γ-tubulin is differentially expressed in mitotic and non-mitotic cardiomyocytes in the regenerating zebrafish heart

This data article contains complementary figures related to the research article entitled, “ A dual epimorphic and compensatory mode of heart regeneration” ([10], http://dx.doi.org/10.1016/j.ydbio.2014.12.002), which presents a spatial and temporal characterization of cardiomyocyte proliferation and dedifferentiation after cryoinjury-induced myocardial infarction. This study demonstrated that mitotic divisions occur in cardiac cells at distinct differentiation status, namely in dedifferentiated cells at the injury border as well as in mature cardiac cells within the remaining intact myocardium. One of the important aspects supporting our conclusions is a characterization of proteins that are upregulated during mitosis in the regenerating hearts. The data presented here reveal a dynamic change in the expression level and in the subcellular distribution of γ-tubulin between mitotic and non-mitotic cardiac cells. We report that in the non-mitotic cells, γ-tubulin expression is restricted to the centrosome. By contrast, during the mitosis, γ-tubulin strongly expands its localization within the spindle apparatus that interacts with the condensed chromosomes. We demonstrated that the differential distribution of γ-tubulin in non-mitotic and mitotic cells requires adjusted image processing for the appropriate visualization of both expression patterns in the same histological specimens.


a b s t r a c t
This data article contains complementary figures related to the research article entitled, " A dual epimorphic and compensatory mode of heart regeneration" ( [10], http://dx.doi.org/10.1016/j. ydbio.2014.12.002), which presents a spatial and temporal characterization of cardiomyocyte proliferation and dedifferentiation after cryoinjury-induced myocardial infarction. This study demonstrated that mitotic divisions occur in cardiac cells at distinct differentiation status, namely in dedifferentiated cells at the injury border as well as in mature cardiac cells within the remaining intact myocardium. One of the important aspects supporting our conclusions is a characterization of proteins that are upregulated during mitosis in the regenerating hearts. The data presented here reveal a dynamic change in the expression level and in the subcellular distribution of γ-tubulin between mitotic and nonmitotic cardiac cells. We report that in the non-mitotic cells, γtubulin expression is restricted to the centrosome. By contrast, during the mitosis, γ-tubulin strongly expands its localization within the spindle apparatus that interacts with the condensed chromosomes. We demonstrated that the differential distribution of γ-tubulin in non-mitotic and mitotic cells requires adjusted image processing for the appropriate visualization of both expression patterns in the same histological specimens. &

Data format
Raw and processed with Adobe Photoshop Experimental factors Cryoinjuries were performed to induce myocardial infarction in transgenic adult zebrafish expressing EGFP under a cardiac specific promoter (cmlc2: EGFP). Hearts were collected at 14 days post cryoinjury, fixed in 2% paraformaldehyde, and sectioned using a cryostat.

Experimental features
Heart sections were analyzed using immunofluorescence against γ-tubulin, phospho-(Ser10)-histone H3, GFP and DAPI. The multiple labeling was analyzed using confocal microscopy. γ-tubulin fluorescent signals were adjusted for the optimal visualization of the subcellular distribution using the levels option of Adobe Photoshop software. Data source location University of Fribourg, Switzerland Data accessibility The data are supplied with this article

Value of the data
Quadruple immunolabeling with a cardiac transgenic marker, phospho-(Ser10)-histone H3, γ-tubulin and DAPI allows unambiguous identification of mitotic cardiomyocytes in the regenerating zebrafish heart.
We analyze a previously uncharacterized distribution of γ-tubulin in the zebrafish adult cardiomyocytes to provide evidence of a differential expression pattern of γ-tubulin in nonmitotic and mitotic cardiac cells.
We describe how to adjust the fluorescence signal intensity of the original confocal data in order to detect γ-tubulin either in the interphase centrosomes or in the mitotic spindle.
γ-tubulin is an evolutionary conserved cytoskeletal protein, which plays essential roles in microtubule organization and nucleation [4,6]. The subcellular distribution of this protein has been previously analyzed in a large variety of model organisms ranging from Aspergillus through Drosophila and mammals. In some of these studies, γ-tubulin was detected only in the centrosomes-related organelles during both the interphase and mitosis [7,11,12]. Other reports revealed the expansion of γ-tubulin during the mitosis within the nearly entire mitotic spindle [5]. To our knowledge, the distribution of γ-tubulin has not yet been analyzed in the zebrafish adult somatic cells. Previous studies revealed that the zebrafish heart regeneration depends on the proliferation of adult cardiomyocytes [8,9]. Here, to understand the mitotic mechanisms associated with zebrafish heart regeneration, we analyzed the expression of both γ-tubulin and phosphohistone H3 (PH3), which demarcates the condensed chromosomes during the nuclear division.
Our data provide evidence for the dynamic γ-tubulin expression during the cell cycle. To distinguish between the interphase/G0 and mitosis, we used phospho-(Ser10)-histone H3 (PH3) immunolabeling that demarcates the condensed chromosomes. To identify cardiomyocytes among other cell types in the heart, we used a transgenic fish line expressing EGFP under a cardiac specific promoter (cmlc2::EGFP), and we performed anti-GFP immunostaining ( Fig. 1A-C; Fig. 3). Analysis of multiple heart sections revealed that in the non-mitotic zebrafish cardiomyocytes, γ-tubulin expression in restricted to a single spot in the vicinity of each nucleus, which corresponds to the centrosome [4]. By contrast, all of the PH3-positive cardiac cells were characterized by an expanded and stronger presence of γ-tubulin that was associated with the condensed chromosomes (n¼17 cells, 5 hearts) (Fig. 1C and E; Fig. 3C and D). Analysis of the red fluorescence with the same image adjustments revealed that this centrosomal pattern of γ-tubulin expression does not derive from background enhancement (Fig. 2). Thus, γ-tubulin is not restricted only to the duplicated centrosomes of the dividing cells, but it covers other domains of the mitotic spindle. Due to the high difference in the intensity of γ-tubulin signals between mitotic and non-mitotic cells, it was not possible to simultaneously display both types of expression patterns on the same image. The original confocal data had to be adjusted using Adobe Photoshop to visualize both aspects of γ-tubulin expression in the separate images of the same original specimen (Fig. 1D and F; Fig. 3C 0 and D 0 ). Our analyses of mitotic cytoskeletal proteins will be helpful to understand the cellular mechanisms underlying the proliferative capacity of adult zebrafish cardiomyocytes.

Animal procedures
The present work was performed with adult fish at the age of 18 months (transgenic fishes: cmlc2:: EGFP zebrafish strains [1]. Cryoinjuries were performed as described previously [2,3]. The experimental research on animals was approved by the cantonal veterinary office of Fribourg.

Immunohistochemistry
The hearts were collected and fixed overnight at 4 1C in 2% paraformaldehyde. They were then rinsed in PBS and equilibrated in 30% sucrose before embedding in Tissue-Tek OCT compound (Sakura Finetek Europe B.V.) and cryosectioned at a thickness of 16 μm.

Image analysis and quantification
After antibody staining, cardiac tissue imaging was performed at different magnifications (20 Â and 63 Â ) with a confocal microscope (Leica TCS-SP5). The following Leica image acquisition parameters were used: