TY - JOUR
T1 - Postembryonic screen for mutations affecting spine development in zebrafish
AU - Gray, Ryan S.
AU - Gonzalez, Roberto
AU - Ackerman, Sarah D.
AU - Minowa, Ryoko
AU - Griest, Johanna F.
AU - Bayrak, Melisa N.
AU - Troutwine, Benjamin
AU - Canter, Stephen
AU - Monk, Kelly R.
AU - Sepich, Diane S.
AU - Solnica-Krezel, Lilianna
N1 - Funding Information:
Thus far our studies of Kif6 in zebrafish define its function in ependymal cell cilia formation and for maintenance of the ventricular system observed as mild hydrocephalus, assembly of the RF, with scoliosis (Buchan et al., 2014; Konjikusic et al., 2018; Troutwine et al., 2020), yet all recessive mutations recovered thus far have been compatible with adult viability. In contrast, loss of Kif6 in mice results in severe progressive hydrocephalus and lethality after birth, but similarly there is a striking loss of ependymal cell cilia (Konjikusic et al., 2018). We speculate that the pathological outcomes when comparing mouse and zebrafish Kif6 mutant animals reflect subtle physiological differences in the essential nature of central canal physiology and the robustness of the zebrafish model to regenerate post embryonic tissues, as compared to mouse. The loss of ependymal cell cilia motility (i.e. beating), which drives near-wall laminar CSF flows (Spassky and Meunier, 2017) has been shown to be essential for maintaining ventricular pressure, leading to hydrocephalus when disrupted in mouse (Lee, 2013). However, many of these genetics findings in mouse have not translated directly to explain hydrocephalus in humans, with some clear exceptions involving strong regulators of multi-ciliated cell differentiation or involving cilia beating (Wallmeier et al., 2019). Regardless, if KIF6 were found to be required for ependymal cell cilia formation in humans, as we and others have clearly defined in mouse and zebrafish models (Buchan et al., 2014; Choksi et al., 2014; Konjikusic et al., 2018), then this might support a model where the loss of laminar CSF flow contributes to the onset of neurological disorders, manifesting as hypotonia and seizures, in humans, which are pathologies associated with deleterious mutations in KIF6 in two humans patients.We thank for the outstanding zebrafish care to the personnel of the fish facilities at Washington University School of Medicine and Dell Pediatric Research Institute. We would like to acknowledge inspiration from the Simpsons - The Quetzlzacatenango (S8E9) for the original pimento locura allele name for adamts9stl316. Research reported here was supported in part by a grant from Spinal Cord Injury/Disease Research Program (SCIDRP; to LSK and late Steve Johnson), the Scoliosis Research Society (to RSG), the National Institute of Child Health and Human Development of the NIH under award number P01HD084387 to LSK and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award number AAR072009 to RSG.
Funding Information:
We thank for the outstanding zebrafish care to the personnel of the fish facilities at Washington University School of Medicine and Dell Pediatric Research Institute. We would like to acknowledge inspiration from the Simpsons - The Quetzlzacatenango (S8E9) for the original pimento locura allele name for adamts9 stl316 . Research reported here was supported in part by a grant from Spinal Cord Injury/Disease Research Program (SCIDRP; to LSK and late Steve Johnson), the Scoliosis Research Society (to RSG), the National Institute of Child Health and Human Development of the NIH under award number P01HD084387 to LSK and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award number AAR072009 to RSG.
Publisher Copyright:
© 2020
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85097774216&partnerID=8YFLogxK
U2 - 10.1016/j.ydbio.2020.11.009
DO - 10.1016/j.ydbio.2020.11.009
M3 - Article
C2 - 33290818
AN - SCOPUS:85097774216
SN - 0012-1606
VL - 471
SP - 18
EP - 33
JO - Developmental Biology
JF - Developmental Biology
ER -