Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

Abstract

BackgroundDevelopment of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution.ResultsIn a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells.ConclusionThis report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells.

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

“…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.…”

Genetic Ablation of Hypocretin Neurons Alters Behavioral State Transitions in Zebrafish

“…In 24% of the larvae, we observed partial ablation, which was indicated by weak EGFP expression in the soma of 4 -10 neurons and undetectable dendrites and axons. This partial ablation is typical of the nitroreductase-metronidazole-based technique and was observed in other ablated tissues as well (Zhao et al, 2009;Montgomery et al, 2010). To further validate the efficacy of MET-induced HCRT-neuron ablation, we performed whole-mount ISH to detect elimination of endogenous hcrt expression under MET treatment.…”

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

“…1 To date, this system has been deployed in several different cell and tissue types in both larval and adult zebrafish. [9][10][11][12][13][14][15][16][17][18] Importantly, prodrugs are easily administered in aqueous media; thus, cell ablation can be induced in thousands of fish simultaneously, facilitating large-scale screens. 19 Accordingly, we recently developed a true high-throughput screening (HTS) methodology for identifying factors that regulate regeneration.…”