(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish [1]

['Haibo Xie', 'Institute Of Evolution', 'Marine Biodiversity', 'Ocean University Of China', 'Qingdao', 'Laboratory For Marine Biology', 'Biotechnology', 'Qingdao National Laboratory For Marine Science', 'Technology', 'Affiliated Hospital Of Guangdong Medical University']

Date: 2023-03 

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Funding: This work was supported by the National Natural Science Foundation of China (Nos. 31991194. 32125015 to C.Z., Nos.91954123. 31972887 to M.C., No. 81972029 to Z.Z., No. 32100661 to H.X. and No.32200415 to Y.K.), the Fundamental Research Funds for Central Universities of China (No. 202113046 to Y.K. and No. 201961016 to Y.L.), the the Natural Science Foundation of Shandong Province of China (No. ZR202111120125) to Y.K., Shanghai Science and Technology Commission (20JC1410100) to M.C., and Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20211800) to M.C.. PCY was supported by the NIH/NIDCR R01 DE018043 and NIH/NIAMS R21 AR065761. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Congenital hydrocephalus is a common phenotype that occurs in several human disorders including PCD [ 34 ]. Intriguingly, PCD patients also exhibit a high prevalence of scoliosis [ 35 ], although it remains unclear how hydrocephalus may result in the development of scoliosis. Similarly, hydrocephalus and scoliosis occur in many zebrafish ciliary mutants. Interestingly, spinal curvature did not develop in these ciliary mutants until approximately 3 weeks postfertilization, a similar stage to the beginning of scoliosis in adolescent idiopathic scoliosis (AIS) patients. The molecular mechanisms of scoliosis development at these stages remains to be elucidated. Here, we have characterized a late-onset zebrafish scoliosis mutant exhibiting loss of function of Ccdc57. Zebrafish ccdc57 mutants develop severe hydrocephalus due to defects in the coordinated beating of multiple cilia. We provide data showing that Ccdc57 regulates ependymal PCP, whose defects are likely the major cause of scoliosis formation in ccdc57 mutants. Moreover, we describe the relationship between spine curvature and abnormal Urotensin expression caused by abnormal CSF circulation, thereby providing important mechanistic clues for the formation of scoliosis.

CSF is produced by the CP, a highly specialized epithelium located in the ventricles of the brain that are in close contact with ependymal cells [ 26 ]. A key feature of brain ventricle ependymal cells is the presence of multiple motile cilia in their apical surface, which need to beat in the same direction to properly propel CSF flow [ 27 ]. Planar cell polarity (PCP) signals are essential to define the distribution of these cilia and ensure the proper direction of ciliary beating [ 28 – 30 ]. Of note, ependymal cells display two types of planar polarity—rotational PCP (rPCP) and translational PCP (tPCP)—based on the orientation and positioning of basal body clusters located within cells and tissues [ 31 , 32 ]. Motile cilia are essential for regulating rPCP, while tPCP is established by primary cilia of radial glial cells during differentiation [ 28 , 31 ]. Defects in ependymal cells, including polarity defects, are often associated with hydrocephalus caused by abnormal CSF circulation [ 29 , 33 ].

Adrenergic signals are critical for the activation of Urotensin neuropeptides in the CSF-cNs. The RF may provide a scaffold or microenvironment to promote the transduction of CSF adrenergic signals to the CSF-cNs [ 11 , 23 ]. Interestingly, the hypomorphic scospondin zebrafish mutants can survive to adulthood and display scoliosis, suggesting a critical role for the RF during later body axis development [ 10 , 19 , 22 ]. Similarly, zebrafish uts2r3 mutants also display severe scoliosis during late development [ 8 ]. These works suggest that defects in the Urotensin signaling pathway contribute to scoliosis formation. The Urotensin signaling pathway appears to be conserved in other vertebrates [ 24 ], and mutations in UTS2R, the human homolog of zebrafish uts2r3, is also associated with human scoliosis [ 25 ].

