Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging

Bek, Jan Willem; Clercq, Adelbert De; Saffel, Hanna De; Soenens, Mieke; Huysseune, Ann; Witten, P. Eckhard; Coucke, Paul; Willaert, Andy

doi:10.1111/jfb.14335

Cited by 6 publications

(12 citation statements)

References 49 publications

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“…For instance, alizarin red staining of the transgenic zebrafish Tg(Ola.sp7:NLS-GFP) zf 132 localized osterix/sp7 positive osteoblasts in the mineralized bone and revealed the absence of osterix/sp7 expression in the anterior notochord region at 8 dpf (104). Similarly, mineral staining in combination with Tg(osx:Kaede) pd64 confirmed the osteoblast independent mineralisation of the notochord (196).…”

Section: Transgenic Lines To Trace Bone Cellsmentioning

confidence: 89%

“…An interesting application of the transgenic reporter lines is their use in combination with a mineral stain, imaged at different fluorescent wavelengths, enabling the combined study of osteoblast dynamics and bone mineralization (196). For instance, alizarin red staining of the transgenic zebrafish Tg(Ola.sp7:NLS-GFP) zf 132 localized osterix/sp7 positive osteoblasts in the mineralized bone and revealed the absence of osterix/sp7 expression in the anterior notochord region at 8 dpf (104).…”

Section: Transgenic Lines To Trace Bone Cellsmentioning

confidence: 99%

“…In vivo imaging of the Tg(sp7:EGFP) b1212 transgenic line during caudal fin regeneration showed the presence of GFP positive cells at the amputation plane starting from 2 days post amputation (dpa) and their association with the formation of newly mineralized matrix by 5 dpa (181). Osteoblast lineage tracing in the Tg(osx:Kaede) pd64 clarified migration and dedifferentiation of scleroblasts during fin regeneration (196). However, the slow rates of regeneration require long-term live imaging to capture dynamic cellular events to improve the understanding of development, homeostasis, and regeneration by stem cell populations (232).…”

Section: Live Imaging Of Bone Regenerationmentioning

confidence: 99%

“…Finally, in order to verify the localization of the reporter protein, the use of dual color analysis in the same transgenic line is recommended (196,199) by for example complementary secondary techniques such as immunohistochemistry or in situ hybridization (169,199).…”

Section: Tips For Transgenic Lines Selectionmentioning

confidence: 99%

“…Zebrafish's ability to repair caudal fin rays and scales has led to the optimization of specific histological protocols for these tissues involving both tissue sectioning as well as whole organ analysis (263). The analysis of histological sections has made clear that during regeneration in the caudal fin rays, cells near the site of injury can dedifferentiate, proliferate and replace the damaged or missing cells (196,264). Furthermore, histological studies have identified a population of Runx2/Sp7 positive chondrocytes involved in bone repair, and have helped to elucidate the ability of periosteal cells to generate cartilage in response to injury in indian hedgehog homolog a (Ihha) mutants (265).…”

Section: Histological Analysis Of Tissue Regenerationmentioning

confidence: 99%

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Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

et al. 2020

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Animal models are essential tools for addressing fundamental scientific questions about skeletal diseases and for the development of new therapeutic approaches. Traditionally, mice have been the most common model organism in biomedical research, but their use is hampered by several limitations including complex generation, demanding investigation of early developmental stages, regulatory restrictions on breeding, and high maintenance cost. The zebrafish has been used as an efficient alternative vertebrate model for the study of human skeletal diseases, thanks to its easy genetic manipulation, high fecundity, external fertilization, transparency of rapidly developing embryos, and low maintenance cost. Furthermore, zebrafish share similar skeletal cells and ossification types with mammals. In the last decades, the use of both forward and new reverse genetics techniques has resulted in the generation of many mutant lines carrying skeletal phenotypes associated with human diseases. In addition, transgenic lines expressing fluorescent proteins under bone cell-or pathway-specific promoters enable in vivo imaging of differentiation and signaling at the cellular level. Despite the small size of the zebrafish, many traditional techniques for skeletal phenotyping, such as x-ray and microCT imaging and histological approaches, can be applied using the appropriate equipment and custom protocols. The ability of adult zebrafish to remodel skeletal tissues can be exploited as a unique tool to investigate bone formation and repair. Finally, the permeability of embryos to chemicals dissolved in water, together with the availability of large numbers of small-sized animals makes zebrafish a perfect model for high-throughput bone anabolic drug screening. This review aims to discuss the techniques that make zebrafish a powerful model to investigate the molecular and physiological basis of skeletal disorders.

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Section: Transgenic Lines To Trace Bone Cellsmentioning

confidence: 89%

Section: Transgenic Lines To Trace Bone Cellsmentioning

confidence: 99%

Section: Live Imaging Of Bone Regenerationmentioning

confidence: 99%

Section: Tips For Transgenic Lines Selectionmentioning

confidence: 99%

Section: Histological Analysis Of Tissue Regenerationmentioning

confidence: 99%

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Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

et al. 2020

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Syntaxin 18 Defects in Human and Zebrafish Unravel Key Roles in Early Cartilage and Bone Development

Guillemyn,

De Saffel,

Bek

et al. 2023

Journal of Bone and Mineral Research

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SNARE proteins comprise a conserved protein family responsible for catalyzing membrane fusion during vesicle traffic. Syntaxin18 (STX18) is a poorly characterized endoplasmic reticulum (ER)‐resident t‐SNARE. Recently, together with TANGO1 and SLY1, its involvement was shown in ER to Golgi transport of collagen II during chondrogenesis. We report a fetus with a severe osteochondrodysplasia in whom we identified a homozygous substitution of the highly conserved p.Arg10 to Pro of STX18. CRISPR/Cas9‐mediated Stx18 deficiency in zebrafish reveals a crucial role for Stx18 in cartilage and bone development. Furthermore, increased expression of multiple components of the Stx18 SNARE complex and of COPI and COPII‐proteins suggests that Stx18 deficiency impairs antero‐ and retrograde vesicular transport in the crispant stx18 zebrafish. Taken together, our studies highlight a new candidate gene for a recessive form of osteochondrodysplasia, thereby possibly broadening the SNAREopathy phenotypic spectrum, and opening new doors towards future research avenues.This article is protected by copyright. All rights reserved.

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A tapt1 knock-out zebrafish line with aberrant lens development and impaired vision models human early-onset cataract

et al. 2023

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Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics.

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Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging

Cited by 6 publications

References 49 publications

Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

Zebrafish: A Resourceful Vertebrate Model to Investigate Skeletal Disorders

Syntaxin 18 Defects in Human and Zebrafish Unravel Key Roles in Early Cartilage and Bone Development

A tapt1 knock-out zebrafish line with aberrant lens development and impaired vision models human early-onset cataract

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