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Novel monoclonal antibodies to study tissue regeneration in planarians.

Ross KG, Omuro KC, Taylor MR, Munday RK, Hubert A, King RS, Zayas RM - BMC Dev. Biol. (2015)

Bottom Line: These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema.These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments.In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, CA, 92182, USA. kel.g.ross@gmail.com.

ABSTRACT

Background: Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians.

Results: We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments.

Conclusions: The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

No MeSH data available.


Smed-6C8 labels intestinal cells and cells near the regeneration blastema. (A-C) Whole-mount view of intact or regenerating planarians immunostained with 6C8 (green) and co-labeled with 6G10 (magenta) in panels B-D and/or counterstained with DAPI (blue) in D and F. (A) 6C8-labeled cells near or within the intestine. (B, C) 6C8 cells are located within the anterior (shown in B) and posterior (shown in C) intestinal branches or in contact with the enteric musculature wall, which is delineated with 6G10 labeling. Examples of cells observed outside the enteric muscle boundary are highlighted with arrowheads. (D) 6C8 labels cell nuclei (observed within the boundary of the enteric musculature). (E, F) 6C8+ cells appear near the anterior and posterior regeneration blastemas at 3 dpa and are detected in the blastema at all later timepoints assayed (examples highlighted with arrowheads). By 5 dpa, examples of 6C8+ cells were detected far from the blastemas (arrows). Higher magnification image of the anterior blastema shown in F. Yellow asterisks indicate non-specific labeling of secretory cells. Dashed boxes indicate the area of the high magnification image shown in the insets. Images are maximum intensity projections except for A and E. Anterior of is to the top in all images. Scale bars: (A, E) 200 μm; (B-D, F) 50 μm.
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Fig7: Smed-6C8 labels intestinal cells and cells near the regeneration blastema. (A-C) Whole-mount view of intact or regenerating planarians immunostained with 6C8 (green) and co-labeled with 6G10 (magenta) in panels B-D and/or counterstained with DAPI (blue) in D and F. (A) 6C8-labeled cells near or within the intestine. (B, C) 6C8 cells are located within the anterior (shown in B) and posterior (shown in C) intestinal branches or in contact with the enteric musculature wall, which is delineated with 6G10 labeling. Examples of cells observed outside the enteric muscle boundary are highlighted with arrowheads. (D) 6C8 labels cell nuclei (observed within the boundary of the enteric musculature). (E, F) 6C8+ cells appear near the anterior and posterior regeneration blastemas at 3 dpa and are detected in the blastema at all later timepoints assayed (examples highlighted with arrowheads). By 5 dpa, examples of 6C8+ cells were detected far from the blastemas (arrows). Higher magnification image of the anterior blastema shown in F. Yellow asterisks indicate non-specific labeling of secretory cells. Dashed boxes indicate the area of the high magnification image shown in the insets. Images are maximum intensity projections except for A and E. Anterior of is to the top in all images. Scale bars: (A, E) 200 μm; (B-D, F) 50 μm.

Mentions: Smed-6C8 (6C8) labeled cells in a punctate pattern throughout the planarian body that resembled the shape of the intestinal branches (Figure 7A). Therefore, we performed co-labeling experiments with 6G10 to visualize the enteric musculature and determine if 6C8 marks intestinal cells. These experiments showed that 6C8+ cells were generally located on the luminal side of the enteric muscle wall in both the anterior and posterior of the animal (Figures 7B and C). However, some 6C8+ cells were detected outside, but still associated with the enteric muscular boundary (arrowheads in Figures 7B and C; Additional file 5: Figure S4). We counterstained samples with DAPI and found that 6C8 labeling was located in the nucleus of cells (Figure 7D, inset). Because 2C4 also labeled an intestinal cell population, we examined if the 2C4 and 6C8 epitopes were expressed in the same cells or in different cell populations. When we performed co-labeling experiments, we noted that 2C4 and 6C8 marked distinct intestinal cell populations (Additional file 6: Figure S5A). Furthermore, 6C8 was not expressed in LCA+ goblet cells (Additional file 6: Figure S5B).Figure 7


Novel monoclonal antibodies to study tissue regeneration in planarians.

