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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.


Related in: MedlinePlus

Smed-1H6 labels CNS and PNS axonal projections. (A-F) Whole-mount view of intact planarians immunostained with 1H6 (green) in conjunction with other antibodies or FISH to genes indicated in the panels. (A) 1H6 labels neural structures in the intact planarian. Arrows mark the anterior end of the ventral nerve cords (VNCs); closed arrowheads mark the VNCs near the pharynx. Open arrowheads highlight transverse and lateral axon branches. (B) Higher magnification image of 1H6 staining in the head region shows labeling in the anterior end of the VNCs (arrow) and in lateral branches (arrowhead). (C) 1H6 labels gpas+ (magenta) brain branches in the head. Arrowheads denote one of the co-labeled branches. (D-F) Planarians double-labeled with1H6 and anti-SYNAPSIN (magenta). (D) High magnification shows that 1H6 labels neuronal projections in close association with SYNAPSIN+ synapses. (E) Co-labeling in the anterior region of the VNCs. (F) 1H6 staining is absent in the neuropil of the cephalic ganglia. Arrows point to examples of 1H6 and anti-SYNAPSIN co-labeling, whereas arrowheads (in F) mark the SYNAPSIN+ neuropil of the cephalic ganglia. (G) 1H6 labels many CRMP2+ (magenta) neurons in the intact planarian (shown in the head region, highlighted with arrowheads). (H) 1H6, 6G10 (magenta), and DAPI (blue, epidermal nuclei) labeling at the anterior tip of the worm demonstrates that 1H6 labels axon projections within the submuscular plexus. Arrowheads mark 1H6+ axons extending between epithelial cells. Images are maximum intensity projections of optical sections, except in A. Anterior is to the left in A, E, and F and to the top in B-D and G-H. Images were taken to the right side of the pharynx (facing the ventral side of the animal) in D and to the left side of the cephalic ganglia in C. Scale bars: (A) 200 μm; (B-G) 20 μm; (H) 50 μm.
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Fig3: Smed-1H6 labels CNS and PNS axonal projections. (A-F) Whole-mount view of intact planarians immunostained with 1H6 (green) in conjunction with other antibodies or FISH to genes indicated in the panels. (A) 1H6 labels neural structures in the intact planarian. Arrows mark the anterior end of the ventral nerve cords (VNCs); closed arrowheads mark the VNCs near the pharynx. Open arrowheads highlight transverse and lateral axon branches. (B) Higher magnification image of 1H6 staining in the head region shows labeling in the anterior end of the VNCs (arrow) and in lateral branches (arrowhead). (C) 1H6 labels gpas+ (magenta) brain branches in the head. Arrowheads denote one of the co-labeled branches. (D-F) Planarians double-labeled with1H6 and anti-SYNAPSIN (magenta). (D) High magnification shows that 1H6 labels neuronal projections in close association with SYNAPSIN+ synapses. (E) Co-labeling in the anterior region of the VNCs. (F) 1H6 staining is absent in the neuropil of the cephalic ganglia. Arrows point to examples of 1H6 and anti-SYNAPSIN co-labeling, whereas arrowheads (in F) mark the SYNAPSIN+ neuropil of the cephalic ganglia. (G) 1H6 labels many CRMP2+ (magenta) neurons in the intact planarian (shown in the head region, highlighted with arrowheads). (H) 1H6, 6G10 (magenta), and DAPI (blue, epidermal nuclei) labeling at the anterior tip of the worm demonstrates that 1H6 labels axon projections within the submuscular plexus. Arrowheads mark 1H6+ axons extending between epithelial cells. Images are maximum intensity projections of optical sections, except in A. Anterior is to the left in A, E, and F and to the top in B-D and G-H. Images were taken to the right side of the pharynx (facing the ventral side of the animal) in D and to the left side of the cephalic ganglia in C. Scale bars: (A) 200 μm; (B-G) 20 μm; (H) 50 μm.

Mentions: Smed-1H6 (1H6) labeled the axonal projections in subsets of cells in both the central and peripheral nervous systems. In the CNS, 1H6 labeled the ventral nerve cords (VNCs) (Figure 3A, closed arrowheads), which are known to extend anteriorly through the head region, beneath the cephalic ganglia [35]. 1H6+ projections were observed in the anterior tip of the VNCs (Figure 3A and 3B, arrows). 1H6 also labeled the transverse axon branches between the VNCs and the lateral axon branches extending from the VNCs (Figure 3A, open arrowheads). We observed 1H6 labeling in the lateral branches of the cephalic ganglia (Figure 3B, arrowhead); these axon projections are known to extend to the sides of the head where they penetrate the epidermis in sensory neuron-rich areas [36,37]. To confirm 1H6 labeling in these branches, we processed planarians for 1H6 immunolabeling and in situ hybridization to G protein α-subunit (gpas), which marks distal lateral branch neurons [7]. We found that 1H6 strongly co-labeled with gpas (arrowheads in Figure 3C), suggesting that 1H6 binds an antigen found in the axonal projections of sensory neurons.Figure 3


