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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-2C4 labels multiple cells with distinct morphologies and anatomical locations. (A-F) Whole-mount staining of intact planarians or regenerating planarians with 2C4 (green) and with either 6G10 (magenta) in panels E and F or Lens culinaris agglutinin lectin (LCA, magenta) in panels G and H. (A) 2C4 labels multiple distinct cell types in the intact worm. Closed arrowhead indicates an example of a 2C4-S cell. Open arrowhead highlights a 2C4-N cell. Arrow indicates a large round 2C4-I cell. (B, C) 2C4 labels the anterior and posterior blastema during regeneration in 2, 3, 4, 5, and 7 dpa trunk regenerates. Higher magnification images of the anterior blastemas are shown in C. Arrowhead highlights an example of the 2C4-S cells seen throughout regeneration. (D) Magnified image of 2C4-N cells. Arrows denote the large cell bodies and arrowheads indicate their projections. (E, F) 2C4-I cells are located within the anterior (shown in E) and posterior (shown in F) intestinal branches (delineated by labeling of the intestinal wall musculature with 6G10). (G, H) 2C4 is expressed in a subset of goblet cells marked with LCA. Strongly labeled LCA+ cells immediately anterior to the pharynx were weakly labeled with 2C4 (shown in G). In contrast, strongly labeled 2C4 cells in anterior secondary branches were weakly labeled with LCA (shown in H). Dashed boxes in E-G indicate the areas shown in the inset images. Images are maximum intensity projections of optical sections except for A and B. Anterior is to the left in A and to the top in B-G. Image D was acquired adjacent to the pharynx. Scale bars: (A, B) 200 μm; (C, E-H) 50 μm; (D) 20 μm.
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Fig4: Smed-2C4 labels multiple cells with distinct morphologies and anatomical locations. (A-F) Whole-mount staining of intact planarians or regenerating planarians with 2C4 (green) and with either 6G10 (magenta) in panels E and F or Lens culinaris agglutinin lectin (LCA, magenta) in panels G and H. (A) 2C4 labels multiple distinct cell types in the intact worm. Closed arrowhead indicates an example of a 2C4-S cell. Open arrowhead highlights a 2C4-N cell. Arrow indicates a large round 2C4-I cell. (B, C) 2C4 labels the anterior and posterior blastema during regeneration in 2, 3, 4, 5, and 7 dpa trunk regenerates. Higher magnification images of the anterior blastemas are shown in C. Arrowhead highlights an example of the 2C4-S cells seen throughout regeneration. (D) Magnified image of 2C4-N cells. Arrows denote the large cell bodies and arrowheads indicate their projections. (E, F) 2C4-I cells are located within the anterior (shown in E) and posterior (shown in F) intestinal branches (delineated by labeling of the intestinal wall musculature with 6G10). (G, H) 2C4 is expressed in a subset of goblet cells marked with LCA. Strongly labeled LCA+ cells immediately anterior to the pharynx were weakly labeled with 2C4 (shown in G). In contrast, strongly labeled 2C4 cells in anterior secondary branches were weakly labeled with LCA (shown in H). Dashed boxes in E-G indicate the areas shown in the inset images. Images are maximum intensity projections of optical sections except for A and B. Anterior is to the left in A and to the top in B-G. Image D was acquired adjacent to the pharynx. Scale bars: (A, B) 200 μm; (C, E-H) 50 μm; (D) 20 μm.

Mentions: We observed multiple distinct cell morphologies (which we refer to as cell types hereafter) throughout the planarian body that labeled with Smed-2C4 (2C4) (Figure 4A). The first cell type (2C4-S cells) had cytoplasmic 2C4 labeling and small cell bodies approximately 4.4 μm in diameter (N = 123 cells measured from 10 worms). We observed that 2C4-S cells were weakly labeled with 2C4 near the epidermis throughout the intact worm (Figure 4A, closed arrowhead; Additional file 4: Figure S3A, arrows) but strongly labeled within the regeneration blastema (Figure 4B and C). To explore whether these cells were secretory, we performed co-labeling experiments with the lectin wheat germ agglutinin (WGA), which labels a subset of secretory cells [20] and found that 2C4-S cells were not WGA+ (Additional file 4: Figure S3A). In order to determine when the protein recognized by 2C4 is expressed during regeneration, we amputated animals anterior and posterior to the pharynx to analyze the presence of 2C4-S cells during 7 days post-amputation. 2C4+ cells were detected in both the anterior and posterior blastemas at all the timepoints we examined (Figure 4B-C).Figure 4


