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An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy.

Collins JJ, King RS, Cogswell A, Williams DL, Newmark PA - PLoS Negl Trop Dis (2011)

Bottom Line: This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system.Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages.The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cell and Developmental Biology, Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

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Related in: MedlinePlus

The protonephridial system of cercariae.(A) Immunofluorescence with an anti-β-tubulin antibody to label the ciliated tufts of the flame cells (yellow arrowheads) and ciliated regions of the protonephridial tubules (white arrows) within the head and tail. Cercariae typically have 10 flame cells, an anterior dorsal pair, a mid-head ventral pair, posterior head dorsal and ventral pairs, and a pair at the anterior tail. Dorsal view is shown to the left and a lateral view is shown to the right. (B) Immunofluorescence with an anti-phospho Y antibody that labels the barrel of the flame cells and the excretory duct. Inserts are magnified views of the indicated regions. Yellow box, flame cells of the tail. Blue box, the protonephridial tubule splits anterior to the point of tail bifurcation. Green box, the protonephridial tubule extends to the nephridiopore at the tip of the tail. Mid-level DIC optical section showing nephridiopore and tegumental structure at tip of tail. (C) Numerous reagents utilized in this study labeled portions of the flame cells providing useful tools for analyzing flame cell morphology. The ciliated tuft stained with various anti-tubulin antibodies (top row). Several lectins showed different staining patterns of the extracellular matrix surrounding the flame cell (middle row). Phospho-specific antibodies (bottom row) also labeled the flame cells: anti-phospho S/T labeled the ciliary rootlet and anti-phosopho Y labeled the flame cell barrel. Differential interference contrast optics also permit observation of flame cell morphology. (D and E) Portions of the protonephridial tubules in the head label with lectin sWGA. (D) Maximum intensity projection of dorsal focal planes showing sWGA labeling of protonephridial tubules (white arrows) leading from the anterior flame cell pair (yellow arrowheads). (E) Cross-section of a maximum intensity projection from animal in D showing the sWGA-labeled protonephridial tubules (arrows) positioned dorsal to the sWGA-labeled acetabular ducts. Abbreviations: Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), succinylated wheat germ agglutinin (sWGA). Scale bars, 10 µm. Anterior faces up in all panels.
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pntd-0001009-g003: The protonephridial system of cercariae.(A) Immunofluorescence with an anti-β-tubulin antibody to label the ciliated tufts of the flame cells (yellow arrowheads) and ciliated regions of the protonephridial tubules (white arrows) within the head and tail. Cercariae typically have 10 flame cells, an anterior dorsal pair, a mid-head ventral pair, posterior head dorsal and ventral pairs, and a pair at the anterior tail. Dorsal view is shown to the left and a lateral view is shown to the right. (B) Immunofluorescence with an anti-phospho Y antibody that labels the barrel of the flame cells and the excretory duct. Inserts are magnified views of the indicated regions. Yellow box, flame cells of the tail. Blue box, the protonephridial tubule splits anterior to the point of tail bifurcation. Green box, the protonephridial tubule extends to the nephridiopore at the tip of the tail. Mid-level DIC optical section showing nephridiopore and tegumental structure at tip of tail. (C) Numerous reagents utilized in this study labeled portions of the flame cells providing useful tools for analyzing flame cell morphology. The ciliated tuft stained with various anti-tubulin antibodies (top row). Several lectins showed different staining patterns of the extracellular matrix surrounding the flame cell (middle row). Phospho-specific antibodies (bottom row) also labeled the flame cells: anti-phospho S/T labeled the ciliary rootlet and anti-phosopho Y labeled the flame cell barrel. Differential interference contrast optics also permit observation of flame cell morphology. (D and E) Portions of the protonephridial tubules in the head label with lectin sWGA. (D) Maximum intensity projection of dorsal focal planes showing sWGA labeling of protonephridial tubules (white arrows) leading from the anterior flame cell pair (yellow arrowheads). (E) Cross-section of a maximum intensity projection from animal in D showing the sWGA-labeled protonephridial tubules (arrows) positioned dorsal to the sWGA-labeled acetabular ducts. Abbreviations: Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), succinylated wheat germ agglutinin (sWGA). Scale bars, 10 µm. Anterior faces up in all panels.

