Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish

Muller glia in the fish retina respond to injury by reprogramming to a stem-cell-like state that enables them to regenerate all of the major retinal cell types. Goldman reviews our current understanding of the mechanisms that regulate this regenerative response and considers how this knowledge might be applied to improve repair in the mammalian retina.

Muller glial cell reprogramming and retina regeneration

https://www.cell.com/trends/genetics/pdf/S0168-9525(13)00113-3.pdf

The zebrafish as a model for complex tissue regeneration

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish.

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sh...

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing a Sharkey FokI Cleavage Domain Variant in Zebrafish

We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.

/pdf/regeneration-of-cone-photoreceptors-when-cell-ablation-is-tipf6e5eij.pdf

Regeneration of Cone Photoreceptors when Cell Ablation Is Primarily Restricted to a Particular Cone Subtype

http://www.biology.ualberta.ca/Allison_Lab/Publications_files/Fleisch,%20Leighton....Allison%202013%20Zf%20Prp%20KO%20ZFN%20Seizures%20NMDA%20b.pdf

Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability

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Evaluating the Mutagenic Activity of Targeted Endonucleases Containing a Sharkey FokI Cleavage Domain Variant in Zebrafish

Regeneration of Cone Photoreceptors when Cell Ablation Is Primarily Restricted to a Particular Cone Subtype

Targeted mutation of the gene encoding prion protein in zebrafish reveals a conserved role in neuron excitability