midbrain primordium (Fig. 1C,E), as was foxj1a (Fig. 1F), a key transcriptional regulator of motile ciliogenesis[37,38,39]
We asked if zebrafish Rsph[9] protein localizes to the ciliary axonemes, using an antibody that recognizes a conserved epitope in Chlamydomonas reinhardtii and human RSPH9 proteins
Summary
Respiratory infections, hearing impairment, infertility, situs inversus and hydrocephalus[16]. Patients with spoke head mutations typically have ciliary transposition defects characterized by loss of the central pair and displacement of an outer microtubule doublet into the center of the axoneme[23,28,31] Airway cilia from these patients show partially penetrant deficits in beat frequency and waveforms, which range from normal (planar) to aberrant (rotational), suggesting that radial spokes are involved in setting the parameters of ciliary motility[23,30]. Due to their association with the central microtubule pair, radial spoke head protein function has been presumed to be restricted to 9 + 2 motile cilia. We show that Rsph[9] is required for the correct motility in both types of motile cilia, and present indirect evidence suggesting a functional role for Rsph[9] in kinocilia-bearing hair cells
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