Mentions: After being shed from their snail host and before locating and penetrating a mammal, cercariae spend a substantial portion of their short lives in freshwater. Cercaria may regulate water balance by controlling permeability through their outer surface (e.g. the tegument and/or glycocalyx) and by excretion of excess fluid by a network of osmoregulatory tubules called protonephridia [44], [45]. Proximally the protonephridia begin as a heavily ciliated cell called a flame cell. Staining of cercariae with antibodies that recognize various tubulin isoforms and modifications showed labeling of the ciliary tufts of flame cells (Figure 3A and C). Anti-tubulin antibodies also labeled several other structures, including portions of the nervous system (see below); proteinase K treatment often abolished this labeling, leaving predominantly protonephridial labeling (Table S1). For example, staining with the anti-β tubulin antibody following Proteinase K treatment provided robust and specific labeling of the flame cells (Figure 3A arrowheads) and ciliated secondary protonephridial tubules (Figure 3A arrows), allowing for easy identification of these cells even by epifluorescence. In contrast to a previous report stating that cercariae have six pairs of flame cells [38], we find that cercariae typically have 5 pairs of flame cells: an anterior-body dorsal pair, a mid-body ventral pair, posterior-body dorsal and ventral pairs, and a pair in the anterior tail (Figure 3A) similar to that described by Skelly and Shoemaker [46]. The base of the flame cell contains a region of tightly packed ciliary rootlets, which label strongly with an anti-phospho S/T antibody, and a nucleus, which is readily identified by DIC microscopy or DAPI staining (Figure 3C). The ciliary tuft of the flame cells sits in a barrel or basket-like structure formed by interdigitation between the flame cell and first tubule cell [38] and labels strongly with an anti-phospho tyrosine (anti-phospho Y) antibody (Figure 3B and 3C). In addition to labeling the flame cell and first tubule cell, anti-phospho Y also strongly labels the protonephridial tubule extending from the bladder to the nephridiopores located at the tips of the tail furci (Figure 3B). The protonephridial tubule splits anterior to the bifurcation of the tail (Figure 3B; blue inset). Although flame cells in the head were labeled by the anti-phospho Y antibody, it was unclear whether the protonephridial tubules were also labeled, since this antibody marked a variety of structures in the head (Table S1 and Movie S3).


An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy.

Collins JJ, King RS, Cogswell A, Williams DL, Newmark PA - PLoS Negl Trop Dis (2011)

The protonephridial system of cercariae.(A) Immunofluorescence with an anti-β-tubulin antibody to label the ciliated tufts of the flame cells (yellow arrowheads) and ciliated regions of the protonephridial tubules (white arrows) within the head and tail. Cercariae typically have 10 flame cells, an anterior dorsal pair, a mid-head ventral pair, posterior head dorsal and ventral pairs, and a pair at the anterior tail. Dorsal view is shown to the left and a lateral view is shown to the right. (B) Immunofluorescence with an anti-phospho Y antibody that labels the barrel of the flame cells and the excretory duct. Inserts are magnified views of the indicated regions. Yellow box, flame cells of the tail. Blue box, the protonephridial tubule splits anterior to the point of tail bifurcation. Green box, the protonephridial tubule extends to the nephridiopore at the tip of the tail. Mid-level DIC optical section showing nephridiopore and tegumental structure at tip of tail. (C) Numerous reagents utilized in this study labeled portions of the flame cells providing useful tools for analyzing flame cell morphology. The ciliated tuft stained with various anti-tubulin antibodies (top row). Several lectins showed different staining patterns of the extracellular matrix surrounding the flame cell (middle row). Phospho-specific antibodies (bottom row) also labeled the flame cells: anti-phospho S/T labeled the ciliary rootlet and anti-phosopho Y labeled the flame cell barrel. Differential interference contrast optics also permit observation of flame cell morphology. (D and E) Portions of the protonephridial tubules in the head label with lectin sWGA. (D) Maximum intensity projection of dorsal focal planes showing sWGA labeling of protonephridial tubules (white arrows) leading from the anterior flame cell pair (yellow arrowheads). (E) Cross-section of a maximum intensity projection from animal in D showing the sWGA-labeled protonephridial tubules (arrows) positioned dorsal to the sWGA-labeled acetabular ducts. Abbreviations: Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), succinylated wheat germ agglutinin (sWGA). Scale bars, 10 µm. Anterior faces up in all panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3050934&req=5

