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The role of Nkx3.2 and Gdf5 during zebrafish skeletal development
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi.
2021 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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. 

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2021. , s. 53
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2002
Nyckelord [en]
Nkx3.2, Gdf5, zebrafish, jaw joint, joints, axial skeleton, appendicular skeleton, fin, enhancer conservation, CRISPR/Cas9, OPT
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
URN: urn:nbn:se:uu:diva-430399ISBN: 978-91-513-1110-4 (tryckt)OAI: oai:DiVA.org:uu-430399DiVA, id: diva2:1515615
Disputation
2021-02-26, Ekmansalen, Evolutionsbiologiskt centrum, Norbyvägen 16, Uppsala, 14:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2021-02-05 Skapad: 2021-01-10 Senast uppdaterad: 2021-03-04
Delarbeten
1. The Broad Role of Nkx3.2 in the Development of the Zebrafish Axial Skeleton
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knock-down and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while little is known about the function of this gene in broader skeletal development. We generated CRISPR/Cas9 knockout of nkx3.2 in zebrafish and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing fusions in bones of the occiput, the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae, and an increased length of the proximal radials of the dorsal and anal fins. These phenotypes are reminiscent of Nkx3.2 knockout phenotypes 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.

Nyckelord
nkx3.2, bapx1, zebrafish, jaw joint, axial skeleton, occipital, basiventral cartilage, fin radials, CRISPR/Cas9 mutant
Nationell ämneskategori
Utvecklingsbiologi
Forskningsämne
Biologi med inriktning mot evolutionär organismbiologi
Identifikatorer
urn:nbn:se:uu:diva-429682 (URN)10.1101/2020.12.30.424496 (DOI)
Tillgänglig från: 2021-01-01 Skapad: 2021-01-01 Senast uppdaterad: 2023-01-09Bibliografiskt granskad
2. An evolutionarily conserved cis-regulatory element of nkx3.2 drives jaw joint-specific expression in zebrafish
Öppna denna publikation i ny flik eller fönster >>An evolutionarily conserved cis-regulatory element of nkx3.2 drives jaw joint-specific expression in zebrafish
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Utvecklingsbiologi
Identifikatorer
urn:nbn:se:uu:diva-430423 (URN)
Forskningsfinansiär
Vetenskapsrådet, 621-2012-4673
Tillgänglig från: 2021-01-10 Skapad: 2021-01-10 Senast uppdaterad: 2023-01-09Bibliografiskt granskad
3. The Role of Gdf5 in the Development of the Zebrafish Fin Endoskeleton
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2022 (Engelska)Ingår i: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 251, nr 9, s. 1535-1549Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
John Wiley & SonsWiley, 2022
Nyckelord
gdf5, fin, joints, zebrafish, appendicular skeleton, CRISPR/Cas9 mutant
Nationell ämneskategori
Utvecklingsbiologi
Identifikatorer
urn:nbn:se:uu:diva-430383 (URN)10.1002/dvdy.399 (DOI)000678743100001 ()
Forskningsfinansiär
Vetenskapsrådet, 621-2012-4673Science for Life Laboratory, SciLifeLab
Tillgänglig från: 2021-01-08 Skapad: 2021-01-08 Senast uppdaterad: 2024-05-07Bibliografiskt granskad
4. zOPT: an open source optical projection tomography system and methods for rapid 3D zebrafish imaging
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2020 (Engelska)Ingår i: Biomedical Optics Express, E-ISSN 2156-7085, Vol. 11, nr 8, s. 4290-4305Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Optical projection tomography (OPT) is a 3D imaging alternative to conventional microscopy which allows imaging of millimeter-sized object with isotropic micrometer resolution. The zebrafish is an established model organism and an important tool used in genetic and chemical screening. The size and optical transparency of the embryo and larva makes them well suited for imaging using OPT. Here, we present an open-source implementation of an OPT platform, built around a customized sample stage, 3D-printed parts and open source algorithms optimized for the system. We developed a versatile automated workflow including a two-step image processing approach for correcting the center of rotation and generating accurate 3D reconstructions. Our results demonstrate high-quality 3D reconstruction using synthetic data as well as real data of live and fixed zebrafish. The presented 3D-printable OPT platform represents a fully open design, low-cost and rapid loading and unloading of samples. Our system offers the opportunity for researchers with different backgrounds to setup and run OPT for large scale experiments, particularly in studies using zebrafish larvae as their key model organism.

Ort, förlag, år, upplaga, sidor
The Optical Society, 2020
Nationell ämneskategori
Medicinsk bildbehandling
Identifikatorer
urn:nbn:se:uu:diva-419799 (URN)10.1364/BOE.393519 (DOI)000577451600016 ()32923043 (PubMedID)
Forskningsfinansiär
Science for Life Laboratory, SciLifeLab
Tillgänglig från: 2020-09-16 Skapad: 2020-09-16 Senast uppdaterad: 2023-02-17Bibliografiskt granskad

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