Development and regeneration dynamics of the Medaka notochord

Seleit, Ali; Gross, Karen; Onistschenko, Jasmin; Woelk, Michaela; Autorino, Camilla; Centanin, Lázaro

doi:10.1016/j.ydbio.2020.03.001

Cited by 15 publications

(8 citation statements)

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“…The cdh2-eGFP KI line shows cellular membrane expression in a variety of neuronal and non-neuronal tissues including the spinal cord, the eye, and the notochord ( Figure 2E , n = 5 embryos) and neuromasts of the lateral line ( Figure 2—figure supplement 3 , n = 5 embryos, Video 7 , n = 3 embryos), in addition to the developing heart (data not shown) ( Chopra et al, 2011 ). The high expression of cdh2 in both differentiated notochord cell types ( Figure 2E , Figure 2—figure supplement 3 ) has not been previously reported in medaka but is not unexpected as this tissue experiences a high level of mechanical stress and requires strong cell–cell adhesion ( Lim et al, 2017 ; Adams et al, 1990 ; Garcia et al, 2017 ; Seleit et al, 2020 ). The cdh2-eGFP KI can be used to study dynamics of n-cadherin distribution in vivo during vertebrate embryogenesis ( Video 8 , n = 2 embryos).…”

Section: Resultssupporting

confidence: 73%

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Seleit

Aulehla

Paix

2021

eLife

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The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

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Section: Resultssupporting

confidence: 73%

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Seleit

Aulehla

Paix

2021

eLife

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“…However, the effect is not as dramatic as that of vacuole fragmentation because vacuolated cell collapse or injury triggers a regenerative response in which sheath cells invade the affected areas of the notochord and differentiate into new vacuolated cells, thereby restoring notochord function (Garcia et al, 2017;Lopez-Baez et al, 2018). A similar response has been reported for the medaka notochord (Seleit et al, 2020), which is essentially identical structurally to that of zebrafish despite the long distance separating them in the phylogenetic tree.…”

Section: Formation and Maintenance Of Vacuolated Cellsmentioning

confidence: 64%

Development of a straight vertebrate body axis

Bagnat

Gray²

2020

Development

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The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis.

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“…The cdh2-eGFP KI line shows cellular membrane expression in a variety of neuronal and non-neuronal tissues including the spinal cord, the notochord (Figure 2E, n=5 embryos) and neuromasts of the lateral line (Figure S5, Supplementary Movie 7, n=5 embryos), in addition to the developing heart (data not shown) (Chopra et al, 2011). The high expression of cdh2 in both differentiated notochord cell types (Figure 2E, Figure S5) has not been previously reported in medaka but is not unexpected as this tissue experiences a high level of mechanical stress and requires strong cell-cell adhesion (Lim et al, 2017, Adams et al, 1990, Garcia et al, 2017, Seleit et al, 2020. The cdh2-eGFP KI is thus the first endogenously tagged cadherin family member in teleosts and can be used to study dynamics of n-cadherin distribution in vivo during vertebrate embryogenesis (Supplementary Movie 8).…”

Section: Cell Adhesion Markermentioning

confidence: 52%

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Seleit

Aulehla

Paix

2021

Preprint

Self Cite

View full text Add to dashboard Cite

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka. We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

show abstract

Development and regeneration dynamics of the Medaka notochord

Cited by 15 publications

References 50 publications

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Development of a straight vertebrate body axis

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

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