pntd-0001009-g003: The protonephridial system of cercariae.(A) Immunofluorescence with an anti-β-tubulin antibody to label the ciliated tufts of the flame cells (yellow arrowheads) and ciliated regions of the protonephridial tubules (white arrows) within the head and tail. Cercariae typically have 10 flame cells, an anterior dorsal pair, a mid-head ventral pair, posterior head dorsal and ventral pairs, and a pair at the anterior tail. Dorsal view is shown to the left and a lateral view is shown to the right. (B) Immunofluorescence with an anti-phospho Y antibody that labels the barrel of the flame cells and the excretory duct. Inserts are magnified views of the indicated regions. Yellow box, flame cells of the tail. Blue box, the protonephridial tubule splits anterior to the point of tail bifurcation. Green box, the protonephridial tubule extends to the nephridiopore at the tip of the tail. Mid-level DIC optical section showing nephridiopore and tegumental structure at tip of tail. (C) Numerous reagents utilized in this study labeled portions of the flame cells providing useful tools for analyzing flame cell morphology. The ciliated tuft stained with various anti-tubulin antibodies (top row). Several lectins showed different staining patterns of the extracellular matrix surrounding the flame cell (middle row). Phospho-specific antibodies (bottom row) also labeled the flame cells: anti-phospho S/T labeled the ciliary rootlet and anti-phosopho Y labeled the flame cell barrel. Differential interference contrast optics also permit observation of flame cell morphology. (D and E) Portions of the protonephridial tubules in the head label with lectin sWGA. (D) Maximum intensity projection of dorsal focal planes showing sWGA labeling of protonephridial tubules (white arrows) leading from the anterior flame cell pair (yellow arrowheads). (E) Cross-section of a maximum intensity projection from animal in D showing the sWGA-labeled protonephridial tubules (arrows) positioned dorsal to the sWGA-labeled acetabular ducts. Abbreviations: Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), succinylated wheat germ agglutinin (sWGA). Scale bars, 10 µm. Anterior faces up in all panels.
Mentions: After being shed from their snail host and before locating and penetrating a mammal, cercariae spend a substantial portion of their short lives in freshwater. Cercaria may regulate water balance by controlling permeability through their outer surface (e.g. the tegument and/or glycocalyx) and by excretion of excess fluid by a network of osmoregulatory tubules called protonephridia [44], [45]. Proximally the protonephridia begin as a heavily ciliated cell called a flame cell. Staining of cercariae with antibodies that recognize various tubulin isoforms and modifications showed labeling of the ciliary tufts of flame cells (Figure 3A and C). Anti-tubulin antibodies also labeled several other structures, including portions of the nervous system (see below); proteinase K treatment often abolished this labeling, leaving predominantly protonephridial labeling (Table S1). For example, staining with the anti-β tubulin antibody following Proteinase K treatment provided robust and specific labeling of the flame cells (Figure 3A arrowheads) and ciliated secondary protonephridial tubules (Figure 3A arrows), allowing for easy identification of these cells even by epifluorescence. In contrast to a previous report stating that cercariae have six pairs of flame cells [38], we find that cercariae typically have 5 pairs of flame cells: an anterior-body dorsal pair, a mid-body ventral pair, posterior-body dorsal and ventral pairs, and a pair in the anterior tail (Figure 3A) similar to that described by Skelly and Shoemaker [46]. The base of the flame cell contains a region of tightly packed ciliary rootlets, which label strongly with an anti-phospho S/T antibody, and a nucleus, which is readily identified by DIC microscopy or DAPI staining (Figure 3C). The ciliary tuft of the flame cells sits in a barrel or basket-like structure formed by interdigitation between the flame cell and first tubule cell [38] and labels strongly with an anti-phospho tyrosine (anti-phospho Y) antibody (Figure 3B and 3C). In addition to labeling the flame cell and first tubule cell, anti-phospho Y also strongly labels the protonephridial tubule extending from the bladder to the nephridiopores located at the tips of the tail furci (Figure 3B). The protonephridial tubule splits anterior to the bifurcation of the tail (Figure 3B; blue inset). Although flame cells in the head were labeled by the anti-phospho Y antibody, it was unclear whether the protonephridial tubules were also labeled, since this antibody marked a variety of structures in the head (Table S1 and Movie S3).

Bottom Line: This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system.Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages.The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cell and Developmental Biology, Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

Show MeSH
Related in: MedlinePlus