PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

Abstract

BackgroundElucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.MethodologyPhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.Conclusions/SignificancePhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.

Clonal analyses in the anterior pre-placodal region: implications for the early lineage bias of placodal progenitors

“…This premise deserves to be further explored by more extensive analyses of the fate of individual cells within the PPR coupled with live imaging to follow the cell movements of the clonal descendants. Technical advances in imaging and improved molecular and computational tools that allow single cells, their progeny and their interactions with their neighbors to be followed over time and space will facilitate such analyses (Dempsey et al, 2012; Sellmyer et al, 2013; Tabansky et al, 2013). Highlighting the significance of such an approach, a recent study utilizing in toto imaging of the zebrafish ventral neural tube, discovered that differentially specified progenitors intermingle with each other as a consequence of heterogeneous responses to a signaling cue prior to being sorted into distinctly defined progenitor domains (Xiong et al, 2013).…”

Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis

“…Spatiotemporal quantitative imaging in vivo is not merely limited to early embryonic development but can also be exploited as a tool to study development and regeneration in whole organisms with potential benefits for future biomedical applications (Dempsey et al, 2012a(Dempsey et al, , 2015. The additional information acquired using these quantitative imaging techniques is critical for our understanding of the regulation of developmental processes in healthy and diseased states such as cancer.…”

“…An alternative labeling strategy to further minimize tracking uncertainties is the recently established PhOTO (photoconvertible optical tracking of) zebrafish. The PhOTO zebrafish method permits instantaneous, non-random cell labeling and tracking with high fidelity in space and time (Dempsey et al, 2012a(Dempsey et al, , 2014. Here, sparse and global cell labeling techniques are combined by the constitutive expression of two distinct fluorescent proteins targeted to either the membrane or the nucleus with one of the two fluorescent proteins being the green-to-red photoconvertible Dendra2 ( Fig.…”

“…Here, sparse and global cell labeling techniques are combined by the constitutive expression of two distinct fluorescent proteins targeted to either the membrane or the nucleus with one of the two fluorescent proteins being the green-to-red photoconvertible Dendra2 ( Fig. 1C) (Dempsey et al, 2012a). The non-invasive, instantaneous photoconversion of membrane-or nucleus-targeted Dendra2 allows for the precise tracking of converted red cells of interest during development.…”

Applications of phototransformable fluorescent proteins for tracking the dynamics of cellular components

“…24 Likewise, cell movement associated with fin regeneration was studied using a new model called PhOTO zebrafish ubiquitously expressing green-to-red Dendra2 PcFP. 25 …”