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-1H6 labels CNS and PNS axonal projections. (A-F) Whole-mount view of intact planarians immunostained with 1H6 (green) in conjunction with other antibodies or FISH to genes indicated in the panels. (A) 1H6 labels neural structures in the intact planarian. Arrows mark the anterior end of the ventral nerve cords (VNCs); closed arrowheads mark the VNCs near the pharynx. Open arrowheads highlight transverse and lateral axon branches. (B) Higher magnification image of 1H6 staining in the head region shows labeling in the anterior end of the VNCs (arrow) and in lateral branches (arrowhead). (C) 1H6 labels gpas+ (magenta) brain branches in the head. Arrowheads denote one of the co-labeled branches. (D-F) Planarians double-labeled with1H6 and anti-SYNAPSIN (magenta). (D) High magnification shows that 1H6 labels neuronal projections in close association with SYNAPSIN+ synapses. (E) Co-labeling in the anterior region of the VNCs. (F) 1H6 staining is absent in the neuropil of the cephalic ganglia. Arrows point to examples of 1H6 and anti-SYNAPSIN co-labeling, whereas arrowheads (in F) mark the SYNAPSIN+ neuropil of the cephalic ganglia. (G) 1H6 labels many CRMP2+ (magenta) neurons in the intact planarian (shown in the head region, highlighted with arrowheads). (H) 1H6, 6G10 (magenta), and DAPI (blue, epidermal nuclei) labeling at the anterior tip of the worm demonstrates that 1H6 labels axon projections within the submuscular plexus. Arrowheads mark 1H6+ axons extending between epithelial cells. Images are maximum intensity projections of optical sections, except in A. Anterior is to the left in A, E, and F and to the top in B-D and G-H. Images were taken to the right side of the pharynx (facing the ventral side of the animal) in D and to the left side of the cephalic ganglia in C. Scale bars: (A) 200 μm; (B-G) 20 μm; (H) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307677&req=5

Fig3: Smed-1H6 labels CNS and PNS axonal projections. (A-F) Whole-mount view of intact planarians immunostained with 1H6 (green) in conjunction with other antibodies or FISH to genes indicated in the panels. (A) 1H6 labels neural structures in the intact planarian. Arrows mark the anterior end of the ventral nerve cords (VNCs); closed arrowheads mark the VNCs near the pharynx. Open arrowheads highlight transverse and lateral axon branches. (B) Higher magnification image of 1H6 staining in the head region shows labeling in the anterior end of the VNCs (arrow) and in lateral branches (arrowhead). (C) 1H6 labels gpas+ (magenta) brain branches in the head. Arrowheads denote one of the co-labeled branches. (D-F) Planarians double-labeled with1H6 and anti-SYNAPSIN (magenta). (D) High magnification shows that 1H6 labels neuronal projections in close association with SYNAPSIN+ synapses. (E) Co-labeling in the anterior region of the VNCs. (F) 1H6 staining is absent in the neuropil of the cephalic ganglia. Arrows point to examples of 1H6 and anti-SYNAPSIN co-labeling, whereas arrowheads (in F) mark the SYNAPSIN+ neuropil of the cephalic ganglia. (G) 1H6 labels many CRMP2+ (magenta) neurons in the intact planarian (shown in the head region, highlighted with arrowheads). (H) 1H6, 6G10 (magenta), and DAPI (blue, epidermal nuclei) labeling at the anterior tip of the worm demonstrates that 1H6 labels axon projections within the submuscular plexus. Arrowheads mark 1H6+ axons extending between epithelial cells. Images are maximum intensity projections of optical sections, except in A. Anterior is to the left in A, E, and F and to the top in B-D and G-H. Images were taken to the right side of the pharynx (facing the ventral side of the animal) in D and to the left side of the cephalic ganglia in C. Scale bars: (A) 200 μm; (B-G) 20 μm; (H) 50 μm.
Mentions: Smed-1H6 (1H6) labeled the axonal projections in subsets of cells in both the central and peripheral nervous systems. In the CNS, 1H6 labeled the ventral nerve cords (VNCs) (Figure 3A, closed arrowheads), which are known to extend anteriorly through the head region, beneath the cephalic ganglia [35]. 1H6+ projections were observed in the anterior tip of the VNCs (Figure 3A and 3B, arrows). 1H6 also labeled the transverse axon branches between the VNCs and the lateral axon branches extending from the VNCs (Figure 3A, open arrowheads). We observed 1H6 labeling in the lateral branches of the cephalic ganglia (Figure 3B, arrowhead); these axon projections are known to extend to the sides of the head where they penetrate the epidermis in sensory neuron-rich areas [36,37]. To confirm 1H6 labeling in these branches, we processed planarians for 1H6 immunolabeling and in situ hybridization to G protein α-subunit (gpas), which marks distal lateral branch neurons [7]. We found that 1H6 strongly co-labeled with gpas (arrowheads in Figure 3C), suggesting that 1H6 binds an antigen found in the axonal projections of sensory neurons.Figure 3

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.


Related in: MedlinePlus