CSF-cNs are a specialized type of neuron that can sense CSF environmental changes, including pH and osmolarity [ 12 , 13 ]. CSF-cNs also respond to the mechanical signals related to CSF flow or tail bending, thus controling the locomotion of zebrafish larvae [ 14 – 17 ]. CSF-cNs contain highly polarized apical protrusions, which help modulate the mechanical sensory functions of these neurons during spinal curvature [ 18 ]. The lumen of the central canal contains a long extracellular thread, the Reissner fiber (RF), which extends from the brain ventricle to the end of the spinal canal. The RF is dynamically formed by the aggregation of SCO-spondin glycoprotein secreted from both the subcommissural organ and the floor plate in zebrafish [ 19 ]. CSF flow is essential for RF assembly and zebrafish mutants with ciliary defects are constantly associated with RF loss [ 20 ]. Recently, extensive studies have demonstrated critical roles for the RF in mediating CSF signaling and controlling body axis development [ 11 , 19 – 22 ]. The RF is located in close vicinity to the apical protrusions of the CSF-cNs, which help transduce the mechanical signals from spinal curvature or CSF flow [ 21 ]. Zebrafish scospondin mutants fail to develop the RF and display severe body curvature defects [ 19 , 20 , 22 ].

In addition to scoliosis, zebrafish ciliary mutants constantly develop body curvature at larval stage [ 7 ]. Studies from our lab and other groups have identified Urotensin as the major signaling pathway that functions downstream of motile cilia to regulate body axis development. Mechanistically, cilia-driven CSF flow transmits adrenergic signals to CSF-contacting neurons (CSF-cNs), promoting the synthesis and secretion of urotensin neuropeptides, Urp1 and Urp2. These neuropeptides further activate their receptor, Uts2r3 (previously named Uts2ra), a Urotensin-2 receptor specifically expressed in dorsal slow-twitch muscle fibers. Thus, signals from CSF finally direct dorsal muscle fiber contraction and control proper body axis straightening during early development [ 8 – 11 ].

Recently, the zebrafish has emerged as a powerful model for human scoliosis based on similar spinal column architecture and vertebral structures in zebrafish and humans [ 2 – 4 ]. Moreover, teleost fish exhibit a natural susceptibility to develop spinal curvatures over time, making zebrafish a reliable model for human IS [ 2 , 4 , 5 ]. Indeed, recent work on several zebrafish scoliosis mutants has provided significant insight into molecular mechanisms regulating scoliosis, including linking scoliosis to cerebrospinal fluid (CSF) flow defects [ 6 ]. CSF is a clear fluid that bathes the brain and spinal cord, is crucial for maintaining homeostasis of the central nervous system (CNS), and is produced by specialized ependymal cells in the choroid plexus (CP) of the ventricles of the brain. CSF flow is propelled by the beating of motile cilia, specialized, tiny organelles protruding from the surface of ependymal cells lining the brain ventricles and spinal canal. Zebrafish mutants exhibiting ciliary motility defects often develop hydrocephalus and progressive late-onset scoliosis [ 6 ].

Idiopathic scoliosis (IS), characterized by the abnormal rotation and curvature of the spine, is the most common spinal deformity, affecting more than 3% of children and adolescents worldwide [ 1 ]. Although more than 80% of scoliosis cases are deemed idiopathic, it is believed that genetic factors make significant contributions to the progression of the disease, based on the high incidence of scoliosis in families and twins. Currently, the pathogenesis of IS remains largely unknown due to insufficient knowledge of its etiology and subsequent disease progression.

Results

Localization of core PCP components in ccdc57 mutants The posterior tilting of spinal canal motile cilia is mainly regulated by the PCP pathway [41]. To further characterize this phenomenon, we examined the localization of two major PCP pathway components, Prickle and Dishevelled 1 (Dvl1), in wild type and ccdc57 mutant fish. Similar to their basal bodies, Dvl1 appeared localized to the apical posterior region of each radial glia cell (Fig 5H), with Dvl1-positive vesicles localized to the region surrounding the basal bodies (Fig 5H). Noticeably, the Dvl1 distribution angles were comparable between ccdc57 mutants and wild type control embryos (Fig 5I and 5J). The localization of Prickle was opposite to that of Dvl1 vesicles, in the anterior apical surface, as demonstrated by GFP-Prickle labeling. The Prickle localization was similar in wild type and ccdc57 mutants (Fig 5K). These data suggested that localization of PCP components in larval zebrafish was not affected by the absence of Ccdc57. Intriguingly, in adult zebrafish, Dvl1 expressing vesicles appeared localized to one side of mature ependymal cells, corresponding to the location where multicilia formed (Fig 5L). In contrast, in adult ccdc57 mutants, Dvl1 expressing vesicles appeared dispersed randomly throughout the entire cell, clearly indicating a late onset cell polarity defect in ccdc57 mutants (Fig 5L).

