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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

Superficial structures and musculature of cercariae.(A and B) Actin-rich spines and sensory cilia. (A) Anterior region of the head as seen by Differential Interference Contrast (DIC) optics, phalloidin staining to visualize actin, and immunofluorescence with an anti-β-tubulin antibody to label cilia. Images are maximum confocal projections. Bottom, overlay showing distribution of actin and β-tubulin. Arrow indicates an actin-rich channel though which a sensory cilium projects. (B) Maximum confocal projection of a cross section though the tail showing a sensory cilium (green) crossing the musculature (magenta) to project to the outside (right). (C and D) Single confocal sections depicting staining with lectin PSA that labels the basement membrane below the tegument. Panels C and D represent distinct confocal sections of the same animal. (E) Single confocal section through the head showing staining with lectins PSA and PNA and with phalloidin to visualize actin. PSA labels the basement membrane between the actin-rich surface spines and the muscle layer (yellow arrow). PNA marks a layer of material at the level of the actin spines which may represent the tegument or the associated glycocalyx (red arrow). (F) Phalloidin staining in various anatomical regions. Magenta box, actin spines and anterior sensory structures. Green box, longitudinal (white arrowhead), circular (yellow arrowhead), and diagonal muscle fibers (magenta arrowhead). Yellow box, acetabulum and the interface between the head and the tail. Note the intense radially symmetric spheres of phalloidin staining (cyan arrowhead); because of the proximity of these structures to longitudinal muscles in the head and tail, we suggest this staining may represent sites of muscle attachment. White box, longitudinal and helical muscles (green arrowhead) of the tail. Whole cercaria image represents a maximum projection derived from tiled stacks. Insets are magnified views of the indicated regions. All images are maximum projections generated from a Z-stack though an entire animal, except for the green inset that was derived from a subset of optical sections. (G and H) Depth projections showing phalloidin staining of the acetabulum and associated musculature. Scale shown below indicates the color-coding of distances from the ventral surface (i.e. the colors transition from red (ventral) to blue (dorsal) moving deeper into the animal). Panel G represents a dorsal view whereas panel H depicts a transverse section with the ventral surface towards the top. (I) Immunofluorescence with an anti-phospho S/T antibody that labels the longitudinal muscles of the tail. Scale bars, 10 µm. Anterior faces up in panel A and to the left in panels C, D, and F.
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pntd-0001009-g001: Superficial structures and musculature of cercariae.(A and B) Actin-rich spines and sensory cilia. (A) Anterior region of the head as seen by Differential Interference Contrast (DIC) optics, phalloidin staining to visualize actin, and immunofluorescence with an anti-β-tubulin antibody to label cilia. Images are maximum confocal projections. Bottom, overlay showing distribution of actin and β-tubulin. Arrow indicates an actin-rich channel though which a sensory cilium projects. (B) Maximum confocal projection of a cross section though the tail showing a sensory cilium (green) crossing the musculature (magenta) to project to the outside (right). (C and D) Single confocal sections depicting staining with lectin PSA that labels the basement membrane below the tegument. Panels C and D represent distinct confocal sections of the same animal. (E) Single confocal section through the head showing staining with lectins PSA and PNA and with phalloidin to visualize actin. PSA labels the basement membrane between the actin-rich surface spines and the muscle layer (yellow arrow). PNA marks a layer of material at the level of the actin spines which may represent the tegument or the associated glycocalyx (red arrow). (F) Phalloidin staining in various anatomical regions. Magenta box, actin spines and anterior sensory structures. Green box, longitudinal (white arrowhead), circular (yellow arrowhead), and diagonal muscle fibers (magenta arrowhead). Yellow box, acetabulum and the interface between the head and the tail. Note the intense radially symmetric spheres of phalloidin staining (cyan arrowhead); because of the proximity of these structures to longitudinal muscles in the head and tail, we suggest this staining may represent sites of muscle attachment. White box, longitudinal and helical muscles (green arrowhead) of the tail. Whole cercaria image represents a maximum projection derived from tiled stacks. Insets are magnified views of the indicated regions. All images are maximum projections generated from a Z-stack though an entire animal, except for the green inset that was derived from a subset of optical sections. (G and H) Depth projections showing phalloidin staining of the acetabulum and associated musculature. Scale shown below indicates the color-coding of distances from the ventral surface (i.e. the colors transition from red (ventral) to blue (dorsal) moving deeper into the animal). Panel G represents a dorsal view whereas panel H depicts a transverse section with the ventral surface towards the top. (I) Immunofluorescence with an anti-phospho S/T antibody that labels the longitudinal muscles of the tail. Scale bars, 10 µm. Anterior faces up in panel A and to the left in panels C, D, and F.

Mentions: Cercaria are covered by a sugar-rich layer, called the glycocalyx, that sits upon the tegument, a modified syncytial epidermis characterized by a trilaminate membrane system [2], [38], [39]. In S. mansoni cercariae spines that face posterior and cover nearly the entire surface of the animal are prominent features of the tegument [20], [38]. Similar to previous reports [20], we find that these spines can be labeled with phalloidin (Figure 1A), consistent with these spines being rich in filamentous actin. Because the goal of this life-cycle stage is to locate and penetrate a suitable host, cercariae also possess a variety of ciliated sensory papillae [38]. We observed ciliated regions of unsheathed papillae scattered along the entire length of the animal by staining with antibodies specific to β-tubulin (Figure 1A, B) and acetylated α-tubulin (Table S1). Additionally, the openings of these ciliated papillae could be visualized as phalloidin-stained discs (Figure 1 A, B) that encircle cilia projecting through the tegument(Figure 1A, B).


