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The Role of Gdf5 in the Development of the Zebrafish Fin Endoskeleton
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, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.ORCID iD: 000-0003-1815-7818
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. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Science for Life Laboratory BioImage Informatics Facility.
Uppsala University, Science for Life Laboratory, SciLifeLab. 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, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Science for Life Laboratory BioImage Informatics Facility.
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2022 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 251, no 9, p. 1535-1549Article in journal (Refereed) Published
Abstract [en]

The development of the vertebrate skeleton requires a complex interaction of multiple factors to facilitate correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5), a member of the transforming growth factor-beta family (TGF-beta) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected within the first pharyngeal arch jaw joint, fin mesenchyme condensations and segmentation zones in median fins, however little is known about the functional role of Gdf5 outside of Amniota. 

We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analysed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish display truncated median fin endoskeletal elements and loss of posterior radials in the pectoral fins. 

These findings are consistent with phenotypes observed in human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

Place, publisher, year, edition, pages
Wiley John Wiley & Sons, 2022. Vol. 251, no 9, p. 1535-1549
Keywords [en]
gdf5, fin, joints, zebrafish, appendicular skeleton, CRISPR/Cas9 mutant
National Category
Developmental Biology
Identifiers
URN: urn:nbn:se:uu:diva-430383DOI: 10.1002/dvdy.399ISI: 000678743100001OAI: oai:DiVA.org:uu-430383DiVA, id: diva2:1515462
Funder
Swedish Research Council, 621-2012-4673Science for Life Laboratory, SciLifeLabAvailable from: 2021-01-08 Created: 2021-01-08 Last updated: 2024-05-07Bibliographically approved
In thesis
1. The role of Nkx3.2 and Gdf5 during zebrafish skeletal development
Open this publication in new window or tab >>The role of Nkx3.2 and Gdf5 during zebrafish skeletal development
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The vertebrate skeleton is composed of bony and cartilaginous structures that are developed under the control of numerous genetic networks. The transcription factor Nkx3.2 and the signaling molecule Gdf5 play a fundamental role during joint development and chondrogenesis, a process whereby mesenchyme cells form precartilaginous condensations followed by chondrocyte differentiation. Mutations in these genes can lead to some rare human skeletal diseases and are furthermore thought to play a role during osteoarthritis, whereby the articular cartilage in synovial joints degrades. Both genes are fairly well studied in amniotes, but their full function and regulation are not completely understood. This thesis focuses on further characterization of Nkx3.2 and Gdf5 function, by using the zebrafish Danio rerio, a small vertebrate, as a model organism.

We generated a CRISPR/Cas9 nkx3.2 mutant zebrafish line and detected broad phenotypes in the axial skeleton. Nkx3.2 deficiency in knockout zebrafish confirms previously reported jaw joint loss, but also revealed new phenotypes in the occipital region, the Weberian apparatus, the vertebrae and some fins.

By identifying a cis-regulatory element of nkx3.2 in zebrafish, we were able to generate a transgenic zebrafish line labelling the developing jaw joint and jaw joint progenitor cells. This line enables detailed documentation of jaw joint development and paves the way for a better understanding of joint development. Knockout of this nkx3.2 enhancer sequence in zebrafish did not result in any phenotypic differences, indicating a redundant function. Besides the identification of a nkx3.2 enhancer in the zebrafish genome, we identified homologous nkx3.2 enhancer sequences in the genomes of multiple gnathostome species and found that they display a high degree of functional conservation.

To study the role of Gdf5, we generated a CRISPR/Cas9 gdf5 mutant line. gdf5 mutant zebrafish displayed abnormalities in endoskeletal elements of all median and the pectoral fins showing truncation of median fin endoskeletal elements and partial absence of pectoral fin radials.

Finally, we developed an optical projection tomography (OPT) based automated workflow to generate 3D reconstructions of in situ and skeletal-stained zebrafish embryos and larvae. The acquired imaging data of skeletal-stained larval zebrafish was subsequently used to quantify phenotypic differences between mutant and wild-type zebrafish groups. This technique allows for the identification of even subtle phenotypic differences at early stages of development.

To conclude, the work presented in this thesis provides further understanding of the role of Nkx3.2 and Gdf5 during skeletogenesis in zebrafish and contributes to the development of zebrafish imaging techniques. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2021. p. 53
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2002
Keywords
Nkx3.2, Gdf5, zebrafish, jaw joint, joints, axial skeleton, appendicular skeleton, fin, enhancer conservation, CRISPR/Cas9, OPT
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-430399 (URN)978-91-513-1110-4 (ISBN)
Public defence
2021-02-26, Ekmansalen, Evolutionsbiologiskt centrum, Norbyvägen 16, Uppsala, 14:15 (English)
Opponent
Supervisors
Available from: 2021-02-05 Created: 2021-01-10 Last updated: 2021-03-04
2. 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)
Opponent
Supervisors
Available from: 2023-09-14 Created: 2023-08-20 Last updated: 2024-05-07

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Waldmann, LauraLeyhr, JakeZhang, HanqingAllalou, AminÖhman, CarolineHaitina, Tatjana

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