Loss of Rsph9 causes neonatal hydrocephalus with abnormal development of motile cilia in mice

Hydrocephalus is a brain disorder triggered by cerebrospinal fluid accumulation in brain cavities. Even though cerebrospinal fluid flow is known to be driven by the orchestrated beating of the bundled motile cilia of ependymal cells, little is known about the mechanism of ciliary motility. RSPH9 is increasingly becoming recognized as a vital component of radial spokes in ciliary “9 + 2” ultrastructure organization. Here, we show that deletion of the Rsph9 gene leads to the development of hydrocephalus in the early postnatal period. However, the neurodevelopment and astrocyte development are normal in embryonic Rsph9−/− mice. The tubular structure of the central aqueduct was comparable in Rsph9−/− mice. Using high-speed video microscopy, we visualized lower beating amplitude and irregular rotation beating pattern of cilia bundles in Rsph9−/− mice compared with that of wild-type mice. And the centriolar patch size was significantly increased in Rsph9−/− cells. TEM results showed that deletion of Rsph9 causes little impact in ciliary axonemal organization but the Rsph9−/− cilia frequently had abnormal ectopic ciliary membrane inclusions. In addition, hydrocephalus in Rsph9−/− mice results in the development of astrogliosis, microgliosis and cerebrovascular abnormalities. Eventually, the ependymal cells sloughed off of the lateral wall. Our results collectively suggested that RSPH9 is essential for ciliary structure and motility of mouse ependymal cilia, and its deletion causes the pathogenesis of hydrocephalus.

www.nature.com/scientificreports/ pair of single microtubules because of the gap between them. Thus, we further investigated the spatiotemporal developmental function of RSPH9 using mouse model. In this study, we generated global knockout mouse models to elucidate the pathogenesis of PCD by targeting the murine Rsph9 locus. We systematically investigated the development of Rsph9 −/− mice to understand the consequence of losing this gene. Our study reveals the role of RSPH9 in hydrocephalus pathogenesis and ependymal cilia motility in the developing mouse brain.

Results
Targeting Rsph9 in mice. RSPH9-associated primary ciliary dyskinesia has a wide phenotypic variability in humans. To target Rsph9 in mice, we used the CRISPR-Cas9 system and the zygote microinjection of a single-guide RNA1 targeting exon1 of Rsph9 (Fig. 1A). The strategy deleted 8 base pairs to produce producing a premature stop codon at the end of exon 1, which significantly truncated the RSPH9 protein (Fig. 1B). The truncated RSPH9 with 61 amino acid residues are much shorter compared with normal RSPH9 with 276 amino acid residues. The generated heterozygous Rsph9 +/− mice were viable and fertile. We backcrossed them with C57BL/6 mice for more than five generations to obtain a more purified genetic background.
Homozygous Rsph9 mutations cause a slower growth rate and postnatal lethality. Rsph9 +/− mice were interbred to obtain homozygous knockout mice and were confirmed by genotyping PCR (Fig. S1A). The genotyping results on the first postnatal day revealed a prospective Mendelian ratio (1:2:1) and no sexspecific differences in survival. RSPH9 is highly expressed in multiciliated cells. Immunostaining with RSPH9 showed deletion of RSPH9 in Rsph9 −/− brain ependymal cilia and tracheal cilia (Fig. 1C,D). However, the knockout pups failed to grow normally, and most died during the weaning phase ( Fig. 1E-G). Only a very few survived into adult (Fig. S1B). These Rsph9 −/− mice are characterized by enlarged dome-shaped skulls and severe neurological symptoms, including lethargy, apathy and muscle weakness.
Rsph9 −/− mice develop progressive hydrocephalus and sinusitis. PCD is usually accompanied by randomized body laterality and respiratory disease. Therefore, we investigated these phenotypes in mice. There was no situs inversus in Rsph9 −/− mice, which shows RSPH9 is not associated with the determination of the left-right axis of visceral organs ( Fig. S2A; n = 6). However, Rsph9 −/− mice developed severe neurological disorders and sinusitis (Fig. S2B). To analyze macrocephaly in Rsph9 −/− mice, the brains were isolated and were found to be enlarged ( Fig. 2A). Magnetic resonance imaging revealed severe thinning of the cerebral cortex and enlarged hemispheres with massive accumulation of CSF in the lateral ventricles (Fig. 2B). The hippocampus and the hypothalamus were severely compressed. All of these are characteristic symptoms of hydrocephalus. To characterize the temporal feature of hydrocephalus in mice with the Rsph9 deletion, we compared sagittal sections of the developing brain between P0 and P7 in wild-type and Rsph9 −/− mouse pups (Fig. 2C). There was no significant difference at P0. However, the area of lateral cerebral ventricular zone was comparatively increased at P3 and P7. Histological analysis of coronal brain sections revealed enlarged ventricles with abnormal brain morphology in P8 Rsph9 −/− mice (Fig. 2D,E). Cerebrospinal fluid analysis showed clear CSF and no visible brain hemorrhage, which indicated that hydrocephalus was not caused by hemorrhaging in the Rsph9 −/− mouse brain. Therefore, RSPH9 is not associated with situs inversus, but sinusitis. Furthermore, deletion of Rsph9 can result in the development of brain dysfunction and progressive hydrocephalus during postnatal development in mice.
