Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies

Abstract

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemicalgenetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR + cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

“…One limitation to the current method is that due to the stochastic and unpredictable nature of cell death, our datasets tend to focus on the later stages of extrusion. To study the formation of the multicellular actin ring and initiation of contraction, future experiments will apply techniques to induce apoptosis in a subset of fluorescently-labeled epithelial cells that are genetically targeted for cell death 10 . Combining this strategy with our method to image actin dynamics in the epidermis should facilitate studies of the early steps leading to apoptotic cell extrusion.…”

Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia

“…This line was generated by using the Tol2kit 33 . The NTR-mCherry 18,19 was cloned under the control of the NBT promoter (5 0 Entry vector, Gateway Invitrogen) and DLexPR (Mid Entry vector; Gateway Invitrogen).…”

A transgenic zebrafish model of targeted oocyte ablation and de novo oogenesis

“…Most published studies report using an Mtz concentration of 10 mM to induce cell ablation (e.g., Pisharath et al, 2007;Curado et al, 2008); however, these studies were conducted primarily in embryonic or larval stages, and employed short-term (e.g., 24-hr) exposure durations. Due to the considerable size of the zebrafish ovary (15-20% of body weight), and its propensity for compensatory regeneration (see Results section), we found in preliminary studies that a similar exposure regime was insufficient to achieve detectable oocyte ablation.…”

“…The innate regenerative capability of this species, combined with its relative ease of genetic manipulation, has consequently led to many novel insights into the molecular and cellular regulation of tissue regeneration (Curado et al, 2007(Curado et al, , 2008Davison et al, 2007;Pisharath, 2007;Pisharath et al, 2007;Moss et al, 2009;Pisharath and Parsons, 2009;Zhao et al, 2009). To date however, regenerative studies have focused primarily on somatic tissues, whereas little is known about the regenerative capacity of reproductive (i.e., gonadal) tissues, which are of central importance for animal reproductive success.…”