Developmental and adult striatal patterning of nociceptin ligand marks striosomal population with direct dopamine projections.

Circuit influences on the midbrain dopamine system are crucial to adaptive behavior and cognition. Recent developments in the study of neuropeptide systems have enabled high-resolution investigations of the intersection of neuromodulatory signals with basal ganglia circuitry, identifying the nociceptin/orphanin FQ (N/OFQ) endogenous opioid peptide system as a prospective regulator of striatal dopamine signaling. Using a prepronociceptin-Cre reporter mouse line, we characterized highly selective striosomal patterning of Pnoc mRNA expression in mouse dorsal striatum, reflecting early developmental expression of Pnoc . In the ventral striatum, Pnoc expression was was clustered across the nucleus accumbens core and medial shell, including in adult striatum. We found that Pnoc tdTomato reporter cells largely comprise a population of dopamine receptor D1 ( Drd1 ) expressing medium spiny projection neurons localized in dorsal striosomes, known to be unique among striatal projections neurons for their direct innervation of midbrain dopamine neurons. These findings provide new understanding of the intersection of the N/OFQ system among basal ganglia circuits with particular implications for developmental regulation or wiring of striatal-nigral circuits.

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.

Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism.

Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96 h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism.

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.

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.

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.

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.

Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

Assessing Emotional Expressions During a Cycling-Based Initiative for Older Care Home Residents Using Video-Based Recordings.

Objective: Through Cycling Without Age, trained volunteers use specially designed trishaws to provide rides for older adults living in care homes and other supported living environments. Qualitative and quantitative research suggests benefits in terms of improvements in mood and wellbeing. Those studies have predominantly been interviews with participants reflecting on previous rides, or as pre-/post-assessments. The current study assessed emotional experiences using video recordings acquired during participants' rides. Methods: Twelve older adults (50% female; 67-92 years old (M = 81.8, SD = 7.4)) living in care homes or supported living environments were recruited. During a Cycling Without Age ride, participants were filmed using an action camera mounted on the trishaw. Recordings were rated using the Facial Expression Coding System by two researchers to assess the frequency, duration and intensity of positive and negative emotions. Results: On average, 23.7 positive emotional expressions were observed per ride, significantly higher than negative emotions (0.4). As well as more frequent, positive emotions were observed over a longer duration in total (139.5 seconds vs. 1.3) and rated as more intense (1.9 out of 5 vs. 0.3). Conclusion: The study supported the value of directly assessing emotional responses during this cycling-based initiative, including minimising the input required from participants. The predominantly positive emotional expressions observed were consistent with both qualitative and quantitative assessments of Cycling Without Age, and suggests a potential pathway by which those benefits manifest. Future studies might adopt a triangulated approach, using in-activity monitoring, quantitative assessments and participant reflections.

Working-Memory, Alpha-Theta Oscillations and Musical Training in Older Age: Research Perspectives for Speech-on-speech Perception.

During the normal course of aging, perception of speech-on-speech or "cocktail party" speech and use of working memory (WM) abilities change. Musical training, which is a complex activity that integrates multiple sensory modalities and higher-order cognitive functions, reportedly benefits both WM performance and speech-on-speech perception in older adults. This mini-review explores the relationship between musical training, WM and speech-on-speech perception in older age (> 65 years) through the lens of the Ease of Language Understanding (ELU) model. Linking neural-oscillation literature associating speech-on-speech perception and WM with alpha-theta oscillatory activity, we propose that two stages of speech-on-speech processing in the ELU are underpinned by WM-related alpha-theta oscillatory activity, and that effects of musical training on speech-on-speech perception may be reflected in these frequency bands among older adults.

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.

Disruptors of AKAP-Dependent Protein-Protein Interactions.

A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen® assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits.

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.

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.

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.

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.

Mutations in KIF7 implicated in idiopathic scoliosis in humans and axial curvatures in zebrafish.

Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.

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.