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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: 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.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-5B1 labels ciliated cells. (A-C) Whole-mount view of intact planarians immunostained with 5B1 (green), and co-labeled with anti-Acetylated Tubulin (magenta) and DAPI (blue) in panel B and anti-Acetylated Tubulin (magenta) in C. (A) 5B1 labels protonephridial tubules. Arrowheads indicate examples of protonephridia. (B) Image of the head region showing that 5B1 labels the protonephridial tubules that are positive for Acetylated Tubulin. Arrows show examples of protonephridia tubules. (C) A higher magnification image of protonephridia. Arrows point to examples of 5B1-labeled tubules. Arrowheads show examples of 5B1 labeling in flame cells. (D) 5B1is shown to label in immediate proximity to anti-Acetylated Tubulin in the dorsal ciliated stripe. Images are maximum intensity projections of optical sections except for in A. The anterior of the animal is to the top in A and B and to the left in D. Scale bars: (A) 200 μm; (B, C) 50 μm; (D) 20 μm.
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Fig5: Smed-5B1 labels ciliated cells. (A-C) Whole-mount view of intact planarians immunostained with 5B1 (green), and co-labeled with anti-Acetylated Tubulin (magenta) and DAPI (blue) in panel B and anti-Acetylated Tubulin (magenta) in C. (A) 5B1 labels protonephridial tubules. Arrowheads indicate examples of protonephridia. (B) Image of the head region showing that 5B1 labels the protonephridial tubules that are positive for Acetylated Tubulin. Arrows show examples of protonephridia tubules. (C) A higher magnification image of protonephridia. Arrows point to examples of 5B1-labeled tubules. Arrowheads show examples of 5B1 labeling in flame cells. (D) 5B1is shown to label in immediate proximity to anti-Acetylated Tubulin in the dorsal ciliated stripe. Images are maximum intensity projections of optical sections except for in A. The anterior of the animal is to the top in A and B and to the left in D. Scale bars: (A) 200 μm; (B, C) 50 μm; (D) 20 μm.

Mentions: Staining planarians with Smed-5B1 (5B1) revealed a pattern strikingly similar to those observed with anti-Acetylated Tubulin and in situ hybridizations against protonephridial markers (arrowheads in Figure 5A) [14,15]. The planarian protonephridial system, analogous to the metanephridial systems found in vertebrates [44], maintains osmoregularity and excretes waste. Protonephridia in planaria consist of fenestrated, ciliated flame cells, which connect to tubules that are ciliated proximal to the flame cells and are non-ciliated distal to the flame cells [14,15]. Protonephridia are located along the majority of the length of the planarian body. Anti-Acetylated Tubulin labels the flame cells and ciliated tubules of protonephridia as well as all other ciliated structures in planarians [25]. Therefore, we tested if 5B1 would co-label with anti-Acetylated Tubulin. We found that 5B1 labeled the tubules of the protonephridia, exterior to the cilia, in a pattern consistent with labeling of the ciliated tubule cells’ cytoplasm or membrane (arrows in Figure 5B and C). Labeling was also observed surrounding Acetylated Tubulin labeling at the flame bulb, which is also highly ciliated (arrowheads in Figure 5C). This observation led us to explore whether 5B1 was associated with other ciliated cell types. Planarians have a high abundance of ciliated cells in the epithelium on their ventral surface and in a discrete stripe running along the dorsal anteroposterior axis; these structures are positive for Acetylated Tubulin [24,45,46]. We detected 5B1 labeling within the dorsal ciliated stripe and the ventral ciliated epithelial cell surface (dorsal staining shown as a representative example in Figure 5D).Figure 5


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-5B1 labels ciliated cells. (A-C) Whole-mount view of intact planarians immunostained with 5B1 (green), and co-labeled with anti-Acetylated Tubulin (magenta) and DAPI (blue) in panel B and anti-Acetylated Tubulin (magenta) in C. (A) 5B1 labels protonephridial tubules. Arrowheads indicate examples of protonephridia. (B) Image of the head region showing that 5B1 labels the protonephridial tubules that are positive for Acetylated Tubulin. Arrows show examples of protonephridia tubules. (C) A higher magnification image of protonephridia. Arrows point to examples of 5B1-labeled tubules. Arrowheads show examples of 5B1 labeling in flame cells. (D) 5B1is shown to label in immediate proximity to anti-Acetylated Tubulin in the dorsal ciliated stripe. Images are maximum intensity projections of optical sections except for in A. The anterior of the animal is to the top in A and B and to the left in D. Scale bars: (A) 200 μm; (B, C) 50 μm; (D) 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Smed-5B1 labels ciliated cells. (A-C) Whole-mount view of intact planarians immunostained with 5B1 (green), and co-labeled with anti-Acetylated Tubulin (magenta) and DAPI (blue) in panel B and anti-Acetylated Tubulin (magenta) in C. (A) 5B1 labels protonephridial tubules. Arrowheads indicate examples of protonephridia. (B) Image of the head region showing that 5B1 labels the protonephridial tubules that are positive for Acetylated Tubulin. Arrows show examples of protonephridia tubules. (C) A higher magnification image of protonephridia. Arrows point to examples of 5B1-labeled tubules. Arrowheads show examples of 5B1 labeling in flame cells. (D) 5B1is shown to label in immediate proximity to anti-Acetylated Tubulin in the dorsal ciliated stripe. Images are maximum intensity projections of optical sections except for in A. The anterior of the animal is to the top in A and B and to the left in D. Scale bars: (A) 200 μm; (B, C) 50 μm; (D) 20 μm.
Mentions: Staining planarians with Smed-5B1 (5B1) revealed a pattern strikingly similar to those observed with anti-Acetylated Tubulin and in situ hybridizations against protonephridial markers (arrowheads in Figure 5A) [14,15]. The planarian protonephridial system, analogous to the metanephridial systems found in vertebrates [44], maintains osmoregularity and excretes waste. Protonephridia in planaria consist of fenestrated, ciliated flame cells, which connect to tubules that are ciliated proximal to the flame cells and are non-ciliated distal to the flame cells [14,15]. Protonephridia are located along the majority of the length of the planarian body. Anti-Acetylated Tubulin labels the flame cells and ciliated tubules of protonephridia as well as all other ciliated structures in planarians [25]. Therefore, we tested if 5B1 would co-label with anti-Acetylated Tubulin. We found that 5B1 labeled the tubules of the protonephridia, exterior to the cilia, in a pattern consistent with labeling of the ciliated tubule cells’ cytoplasm or membrane (arrows in Figure 5B and C). Labeling was also observed surrounding Acetylated Tubulin labeling at the flame bulb, which is also highly ciliated (arrowheads in Figure 5C). This observation led us to explore whether 5B1 was associated with other ciliated cell types. Planarians have a high abundance of ciliated cells in the epithelium on their ventral surface and in a discrete stripe running along the dorsal anteroposterior axis; these structures are positive for Acetylated Tubulin [24,45,46]. We detected 5B1 labeling within the dorsal ciliated stripe and the ventral ciliated epithelial cell surface (dorsal staining shown as a representative example in Figure 5D).Figure 5

Bottom Line: 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.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.