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A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.ORCID iD: 0000-0003-1815-7818
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, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-1746-5157
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2022 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e75749Article in journal (Refereed) Published
Abstract [en]

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression of nkx3.2 and leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

Place, publisher, year, edition, pages
eLife Sciences Publications eLife Sciences Publications Ltd, 2022. Vol. 11, article id e75749
National Category
Developmental Biology
Identifiers
URN: urn:nbn:se:uu:diva-493106DOI: 10.7554/elife.75749ISI: 000961591300001PubMedID: 36377467OAI: oai:DiVA.org:uu-493106DiVA, id: diva2:1725869
Funder
Swedish Research Council, 621-2012-4673Science for Life Laboratory, SciLifeLab, Development Project 2017Science for Life Laboratory, SciLifeLab, Technology Development grant 2018Available from: 2023-01-12 Created: 2023-01-12 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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Available from: 2023-09-14 Created: 2023-08-20 Last updated: 2024-05-07

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Leyhr, JakeWaldmann, LauraFilipek-Górniok, BeataZhang, HanqingHaitina, Tatjana

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