Ross KG, Omuro KC, Taylor MR, Munday RK, Hubert A, King RS, Zayas RM - BMC Dev. Biol. (2015)

Smed-6C8 labels intestinal cells and cells near the regeneration blastema. (A-C) Whole-mount view of intact or regenerating planarians immunostained with 6C8 (green) and co-labeled with 6G10 (magenta) in panels B-D and/or counterstained with DAPI (blue) in D and F. (A) 6C8-labeled cells near or within the intestine. (B, C) 6C8 cells are located within the anterior (shown in B) and posterior (shown in C) intestinal branches or in contact with the enteric musculature wall, which is delineated with 6G10 labeling. Examples of cells observed outside the enteric muscle boundary are highlighted with arrowheads. (D) 6C8 labels cell nuclei (observed within the boundary of the enteric musculature). (E, F) 6C8+ cells appear near the anterior and posterior regeneration blastemas at 3 dpa and are detected in the blastema at all later timepoints assayed (examples highlighted with arrowheads). By 5 dpa, examples of 6C8+ cells were detected far from the blastemas (arrows). Higher magnification image of the anterior blastema shown in F. Yellow asterisks indicate non-specific labeling of secretory cells. Dashed boxes indicate the area of the high magnification image shown in the insets. Images are maximum intensity projections except for A and E. Anterior of is to the top in all images. Scale bars: (A, E) 200 μm; (B-D, F) 50 μm.
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Fig7: Smed-6C8 labels intestinal cells and cells near the regeneration blastema. (A-C) Whole-mount view of intact or regenerating planarians immunostained with 6C8 (green) and co-labeled with 6G10 (magenta) in panels B-D and/or counterstained with DAPI (blue) in D and F. (A) 6C8-labeled cells near or within the intestine. (B, C) 6C8 cells are located within the anterior (shown in B) and posterior (shown in C) intestinal branches or in contact with the enteric musculature wall, which is delineated with 6G10 labeling. Examples of cells observed outside the enteric muscle boundary are highlighted with arrowheads. (D) 6C8 labels cell nuclei (observed within the boundary of the enteric musculature). (E, F) 6C8+ cells appear near the anterior and posterior regeneration blastemas at 3 dpa and are detected in the blastema at all later timepoints assayed (examples highlighted with arrowheads). By 5 dpa, examples of 6C8+ cells were detected far from the blastemas (arrows). Higher magnification image of the anterior blastema shown in F. Yellow asterisks indicate non-specific labeling of secretory cells. Dashed boxes indicate the area of the high magnification image shown in the insets. Images are maximum intensity projections except for A and E. Anterior of is to the top in all images. Scale bars: (A, E) 200 μm; (B-D, F) 50 μm.
Mentions: Smed-6C8 (6C8) labeled cells in a punctate pattern throughout the planarian body that resembled the shape of the intestinal branches (Figure 7A). Therefore, we performed co-labeling experiments with 6G10 to visualize the enteric musculature and determine if 6C8 marks intestinal cells. These experiments showed that 6C8+ cells were generally located on the luminal side of the enteric muscle wall in both the anterior and posterior of the animal (Figures 7B and C). However, some 6C8+ cells were detected outside, but still associated with the enteric muscular boundary (arrowheads in Figures 7B and C; Additional file 5: Figure S4). We counterstained samples with DAPI and found that 6C8 labeling was located in the nucleus of cells (Figure 7D, inset). Because 2C4 also labeled an intestinal cell population, we examined if the 2C4 and 6C8 epitopes were expressed in the same cells or in different cell populations. When we performed co-labeling experiments, we noted that 2C4 and 6C8 marked distinct intestinal cell populations (Additional file 6: Figure S5A). Furthermore, 6C8 was not expressed in LCA+ goblet cells (Additional file 6: Figure S5B).Figure 7

Bottom Line: These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema.These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments.In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, CA, 92182, USA. kel.g.ross@gmail.com.

ABSTRACT

Background: Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians.

Results: We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments.

Conclusions: The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

No MeSH data available.