Motor protein Kif6 regulates cilia motility and polarity in brain ependymal cells.

Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.

The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Variants in the SOX9 transactivation middle domain induce axial skeleton dysplasia and scoliosis.

SOX9 is an essential transcriptional regulator of cartilage development and homeostasis. In humans, dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic and acampomelic dysplasia, and scoliosis. The mechanism of how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four novel pathogenic variants of SOX9 identified in a large cohort of patients with congenital vertebral malformations. Three of these heterozygous variants are in the HMG and DIM domains, and for the first time, we report a pathogenic variant within the transactivation middle (TAM) domain of SOX9 . Probands with these variants exhibit variable skeletal dysplasia, ranging from isolated vertebral malformation to acampomelic dysplasia. We also generated a Sox9 hypomorphic mutant mouse model bearing a microdeletion within the TAM domain ( Sox9 Asp272del ). We demonstrated that disturbance of the TAM domain with missense mutation or microdeletion results in reduced protein stability but does not affect the transcriptional activity of SOX9. Homozygous Sox9 Asp272del mice exhibited axial skeletal dysplasia including kinked tails, ribcage anomalies, and scoliosis, recapitulating phenotypes observed in human, while heterozygous mutants display a milder phenotype. Analysis of primary chondrocytes and the intervertebral discs in Sox9 Asp272del mutant mice revealed dysregulation of a panel of genes with major contributions of the extracellular matrix, angiogenesis, and ossification-related processes. In summary, our work identified the first pathologic variant of SOX9 within the TAM domain and demonstrated that this variant is associated with reduced SOX9 protein stability. Our finding suggests that reduced SOX9 stability caused by variants in the TAM domain may be responsible for the milder forms of axial skeleton dysplasia in humans.

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

Kif6 regulates cilia motility and polarity in brain ependymal cells.

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain ( Kif6 p.G555fs ) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubules in vitro . Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesis in vivo , but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells. Summary statement: We found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

Kif9 is an active kinesin motor required for ciliary beating and proximodistal patterning of motile axonemes.

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair of microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate ortholog, Kif9, is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility and the proper distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro reveal that Xenopus Kif9 is a long-range processive motor, although it does not mediate long-range movement in ciliary axonemes in vivo. Together, our data suggest that Kif9 is integral for ciliary beating and is necessary for proper axonemal distal end integrity.

Genetic animal modeling for idiopathic scoliosis research: history and considerations.

PURPOSE: Idiopathic scoliosis (IS) is defined as a structural lateral spinal curvature ≥ 10° in otherwise healthy children and is the most common pediatric spinal deformity. IS is known to have a strong genetic component; however, the underlying etiology is still largely unknown. Animal models have been used historically to both understand and develop treatments for human disease, including within the context of IS. This intended audience for this review is clinicians in the fields of musculoskeletal surgery and research. METHODS: In this review article, we synthesize current literature of genetic animal models of IS and introduce considerations for researchers. RESULTS: Due to complex genetic and unique biomechanical factors (i.e., bipedalism) hypothesized to contribute to IS in humans, scoliosis is a difficult condition to replicate in model organisms. CONCLUSION: We advocate careful selection of animal models based on the scientific question and introduce gaps and limitations in the current literature. We advocate future research efforts to include animal models with multiple characterized genetic or environmental perturbations to reflect current understanding of the human condition.

The axonemal dynein heavy chain 10 gene is essential for monocilia motility and spine alignment in zebrafish.

Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder worldwide, characterized by atypical spine curvatures in otherwise healthy children. Human genetic studies have identified candidate genes associated with AIS, however, only a few of these have been shown to recapitulate adult-viable scoliosis in animal models. Using an F0 CRISPR screening approach in zebrafish, we demonstrate that disruption of the dynein axonemal heavy chain 10 (dnah10) gene results in recessive adult-viable scoliosis in zebrafish. Using a stably segregating dnah10 mutant zebrafish, we showed that the ependymal monocilia lining the hindbrain and spinal canal displayed reduced beat frequency, which was correlated with the disassembly of the Reissner fiber and the onset of body curvatures. Taken together, these results suggest that monocilia function in larval zebrafish contributes to the polymerization of the Reissner fiber and straightening of the body axis.