CCDC57 encodes a centrosomal satellite protein required for cell polarity To reveal the mechanisms of Ccdc57 regulation of basal body positioning, we further examined the subcellular localization of CCDC57 in RPE-1 cells. Similar to previous reports, CCDC57 mainly localized to centriolar satellites in RPE-1 cells [42]. Flag-tagged CCDC57 protein colocalized with centriolar satellite proteins OFD1 and PCM1 and did not localize to cilia (ARL13B) or to the distal appendage of the basal body (CEP164) (Fig 6A–6C). Mass spectrometry analysis of pulled-down proteins using Anti-FLAG conjugated beads showed that CCDC57 interacted with multiple centrosomal proteins, including CEP170, OFD1, and CP110 (Fig 6D), and we further validated the CCDC57-OFD1 interactions via immunoprecipitation (Fig 6E). Moreover, siRNA knockdown of OFD1 eliminated localization of CCDC57 to the centriolar satellite. In contrast, siRNA knockdown of CCDC57 gene expression had no effect on centrosomal localization of OFD1(Fig 6F). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. CCDC57 is a centrosomal satellite protein required for cell polarity. (A-C) Confocal images showing the relative localization of CCDC57 with OFD1, PCM1, CEP164, and ARL13B in RPE-1 cells. (D) Mass spectrometry analysis of the CCDC57 interacting proteins. (E) Immunoprecipitation results showing the interaction between CCDC57 and OFD1. (F) Confocal images showing the localization of CCDC57 and OFD1 in siRNA knockdown RPE-1 cells. (G) Scratch-wound assay showing the polarized localization of Golgi (GM130, green) during directional cell migration in control and CCDC57 siRNA-treated cells. (H) Model illustrating the statistical analysis of cell polarity by Golgi position. (I) Dot plots showing the angles of Golgi facing the migration edge in control and CCDC57 siRNA-treated cells. (J, K) Images showing the cell migration state and statistical analysis of the migration distance. In all panels, nuclei were counterstained with DAPI in blue. Scale bars: 2.5 μm in panel A; 5 μm in panel B; 3 μm in panel C; 5 μm in panel F; 50 μm in panel G. The data underlying the graphs shown in the figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002008.g006 During migration, cells can establish a front–rear polarity characterized by the polarized distribution of Golgi and centrosomes in the leading edge [43]. We therefore further tested the role of CCDC57 in establishing cell polarity in RPE-1 cells by wound-scratch assay. After scratching, the microtubule cytoskeleton of leading edge RPE-1 cells appeared polarized, with the Golgi facing toward the scratched space to direct cell migration. In control siRNA-treated RPE-1 cells, the majority of leading edge cells contained Golgi apparatus located within 60 degrees of the direction of migration relative to the nucleus (Fig 6G–6I). In contrast, the polarized orientation of Golgi apparatus was significantly compromised in CCDC57 siRNA-treated RPE-1 cells, as demonstrated by significantly increased orientation angles (Fig 6G–6I). Furthermore, migration distance was also reduced in CCDC57 siRNA-treated cells as compared to control siRNA-treated cells (Fig 6J and 6K). Together, these in vitro studies further confirmed the role of the centrosomal protein, CCDC57, in establishing cell polarity.

