The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish

Abstract

At the turn of the 20th century, classical regenerative biology--the study of organismal/tissue/limb regeneration in animals such as crayfish, snails, and planaria--garnered much attention. However, scientific luminaries such as Thomas Hunt Morgan eventually turned to other fields after concluding that inquiries into regenerative mechanisms were largely intractable beyond observational intrigues. The field of regeneration has enjoyed a resurgence in research activity at the turn of the 21st century, in large part due to "the promise" of cultured stem cells regarding reparative therapeutic approaches. Additionally, genomics-based methods that allow sophisticated genetic/molecular manipulations to be carried out in nearly any species have extended organismal regenerative biology well beyond observational limits. Throughout its history, complex paradigms such as limb regeneration--involving multiple tissue/cell types, thus, potentially multiple stem cell subtypes--have predominated the regenerative biology field. Conversely, cellular regeneration--the replacement of specific cell types--has been studied from only a few perspectives (predominantly muscle and mechanosensory hair cells). Yet, many of the degenerative diseases that regenerative biology hopes to address involve the loss of individual cell types; thus, a primary emphasis of the embryonic/induced stem cell field is defining culture conditions which promote cell-specific differentiation. Here we will discuss recent methodological approaches that promote the study of cell-specific regeneration. Such paradigms can reveal how the differentiation of specific cell types and regenerative potential of discrete stem cell niches are regulated. In particular, we will focus on how the nitroreductase (NTR) system of inducible targeted cell ablation facilitates: (1) large-scale genetic and chemical screens for identifying factors that regulate regeneration and (2) in vivo time-lapse imaging experiments aimed at investigating regenerative processes more directly. Combining powerful screening and imaging technologies with targeted ablation systems can expand our understanding of how individual stem cell niches are regulated. The former approach promotes the development of therapies aimed at enhancing regenerative potentials in humans, the latter facilitates investigation of phenomena that are otherwise difficult to resolve, such as the role of cellular transdifferentiation or the innate immune system in regenerative paradigms.

Keywords: Ablation; Nitroreductase; Regeneration; Retina; Stem cells; Time-lapse; Zebrafish.

Figure 1. Kinetics of rod photoreceptor ablation and macrophage activation via MTZ

Confocal time-lapse microscopy (Olympus FV1000) was used to characterize cell loss and neighboring cell responsiveness parameters following MTZ-induced ablation of rod photoreceptor cells in the retina. Two transgenic lines were crossed to label photoreceptors (PR) with a NTR-YFP fusion protein [yellow, Tg(rho:YFP-NTR)gmc700], and macrophages (MP) with RFP [red, Tg(mpeg1:RFP) [63]]. A 6 dpf double transgenic zebrafish was imaged immediately after MTZ treatment. Temporal resolution: a z-stack encompassing the majority of the imaged retina and surrounding tissue was collected every 10 minutes (5 min laser, 5 min ‘dark’). Ablation onset first became evident at ~300min after MTZ exposure – interpreted as separation of outer and inner segments of PR cells. A-B: Arrows indicate MP cells near the eye that transitioned from resting (ramified) state to migratory (activated) state over the course of the study. C-F: (1.5x zoom of ROI, yellow box above): Green/Cyan traces illustrate the migration path of two MP cells within the eye, image acquisition times corresponding to migratory traces are indicated at the lower right. MP#1 was stationary for 630min (C) and migrated from 640min-920min (D-F); MP#2 was stationary for 410min (C) and migrated from 420min-920min – in each case, the onset of migration coincided with the death of nearby PR cells. D-F: Arrowheads indicate an observed phagocytic event (D-E), and subsequent absence of the inner segment of a dead PR (E-F). Scale bar(s), 250μM.

Figure 2. Schematic representing the ‘flow-through’ system

The peristaltic pump (grey) provides consistent media flow. Media proceeds from the peristaltic pump to the water bath (blue) where heat transfer brings the media temperature up to ~28.5°C. Water is circulated through the sample plate on the microscope stage. Media drawn from the sample plate proceeds once again to the peristaltic pump to repeat the cycle indefinitely. The increase in total media volume made possible with this system is believed to underlie temporal increases in specimen viability by diluting the accumulation of reactive oxygen species generated from prolonged periods of time-lapse imaging while maintaining a constant temperature.