Hydrocephalus is not caused by embryonic development defects in Rsph9 −/− mice. Since ciliopathies are a group of genetic disorders closely related to neuronal cell fate, migration, and differentiation, as well as a host of adult behaviors 13 . We investigated whether hydrocephalus originates by embryonic brain developmental disorders. First, BrdU/EdU dual labeling experiments were conducted to confirm whether RSPH9 affects the neurogenesis process. The results showed that there was no significant difference in cell cycle dynamics in embryos at 15 days when comparing wild-type and Rsph9 −/− mice (Fig. 3A,B). Then the cortex markers CUX1 and CTIP2 were used for labeling layer II-IV and layer V of neonatal mice, which showed the typical cortical layer pattern of Rsph9 −/− mice (Fig. 3C,D). Neuronal migration is also not affected by Rsph9 deletion. Immunofluorescent staining of glial fibrillary acidic protein (GFAP) was used to label astrocytes, and we found no significant change in GFAP-positive cell number or expression pattern between P0 Rsph9 +/+ and Rsph9 −/− mice (Fig. 3E,F). Taken together, neurogenesis, neuronal migration and astrocyte development were determined to be undisturbed by Rsph9 deletion in mice. Hydrocephalus was caused by postnatal developmental defects.
CSF flow and circulation are impaired in Rsph9 −/− mice. Cerebrospinal fluid is produced by the choroid plexus in lateral ventricles passing through the foramina of Monro, the 3rd ventricle, the cerebral aqueduct and the 4th ventricle. We injected Evans blue dye into the right lateral ventricle to investigate CSF flow through the ventricular system in Rsph9 −/− mice. In wild-type mice, the tracer could travel through the third ventricle, central aqueduct and fourth ventricle 10 min after injection ( Fig. 4A; the upper row, n = 3). In contrast, no tracer or only very little tracer could be detected at the third ventricle and the fourth ventricle in Rsph9 −/− mice ( Fig. 4A; the lower row, n = 4). Nissl staining results showed that the shape of the central aqueduct in P8 Rsph9 −/− mice was intact, the size was unchanged, and no blockage occurred (Fig. 4B). Thus, these results indicate that the barrier of CSF flow is due to the disrupted activity of the ependymal cells.  www.nature.com/scientificreports/ revealed that the movement of beating cilia bundles is characterized by lower amplitude from the side views in P7 Rsph9 −/− mice compared with that of wild-type mice (Movie 1, 2, Fig. 5A,F). The wild-type cilia bundles moved orderly with planar beating pattern from the top views (Movie 3, Fig. 5B). The Rsph9 −/− cilia bundles, by contrast, moved disorderly with rotation beating pattern (Movie 4, Fig. 5B). Beating frequency was not significantly affected in Rsph9 −/− mice (Fig. 5G). Transmission electron microscopy (TEM) was performed on ependymal cilia to investigate the ciliary axoneme ultrastructure. In wild-type ependymal cilia, all axonemes exhibited a typical "9 + 2" ultrastructure ( Fig. 5C). In Rsph9 −/− ependymal cilia, most axonemes exhibited normal ultrastructure and few exhibited various defects (Fig. 5C,D). The central pair of microtubules and the outer microtubule doublets may turn into single microtubules or may become vacant. Moreover, the Rsph9 −/− cilia frequently had abnormal ectopic ciliary membrane inclusions (Fig. 5E). Thus, RSPH9 has no significant effect on the "9 + 2" arrangement of microtubules. Furthermore, immunofluorescence analysis showed that assembling of RSPH3 into radial spoke head complex is not affected by RSPH9 deletion (Fig. S4). The defects in Rsph9 −/− cilia may due to disorders of sliding motion between adjacent microtubules. Remarkably, centriolar patch size was significantly increased in Rsph9 −/− cells (Fig. 5H,I). The mechanical stress of cilia beating pattern of Rsph9 −/− cells may destroy apical centriolar distribution. These results show that RSPH9 is necessary for coordinated beating Hydrocephalus in Rsph9 −/− mice results in astrogliosis, microgliosis, cerebrovascular abnormality and myelination disorders. Hydrocephalus can damage brain tissue and cause a wide range of symptoms. Here we investigated the pathological characteristics of the thinning of cerebral cortex. The hydrocephalus caused by Rsph9 deletion was accompanied by astrogliosis and microgliosis in the cortex. Immunostaining with GFAP in the P8 Rsph9 −/− cerebral cortex was much stronger than that in the control mice (Fig. 6A,B). Immunostaining with ionized calcium binding adaptor molecule 1 (IBA1) showed that the microglia increased dramatically with the