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Characterization of a col1a1a haploinsufficient zebrafish model for Osteogenesis Imperfecta type I

Pascal Simoens (UGent) , Charlotte Gistelinck (UGent) , Sofie Symoens (UGent) , Christian Vanhove (UGent) , Fransiska Malfait (UGent) , Anne De Paepe (UGent) , Paul Coucke (UGent) and Andy Willaert (UGent)
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Abstract
Introduction: ‘Osteogenesis Imperfecta (OI) is heritable fragile bone disorder, in most cases caused by autosomal dominant mutations in the genes encoding the type I collagen alpha chains. Animal models have proved indispensable for unraveling molecular mechanisms in OI pathogenesis. The zebrafish has recently shown to be a useful vertebrate organism to model OI both at the phenotypic and molecular level. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a, encoding the α1 chain of the type I collagen protein, have been described. They display generalized reduced bone density and misshapen bones with evidence of fractures. However, until now, no zebrafish mutants carrying a nonsense mutation in col1a1a have been described. Such mutant, displaying a quantitative defect of collagen type I synthesis, could serve as a model for the mild OI type I. We have characterized a zebrafish mutant that is heterozygous for a nonsense mutation in the col1a1a gene and evaluated this mutant as a possible model for OI type I. Methodology: The described col1a1a nonsense mutant was generated by the Zebrafish Mutation Project (ZMP). The bone was visualized using alizarin red staining. Adult zebrafish were scanned with a Triumph µCT scanner to determine bone densities. To assess osteoblast activity, embryos were treated with the osteoblast stimulating agent Retinoic Acid (RA) or DMSO from 4 days post fertilization (dpf) to 8 dpf and stained with alizarin complexone in order to evaluate the ossification in the trunk region. Results: Heterozygous col1a1a zebrafish mutants were phenotyped both at larval and adult stages. Larvae displayed no abnormalities in bone structure and geometry or delayed ossification at 8 dpf. The fin folds, appeared to have an overall normal appearance at 3 dpf. Adult mutant fish appeared to have the same body length as their wild type siblings. Bone staining and µCT-scanning revealed no skeletal abnormalities or reduced Bone Mineral Density (BMD). Quantification of type I collagen by western blotting revealed a 25% decrease of the α1(I) chain, in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. Discussion: . Thorough phenotypic analysis of a zebrafish mutant carrying a heterozygous nonsense mutation in the col1a1a gene did not reveal bone fractures or other skeletal malformations. Moreover, no decrease in bone density or osteoblastic activity could be detected Protein analysis in the heterozygous mutants revealed a decrease of only 25 % of the α1(I) chain, a reduction that is considered to be insufficient to give rise to a skeletal phenotype. This reduction of only 25% can be explained by the existence of a paralogue of col1a1a, col1a1b, also encoding for the α1(I) chain Therefore, we are currently creating a double mutant where both col1a1a and col1a1b paralogues are knocked out with the expectation that this will be a more relevant model for OI type I.

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Chicago
Simoens, Pascal, Charlotte Gistelinck, Sofie Symoens, Christian Vanhove, Fransiska Malfait, Anne De Paepe, Paul Coucke, and Andy Willaert. 2015. “Characterization of a Col1a1a Haploinsufficient Zebrafish Model for Osteogenesis Imperfecta Type I.” In 15th Annual Meeting of the Belgian Society of Human Genetics, Abstracts.
APA
Simoens, Pascal, Gistelinck, C., Symoens, S., Vanhove, C., Malfait, F., De Paepe, A., Coucke, P., et al. (2015). Characterization of a col1a1a haploinsufficient zebrafish model for Osteogenesis Imperfecta type I. 15th Annual Meeting of the Belgian Society of Human Genetics, Abstracts. Presented at the 15th Annual Meeting of the Belgian Society of Human Genetics.
Vancouver
1.
Simoens P, Gistelinck C, Symoens S, Vanhove C, Malfait F, De Paepe A, et al. Characterization of a col1a1a haploinsufficient zebrafish model for Osteogenesis Imperfecta type I. 15th Annual Meeting of the Belgian Society of Human Genetics, Abstracts. 2015.
MLA
Simoens, Pascal, Charlotte Gistelinck, Sofie Symoens, et al. “Characterization of a Col1a1a Haploinsufficient Zebrafish Model for Osteogenesis Imperfecta Type I.” 15th Annual Meeting of the Belgian Society of Human Genetics, Abstracts. 2015. Print.
@inproceedings{5865008,
  abstract     = {Introduction: ‘Osteogenesis Imperfecta (OI) is heritable fragile bone disorder, in most cases caused by autosomal dominant mutations in the genes encoding the type I collagen alpha chains. Animal models have proved indispensable for unraveling molecular mechanisms in OI pathogenesis. The zebrafish has recently shown to be a useful vertebrate organism to model OI both at the phenotypic and molecular level. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a, encoding the α1 chain of the type I collagen protein, have been described. They display generalized reduced bone density and misshapen bones with evidence of fractures. However, until now, no zebrafish mutants carrying a nonsense mutation in col1a1a have been described. Such mutant, displaying a quantitative defect of collagen type I synthesis, could serve as a model for the mild OI type I. We have characterized a zebrafish mutant that is heterozygous for a nonsense mutation in the col1a1a gene and evaluated this mutant as a possible model for OI type I.
Methodology: The described col1a1a nonsense mutant was generated by the Zebrafish Mutation Project (ZMP). The bone was visualized using alizarin red staining. Adult zebrafish were scanned with a Triumph µCT scanner to determine bone densities. To assess osteoblast activity, embryos were treated with the osteoblast stimulating agent Retinoic Acid (RA) or DMSO from 4 days post fertilization (dpf) to 8 dpf and stained with alizarin complexone in order to evaluate the ossification in the trunk region. 
Results: Heterozygous col1a1a zebrafish mutants were phenotyped both at larval and adult stages. Larvae displayed no abnormalities in bone structure and geometry or delayed ossification at 8 dpf. The fin folds, appeared to have an overall normal appearance at 3 dpf. Adult mutant fish appeared to have the same body length as their wild type siblings. Bone staining and µCT-scanning revealed no skeletal abnormalities or reduced Bone Mineral Density (BMD). Quantification of type I collagen by western blotting revealed a 25% decrease of the α1(I) chain,  in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. 
Discussion: . Thorough phenotypic analysis of a zebrafish mutant carrying a heterozygous nonsense mutation in the col1a1a gene did not reveal bone fractures or other skeletal malformations. Moreover, no decrease in bone density or osteoblastic activity could be detected  Protein analysis in the heterozygous mutants revealed a decrease of only 25 % of the α1(I) chain, a reduction that is considered to be insufficient to give rise to a skeletal phenotype. This reduction of only 25% can be explained by the existence of a paralogue of col1a1a, col1a1b, also encoding for the α1(I) chain Therefore, we are currently creating a double mutant where both col1a1a and col1a1b paralogues are knocked out with the expectation that this will be a more relevant model for OI type I.},
  author       = {Simoens, Pascal and Gistelinck, Charlotte and Symoens, Sofie and Vanhove, Christian and Malfait, Fransiska and De Paepe, Anne and Coucke, Paul and Willaert, Andy},
  booktitle    = {15th Annual Meeting of the Belgian Society of Human Genetics, Abstracts},
  language     = {eng},
  location     = {Charleroi},
  title        = {Characterization of a col1a1a haploinsufficient zebrafish model for Osteogenesis Imperfecta type I},
  year         = {2015},
}