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.

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.

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.

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 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.

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.

A temporal requirement for Hippo signaling in mammary gland differentiation, growth, and tumorigenesis.

Despite recent progress, the physiological role of Hippo signaling in mammary gland development and tumorigenesis remains poorly understood. Here we show that the Hippo pathway is functionally dispensable in virgin mammary glands but specifically required during pregnancy. In contrast to many other tissues, hyperactivation of YAP in mammary epithelia does not induce hyperplasia but leads to defects in terminal differentiation. Interestingly, loss of YAP causes no obvious defects in virgin mammary glands but potently suppresses oncogene-induced mammary tumors. The selective requirement for YAP in oncogenic growth highlights the potential of YAP inhibitors as molecular targeted therapies against breast cancers.

Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

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.

PRMT5 is necessary to form distinct cartilage identities in the knee and long bone.

During skeletal development, limb progenitors become specified as chondrocytes and subsequently differentiate into specialized cartilage compartments. We previously showed that the arginine dimethyl transferase, PRMT5, is essential for regulating the specification of progenitor cells into chondrocytes within early limb buds. Here, we report that PRMT5 regulates the survival of a separate progenitor domain that gives rise to the patella. Independent of its role in knee development, PRMT5 regulates several distinct types of chondrocyte differentiation within the long bones. Chondrocytes lacking PRMT5 have a striking blockage in hypertrophic chondrocyte differentiation and are marked by abnormal gene expression. PRMT5 remains important for articular cartilage and hypertrophic cell identity during adult stages, indicating an ongoing role in homeostasis of these tissues. We conclude that PRMT5 is required for distinct steps of early and late chondrogenic specialization and is thus a critical component of multiple aspects of long bone development and maintenance.

Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo.

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

Gpr126/Adgrg6 deletion in cartilage models idiopathic scoliosis and pectus excavatum in mice.

Adolescent idiopathic scoliosis (AIS) and pectus excavatum (PE) are common pediatric musculoskeletal disorders. Little is known about the tissue of origin for either condition, or about their genetic bases. Common variants near GPR126/ADGRG6 (encoding the adhesion G protein-coupled receptor 126/adhesion G protein-coupled receptor G6, hereafter referred to as GPR126) were recently shown to be associated with AIS in humans. Here, we provide genetic evidence that loss of Gpr126 in osteochondroprogenitor cells alters cartilage biology and spinal column development. Microtomographic and x-ray studies revealed several hallmarks of AIS, including postnatal onset of scoliosis without malformations of vertebral units. The mutants also displayed a dorsal-ward deflection of the sternum akin to human PE. At the cellular level, these defects were accompanied by failure of midline fusion within the developing annulus fibrosis of the intervertebral discs and increased apoptosis of chondrocytes in the ribs and vertebrae. Molecularly, we found that loss of Gpr126 upregulated the expression of Gal3st4, a gene implicated in human PE, encoding Galactose-3-O-sulfotransferase 4. Together, these data uncover Gpr126 as a genetic cause for the pathogenesis of AIS and PE in a mouse model.

Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.

ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium.

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.

Kinesin family member 6 (kif6) is necessary for spine development in zebrafish.

BACKGROUND: Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. RESULTS: We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6(gw326) ) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6(gw326) mutants. CONCLUSIONS: Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.

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.

A conserved regulation of cell expansion underlies notochord mechanics, spine morphogenesis, and endochondral bone lengthening.

Cell size is a key contributor to tissue morphogenesis 1 . As a notable example, growth plate hypertrophic chondrocytes use cellular biogenesis and disproportionate fluid uptake to expand 10-20 times in size to drive lengthening of endochondral bone 2,3 . Similarly, notochordal cells expand to one of the largest cell types in the developing embryo to drive axial extension 4-6 . In zebrafish, the notochord vacuolated cells undergo vacuole fusion to form a single large, fluid-filled vacuole that fills the cytoplasmic space and contributes to vacuolated cell expansion 7 . When this process goes awry, the notochord lacks sufficient hydrostatic pressure to support vertebral bone deposition resulting in adult spines with misshapen vertebral bones and scoliosis 8 . However, it remains unclear whether endochondral bone and the notochord share common genetic and cellular mechanisms for regulating cell and tissue expansion. Here, we demonstrate that the 5'-inositol phosphatase gene, inppl1a , regulates notochord expansion, spine morphogenesis, and endochondral bone lengthening in zebrafish. Furthermore, we show that inppl1a regulates notochord expansion independent of vacuole fusion, thereby genetically decoupling these processes. We demonstrate that inppl1a -dependent notochord expansion is essential to establish normal mechanical properties of the notochord to facilitate the development of a straight spine. Finally, we find that inppl1a is also important for endochondral bone lengthening in fish, as has been shown in the human INPPL1 -related endochondral bone disorder, Opsismodysplasia 9 . Overall, this work reveals a conserved mechanism of cell size regulation that influences disparate tissues critical for skeletal development and short-stature disorders.

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH Signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded (FFPE) tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of PTHrP but increased IHH signaling throughout the growth plate. Attenuation of Smoothened (SMO)-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

The cartilage growth plate is an essential structure for skeletal growth, however, the mechanisms that govern growth plate homeostasis are still poorly understood. In this study, we showed that an adhesion G protein-coupled receptor (GPCR) named ADGRG6 plays an essential role in maintaining the slow-cycling cells in the resting zone of the growth plate and directing appropriate proliferation and differentiation of the growth plate chondrocytes. Using a technique called spatial transcriptomics, we compared the gene expression profiles in control and Adgrg6 mutant growth plates and found that ADGRG6 prevents premature hypertrophic differentiation of the growth plate chondrocytes by negatively regulating Indian Hedgehog (IHH) signaling. In summary, our findings highlighted the essential role of a cartilage-enriched GPCR in maintaining growth plate homeostasis through IHH signaling.

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.