severity of hydrocephalus (Fig. 6C,D). The microglia were almost all activated characterized by shorter processes and larger soma in P8 Rsph9 −/− mice. This shows that hydrocephalus is proceeded by the www.nature.com/scientificreports/ inflammatory response. The analysis of cerebral vessels showed that vessel density and branching were reduced in P8 Rsph9 −/− mice (Figs. 6E-G and S5). Hydrocephalus in Rsph9 −/− mice also attenuated the expression of myelin basic protein (MBP), which is a marker of myelinating glia, but it enhanced the expression of oligodendrocyte transcription factor 2 (OLIG2), which is a marker of oligodendrocyte progenitor cells and mature oligodendrocytes (Fig. 6H,I). It turned out that myelin is damaged in Rsph9 −/− mice and that enhanced OLIG2 expression may contribute to myelin repair. Immunostaining with an ependymal layer marker (S100β) showed slightly rupture of the ependymal layer in P8 Rsph9 −/− mice (Fig. S6A). And then the ependymal layers were severely damaged and ependymal exfoliation was detected in P12 Rsph9 −/− mice (Fig. S4B). Altogether, these results suggest that in P8 Rsph9 −/− mice, hydrocephalus is associated with severe pathological reactions, inflammation reactions and myelination disorders.

Discussion
RSPH9 is known to be a component of the axonemal radial spoke head complex, which is a thin stalk attached to the outer doublet microtubule in motile cilia. In this study, we show that Rsph9-deficient mice developed severe hydrocephalus with postnatal ventriculomegaly and severe sinusitis. The characteristic feature of hydrocephalus is the excessive CSF in ventricular dilation. Clinically, stenosis and obliteration of the aqueduct often initiate the pathogenesis of hydrocephalus [14][15][16][17] . Several cilia related genes have been accounted for by hydrocephalus. Genetic deficiency of genes, such as Ccdc39 18 , Jhy 19 , Ulk4 20 , Lrrc6 21 , Zmynd10 22 , and Tap73 23 , may lead to defects in ependymal differentiation or obstruction of aqueducts. However, our results showed that neurodevelopment and astrocyte development are normal in embryonic Rsph9 −/− mice. Although the Evans blue dye tracing experiment demonstrated defects in directional CSF flow, the tubular structure of the central aqueduct was comparable in Rsph9 −/− mice. Additionally, as hydrocephalus in murine models of ciliary gene knockouts is background dependent, we backcrossed the first generation with C57BL/6 mice for more than five generations to obtain a more purified genetic background. Ciliated ependymal cells can generate and maintain complex, spatiotemporally regulated flow networks 7 . The beating pattern and beating frequency of ependymal cilia are spatially different 24,25 . Our video microscopy results showed that the movement of beating cilia bundles is characterized by lower amplitude and disorderly rotation beating pattern instead of orderly planar beating pattern in subventricular zone en-face of Rsph9 −/− mice. Liu   27 . This may disrupt the mechanical resistance of the apical actin network around centrioles, in turn, and disrupt centriole stability 28 . Hydrocephalus proceeds with reactive astrogliosis and microgliosis, which lead to the formation of glial scars. Upon activation by injury, active glial cells release chemokines and cytokines, which help recruit of microglia 29,30 . Recruitment facilitates the formation of glial scars, which impede neovascularization and block the growth of neuronal processes 31 . Our experimental data are consistent with previous results 29, [32][33][34] . We have provided further evidence that vessel density and branching frequency are both decreased in mice with hydrocephalus. Furthermore, it has previously been reported that oligodendrocyte precursor migration is associated with the abluminal www.nature.com/scientificreports/ endothelial surface of nearby blood vessels 35 , which may explain the defects of myelin and the accumulation of OLIG2 + cells in the medial ganglionic eminence of Rsph9 −/− mouse brains. Dysfunction of RSPH9 can change the motion pattern of motile cilia. However, we still do not know how this change occur in Rsph9 −/− ciliary motility. Our knowledge of the mechanism of ciliary organization and motility has been very limited. In our experiments, we cannot see the ultrastructure of radial spoke complex clearly using TEM, and there remains questions of that whether RSPH9 affects localization of other radial spoke head proteins. The precise ultrastructural organization and sliding mechanism of radial spoke complex are still need to be investigated.