Abnormal RF assembly and urotensin expression in ccdc57 mutant larvae We next sought to identify the causes of the body curvature observed in ccdc57 mutants. Epinephrine signals are essential for urotensin expression and body straightening [8]. In line with this, epinephrine treatment was also able to rescue body curvature in ccdc57 mutant embryos (S8 Fig). The RF is essential for body axis straightening through transferring the epinephrine signals to the CSF-cNs [20,21]. We found that wheat germ agglutinin (WGA) can be used to label and image the RF (S9A–S9C Fig). In various ciliary mutants, WGA-labeled RF appeared either discontinuous or absent (S9D–S9G Fig). Similarly, WGA staining of the RF was also diminished or absent in ccdc57 mutant larvae (Fig 7A). Interestingly, body curvature severity closely correlated with the severity of RF assembly defects in ccdc57 or kif3a mutants (S9H–S9M Fig). Next, we examined the expression of urotensins in ccdc57 mutant larvae using whole-mount in situ hybridization (WISH) analysis. While urp1 expression appeared relatively normal in the anterior trunk of ccdc57 mutant larvae, the number of urp1-expressing cells was markedly decreased in the posterior trunk (Fig 7B and 7C). Together, these data suggested that Ccdc57 deficiency interrupts assembly of the RF and down-regulates the expression of urotensin genes. The fact that differences in urotensin gene expression mainly occurred in the posterior trunk may explain why ccdc57 mutant larvae exhibit only mild body curvature. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Mutation of ccdc57 results in RF defects and abnormal expression of Urotensins in the spinal cord. (A) Representative images of Reissner fiber (RF, white arrows) in 2 dpf wild type and ccdc57 mutant larvae. cc, central canal; nc, notochord; fp, floor plate. (B) Whole-mount in situ hybridization results showing the expression of urp1 in 24 hpf control and ccdc57 mutant larvae as indicated. The enlarged views of the staining in the posterior region are shown in the bottom. (C) Statistical analysis showing the number of urp1-expressing cells in the anterior and posterior part of the trunk. (D) Confocal images showing RF (white arrow) in wild type and ccdc57 adult mutants. RF was stained with wheat germ agglutinin (WGA, red), and nuclei were counterstained with DAPI. (E) qPCR analysis showing the expression of urotensin genes (urp1 and urp2) in different parts of the adult trunk as illustrated in the diagram. (F) In situ hybridization results showing the expression of urp2 in the brains of adult wild type and ccdc57 mutant as indicated. Arrows point to the expression of urp2 in the posterior part of the brain. (G) In situ hybridization results showing the expression of urp2 in the spinal cord of wild type and ccdc57 mutant as indicated. The purple arrowhead indicates the sites of enriched urp2 expression. (H) The line graphs showing the relative optical density of the expression of urp2 in wild type and ccdc57 mutants. (I) Model illustrating the distribution of urp2 in wild type and ccdc57 mutant. Scale bars: 7.5 μm in panel A; 10 μm in panel D. The data underlying the graphs shown in the figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002008.g007

Ectopic accumulation of Urotensin neuropeptides in ccdc57 adult mutants Next, we attempted to discover the relationship between the development of scoliosis and CSF flow defects. First, we investigated the assembly of the RF in wild type and ccdc57 mutants, based on the previously characterized roles for the RF in regulating body axis development [10,19,22]. In wild type adults, the RF appeared thick and straight (Fig 7D). In contrast, the RF appeared much thinner, discontinuous, and/or absent in adult ccdc57 mutants (Fig 7D). Of note, the severity of RF assembly defects correlated with the level of scoliosis in the mutants (S10A and S10B Fig). Next, we focused on the expression of urotensin neuropeptides. Intriguingly, we found that from a lateral view, adult ccdc57 mutant spines always contained a strong dorsal bending in the anterior trunk and a second dorsal bending in the tail region (Fig 1B and 1E). To better characterize this feature, we dissected adult wild type and ccdc57 mutant trunks into three segments (Head, Middle, and Tail) and isolated total RNA from each segment (Fig 7E). Unexpectedly, qPCR results showed that the expression of urp1 and urp2, the major urotensins regulated by CSF signaling, was increased over approximately 10 times in the Head segments of ccdc57 mutants as compared with those of wild type control siblings (Fig 7E). In contrast, Middle trunk segments displayed lower expression levels of these genes in ccdc57 mutants, while urotensin expression again appeared up-regulated in Tail segments of ccdc57 mutant adult spines (Fig 7E). To further validate these results, we performed ISH analysis on dissected spinal cords for the expression of urp2, one of the major urotensin genes expressed at later stages. Our results showed that the expression of urp2 was dramatically increased in the brainstem region of ccdc57 mutants (Fig 7F). In contrast, urp2 expression was virtually absent in the middle part of the spine, with some urp2 expression observed in the tail region (Fig 7G and 7H). To further examine whether urp2 expression in the tail region correlated with the second dorsal bending of the spine, we dissected the spinal cord at the second bending site and further cut it at the apex into anterior and posterior fragments (S10C Fig). Interestingly, compared with wild type control siblings, urp2 gene expression was up-regulated in the posterior fragment of the dissected spinal cord, while urp2 was not detected in the anterior fragment (S10C Fig). Together, these results suggest an interesting relationship between urp2 expression and spinal curvature (Fig 7I).