An adhesion G protein-coupled receptor is required in cartilaginous and dense connective tissues to maintain spine alignment.

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs, and dense connective tissues (i.e., ligaments and tendons) is essential for the maintenance of spinal alignment. We show that the adhesion G protein-coupled receptor ADGRG6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine alignment in mice. Furthermore, we show that ADGRG6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with a cAMP agonist could mirror aspects of receptor function in culture, thus defining core pathways for regulating these axial cartilaginous and connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis.

The developmental biology of kinesins.

Kinesins are microtubule-based motor proteins that are well known for their key roles in cell biological processes ranging from cell division, to intracellular transport of mRNAs, proteins, vesicles, and organelles, and microtubule disassembly. Interestingly, many of the ~45 distinct kinesin genes in vertebrate genomes have also been associated with specific phenotypes in embryonic development. In this review, we highlight the specific developmental roles of kinesins, link these to cellular roles reported in vitro, and highlight remaining gaps in our understanding of how this large and important family of proteins contributes to the development and morphogenesis of animals.

Postembryonic screen for mutations affecting spine development in zebrafish.

The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

Genomic characterization of the adolescent idiopathic scoliosis-associated transcriptome and regulome.

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is the most common pediatric musculoskeletal disorder, affecting ~3% of the population worldwide. However, its genetic bases and tissues of origin remain largely unknown. Several genome-wide association studies (GWAS) have implicated nucleotide variants in non-coding sequences that control genes with important roles in cartilage, muscle, bone, connective tissue and intervertebral disks (IVDs) as drivers of AIS susceptibility. Here, we set out to define the expression of AIS-associated genes and active regulatory elements by performing RNA-seq and chromatin immunoprecipitation-sequencing against H3 lysine 27 acetylation in these tissues in mouse and human. Our study highlights genetic pathways involving AIS-associated loci that regulate chondrogenesis, IVD development and connective tissue maintenance and homeostasis. In addition, we identify thousands of putative AIS-associated regulatory elements which may orchestrate tissue-specific expression in musculoskeletal tissues of the spine. Quantification of enhancer activity of several candidate regulatory elements from our study identifies three functional enhancers carrying AIS-associated GWAS SNPs at the ADGRG6 and BNC2 loci. Our findings provide a novel genome-wide catalog of AIS-relevant genes and regulatory elements and aid in the identification of novel targets for AIS causality and treatment.

Coding Variants Coupled With Rapid Modeling in Zebrafish Implicate Dynein Genes, dnaaf1 and zmynd10, as Adolescent Idiopathic Scoliosis Candidate Genes.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric spine disorder affecting ∼3% of children worldwide. Human genetic studies suggest a complex polygenic disease model for AIS with large genetic and phenotypic heterogeneity. However, the overall genetic etiology of AIS remains poorly understood. To identify additional AIS susceptibility loci, we performed whole-exome sequencing (WES) on a cohort of 195 Southern Chinese AIS patients. Bioinformatics analysis identified 237 novel rare variants associated with AIS, located in 232 new susceptibility loci. Enrichment analysis of these variants revealed 10 gene families associated with our AIS cohort. We screened these gene families by comparing our candidate gene list with IS candidate genes in the Human Phenotype Ontology (HPO) database and previous reported studies. Two candidate gene families, axonemal dynein and axonemal dynein assembly factors, were retained for their associations with ciliary architecture and function. The damaging effects of candidate variants in dynein genes dnali1, dnah1, dnaaf, and zmynd10, as well as in one fibrillin-related gene tns1, were functionally analyzed in zebrafish using targeted CRISPR/Cas9 screening. Knockout of two candidate genes, dnaaf1 or zmynd10, recapitulated scoliosis in viable adult zebrafish. Altogether, our results suggest that the disruption of one or more dynein-associated factors may correlate with AIS susceptibility in the Southern Chinese population.

