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zOPT: an open source optical projection tomography system and methods for rapid 3D zebrafish imaging
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.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Boije: Zebrafish Neuronal Networks.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Boije: Zebrafish Neuronal Networks.ORCID iD: 0000-0002-4877-9725
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2020 (English)In: Biomedical Optics Express, E-ISSN 2156-7085, Vol. 11, no 8, p. 4290-4305Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
The Optical Society , 2020. Vol. 11, no 8, p. 4290-4305
National Category
Medical Image Processing
Identifiers
URN: urn:nbn:se:uu:diva-419799DOI: 10.1364/BOE.393519ISI: 000577451600016PubMedID: 32923043OAI: oai:DiVA.org:uu-419799DiVA, id: diva2:1467761
Funder
Science for Life Laboratory, SciLifeLabAvailable from: 2020-09-16 Created: 2020-09-16 Last updated: 2023-02-17Bibliographically 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)
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Available from: 2021-02-05 Created: 2021-01-10 Last updated: 2021-03-04

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Zhang, HanqingWaldmann, LauraManuel, RemyBoije, HenrikHaitina, TatjanaAllalou, Amin

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Computerized Image Analysis and Human-Computer InteractionScience for Life Laboratory, SciLifeLabDivision of Visual Information and InteractionEvolution and Developmental BiologyBoije: Zebrafish Neuronal Networks
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