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-2C4 labels multiple cells with distinct morphologies and anatomical locations. (A-F) Whole-mount staining of intact planarians or regenerating planarians with 2C4 (green) and with either 6G10 (magenta) in panels E and F or Lens culinaris agglutinin lectin (LCA, magenta) in panels G and H. (A) 2C4 labels multiple distinct cell types in the intact worm. Closed arrowhead indicates an example of a 2C4-S cell. Open arrowhead highlights a 2C4-N cell. Arrow indicates a large round 2C4-I cell. (B, C) 2C4 labels the anterior and posterior blastema during regeneration in 2, 3, 4, 5, and 7 dpa trunk regenerates. Higher magnification images of the anterior blastemas are shown in C. Arrowhead highlights an example of the 2C4-S cells seen throughout regeneration. (D) Magnified image of 2C4-N cells. Arrows denote the large cell bodies and arrowheads indicate their projections. (E, F) 2C4-I cells are located within the anterior (shown in E) and posterior (shown in F) intestinal branches (delineated by labeling of the intestinal wall musculature with 6G10). (G, H) 2C4 is expressed in a subset of goblet cells marked with LCA. Strongly labeled LCA+ cells immediately anterior to the pharynx were weakly labeled with 2C4 (shown in G). In contrast, strongly labeled 2C4 cells in anterior secondary branches were weakly labeled with LCA (shown in H). Dashed boxes in E-G indicate the areas shown in the inset images. Images are maximum intensity projections of optical sections except for A and B. Anterior is to the left in A and to the top in B-G. Image D was acquired adjacent to the pharynx. Scale bars: (A, B) 200 μm; (C, E-H) 50 μm; (D) 20 μm.
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Fig4: Smed-2C4 labels multiple cells with distinct morphologies and anatomical locations. (A-F) Whole-mount staining of intact planarians or regenerating planarians with 2C4 (green) and with either 6G10 (magenta) in panels E and F or Lens culinaris agglutinin lectin (LCA, magenta) in panels G and H. (A) 2C4 labels multiple distinct cell types in the intact worm. Closed arrowhead indicates an example of a 2C4-S cell. Open arrowhead highlights a 2C4-N cell. Arrow indicates a large round 2C4-I cell. (B, C) 2C4 labels the anterior and posterior blastema during regeneration in 2, 3, 4, 5, and 7 dpa trunk regenerates. Higher magnification images of the anterior blastemas are shown in C. Arrowhead highlights an example of the 2C4-S cells seen throughout regeneration. (D) Magnified image of 2C4-N cells. Arrows denote the large cell bodies and arrowheads indicate their projections. (E, F) 2C4-I cells are located within the anterior (shown in E) and posterior (shown in F) intestinal branches (delineated by labeling of the intestinal wall musculature with 6G10). (G, H) 2C4 is expressed in a subset of goblet cells marked with LCA. Strongly labeled LCA+ cells immediately anterior to the pharynx were weakly labeled with 2C4 (shown in G). In contrast, strongly labeled 2C4 cells in anterior secondary branches were weakly labeled with LCA (shown in H). Dashed boxes in E-G indicate the areas shown in the inset images. Images are maximum intensity projections of optical sections except for A and B. Anterior is to the left in A and to the top in B-G. Image D was acquired adjacent to the pharynx. Scale bars: (A, B) 200 μm; (C, E-H) 50 μm; (D) 20 μm.
Mentions: We observed multiple distinct cell morphologies (which we refer to as cell types hereafter) throughout the planarian body that labeled with Smed-2C4 (2C4) (Figure 4A). The first cell type (2C4-S cells) had cytoplasmic 2C4 labeling and small cell bodies approximately 4.4 μm in diameter (N = 123 cells measured from 10 worms). We observed that 2C4-S cells were weakly labeled with 2C4 near the epidermis throughout the intact worm (Figure 4A, closed arrowhead; Additional file 4: Figure S3A, arrows) but strongly labeled within the regeneration blastema (Figure 4B and C). To explore whether these cells were secretory, we performed co-labeling experiments with the lectin wheat germ agglutinin (WGA), which labels a subset of secretory cells [20] and found that 2C4-S cells were not WGA+ (Additional file 4: Figure S3A). In order to determine when the protein recognized by 2C4 is expressed during regeneration, we amputated animals anterior and posterior to the pharynx to analyze the presence of 2C4-S cells during 7 days post-amputation. 2C4+ cells were detected in both the anterior and posterior blastemas at all the timepoints we examined (Figure 4B-C).Figure 4

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.