Novel roles for the radial spoke head protein 9 in neural and neurosensory cilia

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.

Zebrafish Rfx4 controls dorsal and ventral midline formation in the neural tube

“…5G, H. To determine if motile cilia produced by floor plate cells were also disrupted, we used immunostaining for a component of motile cilia, Rsph9 (Sedykh et al 2016) together with pan-ciliary acetylated tubulin. Despite their aberrant organization, mutant floor plate cells produced long Rsph9-positive cilia (Fig.…”

“…CRISPR was designed to target a site 5 0 -TC TCA GGC GTC CTC ATG CCC-3 0 in exon 8 (minus strand). Mutagenesis and founder identification were carried out as previously described (Sedykh et al, 2016), with HRMA primers 5 0 -TTT CGC AAA AGT CCA GGT AGT G-3 0 (reverse) and 5 0 -TGG GTG TGT GTG ACT CCA TAT TG-3 0 (forward).…”

Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis

“…Design of zic2b CRISPR site 5′-GGTGGAGTTAAAAGTGGAGC-3′ in exon1, mutagenesis and founder identifications were carried out as previously described ((Sedykh et al, 2016), with the following HRMA primers pairs: TALEN site - 5′-TGGACAACACCCCATCTTCA-3′ and 5′-GGATGTTTGGAGAGCCGTGAT-3; CRISPR site - 5′-TATTCTGCGGCCGCTCTT3′ and 5′-GGAGTCGAATCCCCAAATC-3′.…”

Radial spoke proteins regulate otolith formation during early zebrafish development

“…A search of the zebrafish genome failed to identify a duplicate gene, which suggests that the components of the radial spoke proteins may function redundantly to regulate cilia motility. These phenotypes differed from that of another radial spoke mutant published recently, which had ciliary motility defects in both the olfactory placode and ventral spinal canal (36). This discrepancy may be caused by the specific roles of Rsph9, which functions in both motile and immotile cilia (kinocilia) (36).…”