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)

Superficial structures and musculature of cercariae.(A and B) Actin-rich spines and sensory cilia. (A) Anterior region of the head as seen by Differential Interference Contrast (DIC) optics, phalloidin staining to visualize actin, and immunofluorescence with an anti-β-tubulin antibody to label cilia. Images are maximum confocal projections. Bottom, overlay showing distribution of actin and β-tubulin. Arrow indicates an actin-rich channel though which a sensory cilium projects. (B) Maximum confocal projection of a cross section though the tail showing a sensory cilium (green) crossing the musculature (magenta) to project to the outside (right). (C and D) Single confocal sections depicting staining with lectin PSA that labels the basement membrane below the tegument. Panels C and D represent distinct confocal sections of the same animal. (E) Single confocal section through the head showing staining with lectins PSA and PNA and with phalloidin to visualize actin. PSA labels the basement membrane between the actin-rich surface spines and the muscle layer (yellow arrow). PNA marks a layer of material at the level of the actin spines which may represent the tegument or the associated glycocalyx (red arrow). (F) Phalloidin staining in various anatomical regions. Magenta box, actin spines and anterior sensory structures. Green box, longitudinal (white arrowhead), circular (yellow arrowhead), and diagonal muscle fibers (magenta arrowhead). Yellow box, acetabulum and the interface between the head and the tail. Note the intense radially symmetric spheres of phalloidin staining (cyan arrowhead); because of the proximity of these structures to longitudinal muscles in the head and tail, we suggest this staining may represent sites of muscle attachment. White box, longitudinal and helical muscles (green arrowhead) of the tail. Whole cercaria image represents a maximum projection derived from tiled stacks. Insets are magnified views of the indicated regions. All images are maximum projections generated from a Z-stack though an entire animal, except for the green inset that was derived from a subset of optical sections. (G and H) Depth projections showing phalloidin staining of the acetabulum and associated musculature. Scale shown below indicates the color-coding of distances from the ventral surface (i.e. the colors transition from red (ventral) to blue (dorsal) moving deeper into the animal). Panel G represents a dorsal view whereas panel H depicts a transverse section with the ventral surface towards the top. (I) Immunofluorescence with an anti-phospho S/T antibody that labels the longitudinal muscles of the tail. Scale bars, 10 µm. Anterior faces up in panel A and to the left in panels C, D, and F.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001009-g001: Superficial structures and musculature of cercariae.(A and B) Actin-rich spines and sensory cilia. (A) Anterior region of the head as seen by Differential Interference Contrast (DIC) optics, phalloidin staining to visualize actin, and immunofluorescence with an anti-β-tubulin antibody to label cilia. Images are maximum confocal projections. Bottom, overlay showing distribution of actin and β-tubulin. Arrow indicates an actin-rich channel though which a sensory cilium projects. (B) Maximum confocal projection of a cross section though the tail showing a sensory cilium (green) crossing the musculature (magenta) to project to the outside (right). (C and D) Single confocal sections depicting staining with lectin PSA that labels the basement membrane below the tegument. Panels C and D represent distinct confocal sections of the same animal. (E) Single confocal section through the head showing staining with lectins PSA and PNA and with phalloidin to visualize actin. PSA labels the basement membrane between the actin-rich surface spines and the muscle layer (yellow arrow). PNA marks a layer of material at the level of the actin spines which may represent the tegument or the associated glycocalyx (red arrow). (F) Phalloidin staining in various anatomical regions. Magenta box, actin spines and anterior sensory structures. Green box, longitudinal (white arrowhead), circular (yellow arrowhead), and diagonal muscle fibers (magenta arrowhead). Yellow box, acetabulum and the interface between the head and the tail. Note the intense radially symmetric spheres of phalloidin staining (cyan arrowhead); because of the proximity of these structures to longitudinal muscles in the head and tail, we suggest this staining may represent sites of muscle attachment. White box, longitudinal and helical muscles (green arrowhead) of the tail. Whole cercaria image represents a maximum projection derived from tiled stacks. Insets are magnified views of the indicated regions. All images are maximum projections generated from a Z-stack though an entire animal, except for the green inset that was derived from a subset of optical sections. (G and H) Depth projections showing phalloidin staining of the acetabulum and associated musculature. Scale shown below indicates the color-coding of distances from the ventral surface (i.e. the colors transition from red (ventral) to blue (dorsal) moving deeper into the animal). Panel G represents a dorsal view whereas panel H depicts a transverse section with the ventral surface towards the top. (I) Immunofluorescence with an anti-phospho S/T antibody that labels the longitudinal muscles of the tail. Scale bars, 10 µm. Anterior faces up in panel A and to the left in panels C, D, and F.
Mentions: Cercaria are covered by a sugar-rich layer, called the glycocalyx, that sits upon the tegument, a modified syncytial epidermis characterized by a trilaminate membrane system [2], [38], [39]. In S. mansoni cercariae spines that face posterior and cover nearly the entire surface of the animal are prominent features of the tegument [20], [38]. Similar to previous reports [20], we find that these spines can be labeled with phalloidin (Figure 1A), consistent with these spines being rich in filamentous actin. Because the goal of this life-cycle stage is to locate and penetrate a suitable host, cercariae also possess a variety of ciliated sensory papillae [38]. We observed ciliated regions of unsheathed papillae scattered along the entire length of the animal by staining with antibodies specific to β-tubulin (Figure 1A, B) and acetylated α-tubulin (Table S1). Additionally, the openings of these ciliated papillae could be visualized as phalloidin-stained discs (Figure 1 A, B) that encircle cilia projecting through the tegument(Figure 1A, B).

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