Methods
Mice. All mice were housed in specific pathogen-free conditions and maintained on a 12:12 h light-dark lighting cycle, with lights off at 19:00. Animal procedures were performed in accordance with experimental protocols and approved by Animal Care and Use Committees of the Institute of Zoology, Chinese Academy of Sciences. The Experimental Animal Center of the Institute of Zoology generated the Rsph9 knockout mice. The Rsph9 knockout mice were generated by C57Bl/6 × 129/SvEv zygote microinjection with CRISPR-Cas9 system. Heterozygous Rsph9 +/− mice were back-crossed to C57BL/6 mice for at least five generations.
Histology and immunofluorescence confocal microscopy. Tissues were fixed in 4% paraformaldehyde (PFA) overnight and dehydrated in 30% sucrose, and 15 μm-thick cryosections were prepared. For Nissl staining, 0.1% Cresyl Violet solution was used. For immunofluorescence, sections were blocked by 5% boveine serum albumin/0.1% Triton X-100/PBS for 1 h, incubated with primary antibodies at 4 °C overnight and fluorophore-conjugated secondary antibodies for 2 h. Images were taken with a Zeiss LSM780 laser scanning confocal microscope and Leica Aperio VESA8 microscope.
Transmission electron microscopy (TEM). The medial walls of P6 forebrains were fixed in 4% PFA. The samples were cut into 300 μm thick by Leica vibratome. The brain slices were shaped into cubes and fixed in electron microscopy grade 2% PFA and 2.5% glutaraldehyde in PBS (pH 7.4) at 4 °C overnight. Tissues were washed with PBS three times, and post-fixed in 1% osmium oxide for 1 h. The samples were thoroughly washed in PBS and dehydrated through an ethanol series (30%, 50%, 70%, 80%, 90%, 95%, 100%). The samples were washed twice in 100% acetone. Soak the samples in 1:1 ratio of acetone to epoxy resin for 1 h, then in 1:3 ratio of acetone to epoxy resin for 3 h, and in pure epoxy resin for more than 5 h. The samples were embedded and polymerized in epoxy resin at 60 °C for 48 h. Ultra-thin sections of 60 nm were obtained with Leica UC7 ultramicrotome, stained with uranyl acetate and Reynold's lead citrate, and imaged using a transmission electron microscope (Tecnai G2 F20 TWIN TMP).
Video microscopy of ciliary motion. P7 wild-type and Rsph9 −/− brains were collected and dissected in DMEM/F12 supplemented with l-glutamine and 2% B27 (Invitrogen) at room temperature. 200 μm-thick sections including ventromedial walls of lateral ventricle was acquired using a Leica vibratome. Images were acquired by a customized microscopy with a Nikon S Plan Fluro 40 × objective. The motion of cilia was captured for 7 s (380 frames/s) with a high-speed CMOS camera (PDV, MV-500C). Time-series images were captured at a resolution of 640 by 480 pixels and saved in raw format with timing information. A MATLAB (Mathworks, ver 2015b) script was used to select the region of interest (ROI) and export the ROI into PNG format. The in plane Immunofluorescence staining with antibodies for GFAP (red, astrocyte marker) and staining for DAPI (blue, nuclear marker) in P8 mouse brains. Scale bars, 100 μm. (B) Quantification of reactive gliosis in Rsph9 +/+ and Rsph9 −/− mice (n = 5 mice per group; ***P < 0.001; and data are expressed as the means ± SEMs). (C) Immunofluorescence staining with antibodies for IBA1 (red, microglia marker) and staining for DAPI (blue, nuclear marker) in P8 mouse brains. Scale bars, 50 μm. (D) Quantification of total microglia and reactive microglia from Rsph9 +/+ and Rsph9 −/− mice (n = 4 mice per group; ***P < 0.001; and data are expressed as the means ± SEMs). (E) Immunofluorescence staining with antibodies for IB4 (red, vessel marker) and staining for DAPI (blue, nuclear marker) in P8 mouse brains. Scale bars, 100 μm. (F,G) Quantification of vessel density and branching frequency from Rsph9 +/+ and Rsph9 −/− mice (n = 4 mice per group; **P < 0. 01; ***P < 0.001; and data are expressed as the means ± SEMs). (H) Immunofluorescence staining for MBP (red, myelinating glia marker), OLIG2 (green, mature oligodendrocyte marker) and DAPI (blue, nuclear marker) in P8 mouse brains. Scale bar, 500 μm. (I) Quantification of OLIG2 + cells in P8 Rsph9 +/+ and Rsph9 −/− mouse brains (n = 3 mice per group; **P < 0. 01; and data are expressed as the means ± SEMs). SEM is the standard deviation divided by the square root of the sample size. Student's t test. ◂