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.
Highlights
Extensive migratory events, morphological changes, and cell divisions coordinated by cell signaling drive complex vertebrate patterning events during embryonic development [1] as well as during epimorphic tissue regeneration [2]
Establishing the PhOTO-N and PhOTO-M transgenic lines In order to best address the requirements for long-term imaging of complex vertebrate development and regeneration, we constructed the PhOTO vector (Figure 1A), which allows for constitutive b-actin2 [16] driven expression of two fluorescent protein (FP) – green-tored photoconvertible Dendra2 [17] and the blue FP Cerulean [18] – targeted to either (1) the nucleus of the cell by means of an H2B fusion or (2) the membrane (‘‘memb’’) via a palmitoylation and myristoylation fatty acid substrate sequence included at the N
Founders were established for each line (7 for PhOTO-N; 5 for PhOTO-M), and only offspring with ubiquitous, bright FP expression in all cells were considered for this analysis
Summary
Morphological changes, and cell divisions coordinated by cell signaling drive complex vertebrate patterning events during embryonic development [1] as well as during epimorphic tissue regeneration [2]. Two major considerations are required for this tracking strategy: first, a precise, indelible mark must be placed within cells of interest at an early stage of development or regeneration; and second, after sufficient time, the final location of labeled cells and their progeny must be scored accurately [3]. Elucidating 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
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