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The broad role of Nkx3.2 in the development of the zebrafish axial skeleton
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. BioImage Informatics Facility, Uppsala, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Applied Material Science.ORCID iD: 0000-0003-2709-9541
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2021 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 16, no 8, article id e0255953Article in journal (Refereed) Published
Abstract [en]

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.

Place, publisher, year, edition, pages
Public Library of Science (PLoS) Public Library of Science (PLoS), 2021. Vol. 16, no 8, article id e0255953
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-455491DOI: 10.1371/journal.pone.0255953ISI: 000686373300086PubMedID: 34411150OAI: oai:DiVA.org:uu-455491DiVA, id: diva2:1603818
Funder
Swedish Research Council, 621-2012-4673Available from: 2021-10-18 Created: 2021-10-18 Last updated: 2024-01-15Bibliographically approved
In thesis
1. Musculoskeletal Development in Jawed Vertebrates: Gene function, cis-regulation, and 3D phenotypes in zebrafish
Open this publication in new window or tab >>Musculoskeletal Development in Jawed Vertebrates: Gene function, cis-regulation, and 3D phenotypes in zebrafish
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhancer with different species-dependent activity patterns. In summary, this thesis contributes to our understanding of the derived and conserved functions of Nkx3.2, Gdf5, and Mkx in the development of the vertebrate skeleton and associated connective tissues, and provides a novel high-resolution 3D imaging method for future studies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2023. p. 107
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2298
Keywords
Nkx3.2, Gdf5, Mkx, zebrafish, jaw joint, skeleton, fin development, cartilage, tendon, ligament, enhancer, CRISPR/Cas9, mutant, microCT, synchrotron
National Category
Developmental Biology Evolutionary Biology
Research subject
Biology with specialization in Evolutionary Organismal Biology
Identifiers
urn:nbn:se:uu:diva-509354 (URN)978-91-513-1879-0 (ISBN)
Public defence
2023-10-06, Ekmansalen, Evolutionsbiologiskt centrum, Norbyvägen 14, Uppsala, 13:15 (English)
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Supervisors
Available from: 2023-09-14 Created: 2023-08-20 Last updated: 2024-05-07

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Waldmann, LauraLeyhr, JakeZhang, HanqingÖhman, CarolineAllalou, AminHaitina, Tatjana

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