Correlation between urotensin expression and spine curvature in ciliary mutants To further gauge the relationship between urotensin expression and spinal curvature, we evaluated spine curvature phenotypes in several zebrafish scoliosis mutants. Both tmem67 and ofd1 mutants displayed scoliosis. Micro-CT results showed that these mutants also displayed initial dorsal bending in the anterior spine (Fig 8A). The observed scoliosis in ofd1 and ccdc57 mutants further demonstrated the interactions between Ofd1 and Ccdc57 that were revealed by our IP pulldown experiments (Fig 6E). Of note, qPCR results confirmed the enhanced expression of urp1 and urp2 in the anterior spines of ccdc57, tmem67, and ofd1 mutants (Fig 8B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. Ectopic accumulation of urotensin neuropeptides is associated with spine curvature in zebrafish ciliary mutants. (A) Representative images of wild type and scoliosis zebrafish mutants. Micro-CT images are shown on the bottom. (B) qPCR analysis showing the expression of urotensin genes in the heads of wild type and scoliosis mutants. (C) Enlarged views of the head regions of wild type, ccdc57, and uts2r3 mutants. (D, E) Statistical analysis of the dorsal curvature angles in different mutants as indicated. The angles were measured between the direction of the parasphenoid bone and the Weberian vertebrae as illustrated in the diagram (D). (F) In situ hybridization results showing the expression of urp2 in the brains of wild type and uts2r3 mutant as indicated. (G) Expression of urp2 in the spinal cords of wild type and uts2r3 mutant. The strongly increased expression of the urp2 in the tail region was shown on the enlarged views. (H) Dissected spinal cords from wild type and uts2r3 mutants as indicated. The two boxed regions correspond to those in panel G. Scale bars: 1 cm in panels A and H. The data underlying the graphs shown in the figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002008.g008 We have previously shown that mutation of uts2r3, the major Urotensin receptor, leads to scoliosis. Noticeably, the spinal curvature of uts2r3 mutants was clearly different from that of ciliary mutants. The anterior dorsal bending phenotype appeared relatively minor in uts2r3 mutants, while all uts2r3 mutants bent to the ventral side first, and displayed strong dorsal bending in the posterior portion of the trunk (Fig 8A). To better characterize spinal bending, we measured the angle between the parasphenoid bone and the Weberian vertebrae orientation in wild type, ccdc57, and uts2r3 mutants (Fig 8C). The measured angles of ccdc57 mutants were significantly larger than those in control and uts2r3 mutants (Fig 8C–8E). Consistent with these results, urp2 expression levels were dramatically increased in the anterior spines of ccdc57 mutants, while only a slight increase was observed in uts2r3 mutants (Fig 8B), as validated via WISH assay (Fig 8F). Noticeably, the location of increased urp2 expression in uts2r3 mutant spines also correlated with the observed second bending in uts2r3 mutant spines (Fig 8G and 8H). Together, these data provide strong evidence that spinal bending closely correlates with the activation of Urotensin signaling.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002008

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/