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PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration.

Dempsey WP, Fraser SE, Pantazis P - PLoS ONE (2012)

Bottom Line: To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.PhOTO 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 ∼100 µm 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Beckman Institute (139-74), California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT

Background: 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.

Methodology: PhOTO 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 ∼100 µm 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/significance: PhOTO 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.

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Description of PhOTO Vector and PhOTO Transgenics.(A) Depicted is a schematic of the general PhOTO vector. The major components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on the outside of the plasmid circle. Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100µm.
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pone-0032888-g001: Description of PhOTO Vector and PhOTO Transgenics.(A) Depicted is a schematic of the general PhOTO vector. The major components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on the outside of the plasmid circle. Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100µm.

Mentions: 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 β-actin2[16] driven expression of two FPs – green-to-red 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-terminus of the protein [19]. Self-cleavage of a highly efficient Thosea asigna virus (TaV) 2A sequence between the two FPs at the ribosome separates the two proteins to their respective target locations within the cell at a 1∶1 stoichiometric ratio [20]. Two stable transgenic lines (Tg(βactin2:memb-Cerulean-2A-H2B-Dendra2)pw1 and Tg(βactin2:memb-Dendra2-2A-H2B-Cerulean)pw2) were established after Tol2 mediated [21] genome integration. PhOTO-N zebrafish constitutively express H2B-Dendra2 to label all nuclei and memb-Cerulean to label all membranes, and the complementary PhOTO-M zebrafish line expresses H2B-Cerulean and memb-Dendra2. Efficient cleavage and proper FP localization were verified by western blot (Figure S1) and confocal microscopy (Figure 1B). 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.


PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration.

Dempsey WP, Fraser SE, Pantazis P - PLoS ONE (2012)

Description of PhOTO Vector and PhOTO Transgenics.(A) Depicted is a schematic of the general PhOTO vector. The major components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on the outside of the plasmid circle. Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303793&req=5

pone-0032888-g001: Description of PhOTO Vector and PhOTO Transgenics.(A) Depicted is a schematic of the general PhOTO vector. The major components of the vector, including the promoter, the TaV 2A sequence, the Tol2 transposable elements, and the protein locations are indicated on the outside of the plasmid circle. Each vector was designed such that each of the components (e.g. the promoter, the FPs, etc.) may be easily switched out for alternate protein fusions, etc. using the listed restriction enzymes (inside the circle) and an appropriate subcloning procedure. Note that the sizes of the blocked regions indicating coding sequences are not to scale. (B) Representative heterozygous PhOTO-N expression in an 18–19 hour post fertilization F1 transgenic zebrafish embryo. The top left panel of (B) depicts memb-Cerulean (blue); the top right panel depicts unconverted H2B-Dendra2 (green); the bottom left panel depicts photoconverted H2B-Dendra2 (red) in 4 somites, the tip of the tail, a subset of cells in the eye, and a subset of cells atop the yolk; and the bottom right panel depicts a merged image of all three colors. Scale bar is 100µm.
Mentions: 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 β-actin2[16] driven expression of two FPs – green-to-red 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-terminus of the protein [19]. Self-cleavage of a highly efficient Thosea asigna virus (TaV) 2A sequence between the two FPs at the ribosome separates the two proteins to their respective target locations within the cell at a 1∶1 stoichiometric ratio [20]. Two stable transgenic lines (Tg(βactin2:memb-Cerulean-2A-H2B-Dendra2)pw1 and Tg(βactin2:memb-Dendra2-2A-H2B-Cerulean)pw2) were established after Tol2 mediated [21] genome integration. PhOTO-N zebrafish constitutively express H2B-Dendra2 to label all nuclei and memb-Cerulean to label all membranes, and the complementary PhOTO-M zebrafish line expresses H2B-Cerulean and memb-Dendra2. Efficient cleavage and proper FP localization were verified by western blot (Figure S1) and confocal microscopy (Figure 1B). 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.

Bottom Line: To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.PhOTO 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 ∼100 µm 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Beckman Institute (139-74), California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT

Background: 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.

Methodology: PhOTO 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 ∼100 µm 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/significance: PhOTO 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.

Show MeSH
Related in: MedlinePlus