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Kinesin Family Member 6 (Kif6) Is Necessary for Spine Development in Zebrafish 8 9 10 11 12 Jillian G

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Kinesin Family Member 6 (Kif6) Is Necessary for Spine Development in Zebrafish 8 9 10 11 12 Jillian G Page 1 of 40 Developmental Dynamics 1 2 3 4 5 6 7 Kinesin family member 6 (kif6) is necessary for spine development in zebrafish 8 9 10 11 12 Jillian G. Buchan1, Ryan S. Gray2, John M. Gansner6, David M. Alvarado3, Lydia Burgert4, 13 14 Jonathan D. Gitlin7, Christina A. Gurnett3,4,5, Matthew I. Goldsmith1,4,* 15 16 17 Departments of 1Genetics, 2Developmental Biology, 3Orthopaedic Surgery, 4Pediatrics, 18 5 6 19 Neurology, Washington University School of Medicine, St. Louis, MO, 63110; Dana-Farber 20 Cancer Institute, Boston, MA, 02215; 7Eugene Bell Center for Regenerative Biology, Marine 21 Biological Laboratory, Woods Hole, MA, 02543 22 23 24 25 26 *Corresponding author: Matthew I. Goldsmith, MD 27 28 Department of Pediatrics 29 Washington University School of Medicine 30 [email protected] 31 660 S Euclid Ave, Campus Box 8208 32 St Louis, MO 63110 33 Phone: (314) 286-2769 34 35 Fax: (314) 286-2784 36 37 38 39 Running Title: Kif6 in zebrafish spine development 40 41 Keywords: kinesin, scoliosis, Danio rerio 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Developmental Dynamics Page 2 of 40 1 2 3 ABSTRACT 4 5 6 Background: Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and 7 8 results in curvature of the spine without structural defects of the vertebral units. The 9 10 11 pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a 12 13 relevant animal model. Results: We performed a forward mutagenesis screen in zebrafish to 14 15 identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called 16 17 18 skolios, which develops isolated spinal curvature that arises independent of vertebral 19 20 malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense 21 22 mutation in kinesin family member 6 (kif6gw326) unique to skolios mutants. Three additional kif6 23 24 25 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like 26 27 effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 28 29 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, 30 31 32 confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no 33 34 evidence for cilia dysfunction was seen in kif6gw326 mutants. Conclusions: Overall, these 35 36 37 findings demonstrate a novel role for kif6 in spinal development and identify a new candidate 38 39 gene for human idiopathic scoliosis. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Page 3 of 40 Developmental Dynamics 1 2 3 4 INTRODUCTION 5 6 7 Idiopathic scoliosis is a multifaceted genetic disorder that causes curvature of the spine in 8 9 2-3% of humans, with about 10% of patients progressing to deformity warranting bracing or 10 11 surgical treatment (Miller, 1999). Although idiopathic scoliosis is defined by a deviation of the 12 13 14 spine from the midline in the medial-lateral plane, curvature is typically complex and causes a 15 16 three dimensional deformity affecting all three planes (medial-lateral, dorsal-ventral and 17 18 transverse). Unlike secondary scoliosis that arises from congenital vertebral malformations or 19 20 21 scoliosis arising from other syndromic conditions, idiopathic scoliosis is poorly understood and 22 23 no obvious structural vertebral abnormalities are present. A major challenge in determining the 24 25 pathogenesis of idiopathic scoliosis has been the lack of a relevant animal model. Animal 26 27 28 models of idiopathic scoliosis have been primarily limited to experimental forms of scoliosis, 29 30 where scoliosis is induced by invasive surgical procedures, immobilization or the use of the 31 32 33 systemic agents (reviewed in (Janssen et al., 2011)). Because spinal deformity develops 34 35 secondary to harsh interventions, experimentally induced forms of scoliosis are unlikely to 36 37 recapitulate many features of human idiopathic scoliosis. 38 39 40 With the exception of humans, naturally occurring scoliosis has been described only 41 42 rarely in the animal kingdom (Ouellet and Odent, 2013). Humans ambulate in an upright 43 44 position with their center of gravity over the pelvis, a characteristic that is unique even among 45 46 47 other primates (D'Aout et al., 2002). This erect, bipedal posture significantly alters the dorsal 48 49 shear loads of the spine, which has been proposed as an important prerequisite for scoliosis 50 51 52 development (Castelein et al., 2005). In support of this model, pinealectomized rats develop 53 54 scoliosis when also made bipedal, whereas similarly treated quadrupedal rats do not (Machida et 55 56 al., 2005). Although pinealectomy and bipedalism may be important prerequisites for scoliosis 57 58 59 60 3 Developmental Dynamics Page 4 of 40 1 2 3 in some species, pinealectomy in bipedal nonhuman primates does not cause spinal deformity 4 5 6 (Cheung et al., 2005). Moreover, pinealectomy in guppy (Gorman et al., 2007) and salmon 7 8 (Fjelldal et al., 2004) induces spinal curvature, demonstrating that scoliosis induced by 9 10 11 pinealectomy does not require bipedalism. 