Conditional targeted cell ablation in zebrafish: A new tool for regeneration studies

Abstract

Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal-and spatial-specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross-linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein-NTR (CFP-NTR) under control of tissue-specific promoters allowed us to induce the death of cardiomyocytes, pancreatic ␤-cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration. Developmental Dynamics 236:1025-1035, 2007.

Hydrogen peroxide primes heart regeneration with a derepression mechanism

“…Importantly, DPI-treated hearts failed to regenerate even at 60 dpa (Supplementary information, Figure S12), suggesting a permanent arrest rather than a delay of regenerative process. In addition, exogenous H 2 O 2 partially rescued the inhibitory effect of DPI on embryonic heart regeneration (Supplementary information, Figure S13A and S13B) after a large number of cardiomyocytes were damaged by incubating Tg(myl7:CFP-NTR) transgenic embryos with metronidazole (Mtz), as Mtz can be converted into a toxic DNA crosslinking agent by nitroreductase (NTR) as previously described [35] …”

“…The zebrafish myl7 promoter was provided by Dr C Geoff Burns [52]; pHyPer-Cyto cDNA clone was purchased from Evrogen (Cat No. FP941); zebrafish catalase cDNA (CDS) was isolated from 2-day embryos according to catalase sequence (NM_130912); rat Dusp6 cDNA was obtained from Dr Zhi-Xin Wang [53]; and CFP-NTR was provided by Dr Ling-Fei Luo [35]. These expression constructs were cloned into the Tol2 backbone [50].…”

Tissue regeneration after injury in adult zebrafish: The regenerative potential of the caudal fin

“…Increase of msxb expression is required for tissue outgrowth during regeneration (Thummel et al, 2010). Genetic control of organ regeneration has been reported in other zebrafish organs, such as heart, spinal cord, and retina (Curado et al, 2007;Schoenebeck et al, 2007;Qin et al, 2009;Jopling et al, 2010;Montgomery et al, 2010).…”

The Hydra model: disclosing an apoptosis-driven generator of Wnt-based regeneration

“…Finally genetic approaches need to be combined to highly reliable analyses of cell lineages and cell behaviors, which is rather difficult when these behaviors are as transient as apoptosis 76 . Significant advances were recently made in that direction in the salamanders 72 , the Xenopus 73 and the zebrafish [77][78][79][80] . In non-classical invertebrates (planarians, Hydra but also crustaceans 81 , cricket 82 ) timely gene silencing by RNAi offers the possibility to finely dissect the molecular mechanisms underlying the cellular remodeling linked to regeneration.…”