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Characterization of a COL1A1A haploinsufficient zebrafish model for Osteogenesis Imperfecta type I

Charlotte Gistelinck (UGent) , Pascal Simoens (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: Animal models for OI 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. Forward genetic screens for skeletal dysplasia in adult zebrafish have identified mutations in bmp1a and col1a1a that accurately model OI. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a 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 in live zebrafish embryos using alizarin complexone and in fixed fish using alizarin red. Adult zebrafish were scanned using a Triumph µCT scanner and the data was analyzed using AMIDE software in order 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. To quantify type I collagen a western blot was performed using protein lysates prepared form whole embryos at 4 dpf and from adult fish by caudal fin amputation. 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 chain in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. Discussion: We have characterized a zebrafish mutant carrying a nonsense mutation in the col1a1a gene. Phenotypic analysis of heterozygous mutants, both in larvae and adults, didn’t show a decrease in BMD, presence of bone fractures or other skeletal malformations. Moreover, osteoblast activity is not affected as shown by RA treatment. Western blotting for type I collagen revealed a decrease of only 25 % of the α1 chain, a reduction that might be insufficient to give rise to a skeletal phenotype. Moreover, bone defects tend to be milder when modeled in zebrafish, as the mechanical load on the skeleton is less due to the aqueous environment. Also, the paralogues col1a1a and col1a1b might have redundant functions, explaining the absence of a bone phenotype in the col1a1a mutant. Therefore, we are 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. References: Asharani et al., Am J Hum Genet. 2012 Apr 6;90(4):661-74 Fisher et al., Dev Biol. 2003 Dec 1;264(1):64-76

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Chicago
Gistelinck, Charlotte, Pascal Simoens, Sofie Symoens, Christian Vanhove, Fransiska Malfait, Anne De Paepe, Paul Coucke, and Andy Willaert. 2014. “Characterization of a COL1A1A Haploinsufficient Zebrafish Model for Osteogenesis Imperfecta Type I.” In 12th International Conference on Osteogenesis Imperfecta, Abstracts.
APA
Gistelinck, C., Simoens, P., Symoens, S., Vanhove, C., Malfait, F., De Paepe, A., Coucke, P., et al. (2014). Characterization of a COL1A1A haploinsufficient zebrafish model for Osteogenesis Imperfecta type I. 12th International Conference on Osteogenesis Imperfecta, Abstracts. Presented at the 12th International Conference on Osteogenesis Imperfecta.
Vancouver
1.
Gistelinck C, Simoens P, Symoens S, Vanhove C, Malfait F, De Paepe A, et al. Characterization of a COL1A1A haploinsufficient zebrafish model for Osteogenesis Imperfecta type I. 12th International Conference on Osteogenesis Imperfecta, Abstracts. 2014.
MLA
Gistelinck, Charlotte, Pascal Simoens, Sofie Symoens, et al. “Characterization of a COL1A1A Haploinsufficient Zebrafish Model for Osteogenesis Imperfecta Type I.” 12th International Conference on Osteogenesis Imperfecta, Abstracts. 2014. Print.
@inproceedings{5864924,
  abstract     = {Introduction: Animal models for OI 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. Forward genetic screens for skeletal dysplasia in adult zebrafish have identified mutations in bmp1a and col1a1a that accurately model OI. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a 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 in live zebrafish embryos using alizarin complexone and in fixed fish using alizarin red. Adult zebrafish were scanned using a Triumph µCT scanner and the data was analyzed using AMIDE software in order 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. To quantify type I collagen a western blot was performed using protein lysates prepared form whole embryos at 4 dpf and from adult fish by caudal fin amputation. 
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 chain in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. 
Discussion: We have characterized a zebrafish mutant carrying a nonsense mutation in the col1a1a gene. Phenotypic analysis of heterozygous mutants, both in larvae and adults, didn’t show a decrease in BMD, presence of bone fractures or other skeletal malformations. Moreover, osteoblast activity is not affected as shown by RA treatment.  Western blotting for type I collagen revealed a decrease of only 25 % of the α1 chain, a reduction that might be insufficient to give rise to a skeletal phenotype. Moreover, bone defects tend to be milder when modeled in zebrafish, as the mechanical load on the skeleton is less due to the aqueous environment. Also, the paralogues col1a1a and col1a1b might have redundant functions, explaining the absence of a bone phenotype in the col1a1a mutant. Therefore, we are 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. 
References: 
Asharani et al., Am J Hum Genet. 2012 Apr 6;90(4):661-74
Fisher et al., Dev Biol. 2003 Dec 1;264(1):64-76},
  author       = {Gistelinck, Charlotte and Simoens, Pascal and Symoens, Sofie and Vanhove, Christian and Malfait, Fransiska and De Paepe, Anne and Coucke, Paul and Willaert, Andy},
  booktitle    = {12th International Conference on Osteogenesis Imperfecta, Abstracts},
  language     = {eng},
  location     = {Wilmington, Delaware},
  title        = {Characterization of a COL1A1A haploinsufficient zebrafish model for Osteogenesis Imperfecta type I},
  year         = {2014},
}