Development of a straight vertebrate body axis.

The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis.

The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine.

Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development [3]. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber in vivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology. VIDEO ABSTRACT.

A comparative study of the turnover of multiciliated cells in the mouse trachea, oviduct, and brain.

BACKGROUND: In mammals, multiciliated cells (MCCs) line the lumen of the trachea, oviduct, and brain ventricles, where they drive fluid flow across the epithelium. Each MCC population experiences vastly different local environments that may dictate differences in their lifetime and turnover rates. However, with the exception of MCCs in the trachea, the turnover rates of these multiciliated epithelial populations at extended time scales are not well described. RESULTS: Here, using genetic lineage-labeling techniques we provide a direct comparison of turnover rates of MCCs in these three different tissues. CONCLUSION: We find that oviduct turnover is similar to that in the airway (~6 months), while multiciliated ependymal cells turnover more slowly.

The cartilage matrisome in adolescent idiopathic scoliosis.

The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called "matrisome" and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility.

Zebrafish: An Emerging Model for Orthopedic Research.

Advances in next-generation sequencing have transformed our ability to identify genetic variants associated with clinical disorders of the musculoskeletal system. However, the means to functionally validate and analyze the physiological repercussions of genetic variation have lagged behind the rate of genetic discovery. The zebrafish provides an efficient model to leverage genetic analysis in an in vivo context. Its utility for orthopedic research is becoming evident in regard to both candidate gene validation as well as therapeutic discovery in tissues such as bone, tendon, muscle, and cartilage. With the development of new genetic and analytical tools to better assay aspects of skeletal tissue morphology, mineralization, composition, and biomechanics, researchers are emboldened to systematically approach how the skeleton develops and to identify the root causes, and potential treatments, of skeletal disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:925-936, 2020.

Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis.

Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect affecting children worldwide, and is classified by age of onset, location and degree of spine curvature. Although rare, IS with onset during infancy is the more severe and rapidly progressive form of the disease, associated with increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remains elusive. Here, we demonstrate the role of PRMT5 in the infantile IS phenotype in mouse. Conditional genetic ablation of PRMT5 in osteochondral progenitors results in impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of these several markers of endochondral ossification revealed increased type X collagen (COLX) and Ihh expression, coupled with a dramatic reduction in Mmp13 and RUNX2 expression, in the vertebral growth plate and in regions of the intervertebral disc in the Prmt5 conditional mutant mice. We also demonstrate that PRMT5 has a continuous role in the intervertebral disc and vertebral growth plate in adult mice. Altogether, our results establish PRMT5 as a critical promoter of terminal hypertrophic chondrocyte differentiation and endochondral bone formation during spine development and homeostasis.This article has an associated First Person interview with the first author of the paper.

Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice.

Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide and has been attributed primarily to trauma and the accumulation of pathology during aging. While genetic defects have also been associated with disc degeneration, the precise mechanisms driving the initiation and progression of disease have remained elusive due to a paucity of genetic animal models. Here, we discuss a novel conditional mouse genetic model of endplate-oriented disc herniations in adult mice. Using conditional mouse genetics, we show increased mechanical stiffness and reveal dysregulation of typical gene expression profiles of the IVD in adhesion G-protein coupled receptor G6 (Adgrg6) mutant mice prior to the onset of endplate-oriented disc herniations in adult mice. We observed increased STAT3 activation prior to IVD defects and go on to demonstrate that treatment of Adgrg6 conditional mutant mice with a small molecule inhibitor of STAT3 activation ameliorates endplate-oriented herniations. These findings establish ADGRG6 and STAT3 as novel regulators of IVD endplate and growth plate integrity in the mouse, and implicate ADGRG6/STAT3 signaling as promising therapeutic targets for endplate-oriented disc degeneration.