Postembryonic screen for mutations affecting spine development in zebrafish

Gray, Ryan S.; González, R. Gilberto; Ackerman, Sarah D.; Minowa, Ryoko; Griest, Johanna F.; Bayrak, Melisa N.; Troutwine, Benjamin R; Canter, Stephen; Monk, Kelly R.; Sepich, Diane S.; Solnica‐Krezel, Lilianna

doi:10.1016/j.ydbio.2020.11.009

Cited by 30 publications

(28 citation statements)

References 78 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Embryonic and larval zebrafish are traditionally employed to probe vertebrate embryogenesis ( Streisinger et al 1981 ; Eisen 1996 ; Schier and Talbot 2005 ). But more recently, adult zebrafish have been eminently used to model tissue physiology and disease mechanisms, including arthritis, scoliosis, cancer, blood disorders, and undiagnosed diseases ( Langenau et al 2003 ; Askary et al 2016 ; Kaufman et al 2016 ; Wangler et al 2017 ; Van Gennip et al 2018 ; Gray et al 2021 ). Due to their renowned regenerative capacity, adult zebrafish are widely used to uncover injury responses and repair mechanisms in multiple tissues such as fin, heart, and pancreas ( Vihtelic and Hyde 2000 ; Poss et al 2002b ; Yurco and Cameron 2005 ; Moss et al 2009 ; Kroehne et al 2011 ; Tu and Johnson 2011 ).…”

Section: Introductionmentioning

confidence: 99%

“…A number of genetic screens have defied the challenges of large-scale genetics in adult zebrafish. These screens preferentially targeted accessible tissues such as skin pigmentation or fin regeneration, or sought easily discernable phenotypes in less accessible tissues such as sterility or scoliosis ( Haffter et al 1996 ; Maderspacher and Nusslein-Volhard 2003 ; Dosch et al 2004 ; Wagner et al 2004 ; Henke et al 2017 ; Gray et al 2021 ). Notably, temperature-sensitive adult genetic screens provided an additional advantage, by screening for mutations that bypass early development at permissive temperature but impair fin regeneration at nonpermissive temperature ( Johnson and Weston 1995 ; Poss et al 2002a ; Oppedal and Goldsmith 2010 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Shaw

Mokalled

2021

G3 Genes|Genomes|Genetics

View full text Add to dashboard Cite

Adult zebrafish are increasingly used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 ‘crispants’. We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Shaw

Mokalled

2021

G3 Genes|Genomes|Genetics

View full text Add to dashboard Cite

show abstract

“…Every family within our dataset had at least one cytoskeletal genetic variant, and 20/23 families showed an enriched cytoskeletal GO term ( Supplementary Files S2 ). Variants in the centriolar protein POC5 have been associated with scoliosis through human and animal studies [ 90 , 91 , 92 ]. The cytoskeletal kinesin kif6 was shown to be necessary for proper spine development in zebrafish [ 34 ], and mutations in kif6 also appeared in a zebrafish ENU genetic screen for scoliosis [ 93 ].…”

Section: Discussionmentioning

confidence: 99%

Whole Exome Sequencing of 23 Multigeneration Idiopathic Scoliosis Families Reveals Enrichments in Cytoskeletal Variants, Suggests Highly Polygenic Disease

Terhune

Wethey

Cuevas

et al. 2021

Genes

View full text Add to dashboard Cite

Adolescent idiopathic scoliosis (AIS) is a lateral spinal curvature >10° with rotation that affects 2–3% of healthy children across populations. AIS is known to have a significant genetic component, and despite a handful of risk loci identified in unrelated individuals by GWAS and next-generation sequencing methods, the underlying etiology of the condition remains largely unknown. In this study, we performed exome sequencing of affected individuals within 23 multigenerational families, with the hypothesis that the occurrence of rare, low frequency, disease-causing variants will co-occur in distantly related, affected individuals. Bioinformatic filtering of uncommon, potentially damaging variants shared by all sequenced family members revealed 1448 variants in 1160 genes across the 23 families, with 132 genes shared by two or more families. Ten genes were shared by >4 families, and no genes were shared by all. Gene enrichment analysis showed an enrichment of variants in cytoskeletal and extracellular matrix related processes. These data support a model that AIS is a highly polygenic disease, with few variant-containing genes shared between affected individuals across different family lineages. This work presents a novel resource for further exploration in familial AIS genetic research.

show abstract

“…For example, limitations from subjective radiographic evaluations may necessitate larger pedigrees to confidently map disease-causing ENU alleles or sexually dimorphic alleles. A recent saturation mutagenesis screen in zebrafish reported similarly low occurrence of pedigrees with spine deformity and with mapped alleles (Gray et al, 2020), suggesting such challenges may not be unique to mouse or other quadruped species. Although selected examples are presented here, results from the entire skeletal screen are deposited in the public online Mutagenetix database, which complements ongoing efforts of the IMPC (mousephenotype.org) to advance discovery of genes required for proper spine development in mice and, potentially, novel mouse models of human disease.…”

Section: Discussionmentioning

confidence: 99%

“…Other mutagenesis efforts have been undertaken to identify alleles associated with spine deformity in non-rodent model systems, such as zebrafish (Gray et al, 2020;Henke et al, 2017). Such large-scale mutagenesis efforts can rapidly resolve causal alleles with the integration of next-generation sequencing (Andrews et al, 2012;Wang et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Saturation mutagenesis defines novel mouse models of severe spine deformity

Rios

Denton

et al. 2021

Disease Models &Amp; Mechanisms

View full text Add to dashboard Cite

Embryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular, and nervous systems, which may result in deformities such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1,275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1,275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated ENU alleles in 21% of pedigrees. Our study provides proof-of-concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural, and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced trans-activation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.

show abstract

Postembryonic screen for mutations affecting spine development in zebrafish

Cited by 30 publications

References 78 publications

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Whole Exome Sequencing of 23 Multigeneration Idiopathic Scoliosis Families Reveals Enrichments in Cytoskeletal Variants, Suggests Highly Polygenic Disease

Saturation mutagenesis defines novel mouse models of severe spine deformity

Contact Info

Product

Resources

About