12 13 A mutant guppy strain, curveback, is one of the few examples of an animal model with 14 15 non-induced spinal deformity that parallels human idiopathic scoliosis (Gorman et al., 2007). 16 17 18 curveback mutants develop a spinal curvature sporadically in the dorsal-ventral and medial- 19 20 lateral plane that is not due to vertebral malformations. Sporadic spinal deformity has also been 21 22 noted in other teleosts, including medaka and swordtail, suggesting that teleosts may be ideal 23 24 25 animal models to identify and characterize naturally occurring scoliosis (reviewed in (Gorman 26 27 and Breden, 2007)). Of the teleosts, zebrafish (Danio rerio) are particularly well-suited model 28 29 organisms, as zebrafish are highly tractable and have extensive genomic resources. The 30 31 32 zebrafish mutant leviathan is caused by recessive mutations in collagen type VIII alpha1a 33 34 (col8a1a) and develops scoliosis (Gray et al., 2014). However, leviathan mutants also develop 35 36 37 notochord defects and congenital vertebral malformations and it remains unknown whether 38 39 zebrafish are susceptible to naturally occurring scoliosis without vertebral malformations. 40 41 Therefore, we sought to identify novel zebrafish scoliosis mutants that could serve as a model for 42 43 44 human idiopathic scoliosis. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis forward genetic 45 46 screen, we identify and describe skolios, a zebrafish mutant with recessively inherited scoliosis 47 48 that shares several characteristics with human idiopathic scoliosis. 49 50 51 52 53 54 55 56 57 58 59 60 4 Page 5 of 40 Developmental Dynamics 1 2 3 RESULTS 4 5 6 7 Identification of skolios mutant 8 9 To identify zebrafish mutants with scoliosis, we performed an ENU mutagenesis screen 10 11 on wild-type (WT) AB zebrafish. From this screen, we identified a new zebrafish mutant, 12 13 14 skolios, which develops recessively inherited curvature of the body axis. 15 16 Body curvature first develops in skolios mutants during embryonic hatching stages (2-3 17 18 days post-fertilization [dpf]), causing a moderate ventral curvature of the body axis that is 19 20 21 frequently limited to the distal end of the tail (Figure 1A). This early curvature arises without 22 23 observable defects in the notochord (e.g. kinking) and is incompletely penetrant with variable 24 25 26 expressivity. Approximately 20% of homozygous skolios embryos appear normal at 3 dpf, while 27 28 the majority exhibit either moderate (~70%) or severe (~10%) ventral body curvature (Figure 29 30 1A, Figure 1B). 31 32 33 Unlike the incompletely penetrant ventral body curvature first seen during hatching 34 35 stages, curvature in the medial-lateral plane becomes fully penetrant during early larval stages 36 37 (4-5 dpf) and progresses through later larval and juvenile stages (5-89 dpf) (Figure 1C). 38 39 40 Although there is large phenotypic variability in the appearance and severity of the curve, body 41 42 curvature defects are easily observed in both the dorsal-ventral and medial-lateral planes of all 43 44 adult skolios mutants (Figure 1D). Despite the severity of the deformity, adult skolios mutants 45 46 47 are both viable and fertile. Heterozygous mutants exhibit no overt embryonic or post-embryonic 48 49 phenotype. 50 51 52 53 54 Characterization of spinal curvature in skolios mutants 55 56 57 58 59 60 5 Developmental Dynamics Page 6 of 40 1 2 3 MicroCT imaging of a skolios mutant and WT zebrafish was performed at 50 dpf to 4 5 6 determine if vertebral abnormalities underlie the curved body axis in skolios. Compared to WT, 7 8 the skolios mutant developed a marked curvature of the abdominal and caudal spine in both the 9 10 11 dorsal-ventral and medial-lateral planes (Figure 1E). To evaluate the temporal onset of curvature 12 13 and to screen for vertebral defects, we performed a two-staining protocol using alcian blue, a 14 15 positively charged dye that is thought to react with the acidic mucopolysaccharides in cartilage, 16 17 18 and alizarin red, a dye that binds to mineralized bone (Walker and Kimmel, 2007). Vertebral 19 20 morphology was evaluated at several developmental time points (2-18 months post-fertilization 21 22 [mpf]) for WT and skolios mutants. To track the progression of the spinal curve, curves were 23 24 25 measured in the dorsal-ventral plane as shown in Figure 2A.
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