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The twisted pharynx phenotype in C. elegans.

Axäng C, Rauthan M, Hall DH, Pilon M - BMC Dev. Biol. (2007)

Bottom Line: The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development.We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges.The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Chemical and Biological Engineering, Lundberg Laboratory, Chalmers University, Göteborg, Sweden. claes.axang@molbiotech.chalmers.se <claes.axang@molbiotech.chalmers.se>

ABSTRACT

Background: The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.

Results: We find that the twisting phenotype worsens throughout larval development, that in most mutants the pharynx retains its twist when dissected away from the worm body, and that double mutants between mnm-4 and mutants with thickened pharyngeal domains (pha-2 and sma-1) have less twisting in these regions. We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges. Within twisted pharynges, actin filaments also show twisting and are longer than in controls. In a mini screen of adhesionmolecule mutants, we also identified one more twisting pharynx mutant, sax-7.

Conclusion: Defects in pharyngeal cytoskeleton length or its anchor points to the extracellular matrix are proposed as the actual source of the twisting force. The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.

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The twisted pharynx phenotype in mnm-4;etIs2 worms of different stages, and in adult mnm-4;unc-61. (A) An example of a twisted pharynx and the three measurements that can be obtained by DIC microscopy to estimate the actual degree of twist within the isthmus using the formula shown on the right side (D is diameter; L is isthmus length; and θ is the angle between the torsion lines and the pharyngeal axis). (B) Analysis of etIs2 [pRF4 pRIC-19::GFP] transgenic worms [12]: DIC images (left column), M2 neurons of the same worms visualized via their GFP expression (middle column), and the average degree of twist within the isthmus for at least three similar worms scored using confocal microscopy (pie charts). Genotypes and stages are as indicated in the DIC images. See Table 1 for actual numerical data and list of alleles. For the confocal microscopy analysis, worms were mounted on dried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.
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Figure 1: The twisted pharynx phenotype in mnm-4;etIs2 worms of different stages, and in adult mnm-4;unc-61. (A) An example of a twisted pharynx and the three measurements that can be obtained by DIC microscopy to estimate the actual degree of twist within the isthmus using the formula shown on the right side (D is diameter; L is isthmus length; and θ is the angle between the torsion lines and the pharyngeal axis). (B) Analysis of etIs2 [pRF4 pRIC-19::GFP] transgenic worms [12]: DIC images (left column), M2 neurons of the same worms visualized via their GFP expression (middle column), and the average degree of twist within the isthmus for at least three similar worms scored using confocal microscopy (pie charts). Genotypes and stages are as indicated in the DIC images. See Table 1 for actual numerical data and list of alleles. For the confocal microscopy analysis, worms were mounted on dried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.

Mentions: By measuring the torsion lines seen in a twisted pharynx, one can calculate the degree of twist (Fig. 1A). This is useful because it allows us to quantitatively measure pharyngeal twisting, even in live animals as they mature. We verified the validity of this mathematical relationship using 3D reconstructions obtained by confocal microscopy of pharynges that expressed GFP in the M2 pharyngeal neurons (Fig. 1B, Table 1; see also Additional files 1 and 2). Note that the degree of twist in the examined mutants was the same between non-transgenic worms and worms in which rol-6 (su1006dm) or dpy-20 (+) were used as phenotypic markers to establish the transgenic lines (Table 1): these markers therefore do not in themselves affect intrinsic pharyngeal twisting.


The twisted pharynx phenotype in C. elegans.

Axäng C, Rauthan M, Hall DH, Pilon M - BMC Dev. Biol. (2007)

The twisted pharynx phenotype in mnm-4;etIs2 worms of different stages, and in adult mnm-4;unc-61. (A) An example of a twisted pharynx and the three measurements that can be obtained by DIC microscopy to estimate the actual degree of twist within the isthmus using the formula shown on the right side (D is diameter; L is isthmus length; and θ is the angle between the torsion lines and the pharyngeal axis). (B) Analysis of etIs2 [pRF4 pRIC-19::GFP] transgenic worms [12]: DIC images (left column), M2 neurons of the same worms visualized via their GFP expression (middle column), and the average degree of twist within the isthmus for at least three similar worms scored using confocal microscopy (pie charts). Genotypes and stages are as indicated in the DIC images. See Table 1 for actual numerical data and list of alleles. For the confocal microscopy analysis, worms were mounted on dried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904197&req=5

Figure 1: The twisted pharynx phenotype in mnm-4;etIs2 worms of different stages, and in adult mnm-4;unc-61. (A) An example of a twisted pharynx and the three measurements that can be obtained by DIC microscopy to estimate the actual degree of twist within the isthmus using the formula shown on the right side (D is diameter; L is isthmus length; and θ is the angle between the torsion lines and the pharyngeal axis). (B) Analysis of etIs2 [pRF4 pRIC-19::GFP] transgenic worms [12]: DIC images (left column), M2 neurons of the same worms visualized via their GFP expression (middle column), and the average degree of twist within the isthmus for at least three similar worms scored using confocal microscopy (pie charts). Genotypes and stages are as indicated in the DIC images. See Table 1 for actual numerical data and list of alleles. For the confocal microscopy analysis, worms were mounted on dried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.
Mentions: By measuring the torsion lines seen in a twisted pharynx, one can calculate the degree of twist (Fig. 1A). This is useful because it allows us to quantitatively measure pharyngeal twisting, even in live animals as they mature. We verified the validity of this mathematical relationship using 3D reconstructions obtained by confocal microscopy of pharynges that expressed GFP in the M2 pharyngeal neurons (Fig. 1B, Table 1; see also Additional files 1 and 2). Note that the degree of twist in the examined mutants was the same between non-transgenic worms and worms in which rol-6 (su1006dm) or dpy-20 (+) were used as phenotypic markers to establish the transgenic lines (Table 1): these markers therefore do not in themselves affect intrinsic pharyngeal twisting.

Bottom Line: The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development.We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges.The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Chemical and Biological Engineering, Lundberg Laboratory, Chalmers University, Göteborg, Sweden. claes.axang@molbiotech.chalmers.se <claes.axang@molbiotech.chalmers.se>

ABSTRACT

Background: The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.

Results: We find that the twisting phenotype worsens throughout larval development, that in most mutants the pharynx retains its twist when dissected away from the worm body, and that double mutants between mnm-4 and mutants with thickened pharyngeal domains (pha-2 and sma-1) have less twisting in these regions. We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges. Within twisted pharynges, actin filaments also show twisting and are longer than in controls. In a mini screen of adhesionmolecule mutants, we also identified one more twisting pharynx mutant, sax-7.

Conclusion: Defects in pharyngeal cytoskeleton length or its anchor points to the extracellular matrix are proposed as the actual source of the twisting force. The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.

Show MeSH
Related in: MedlinePlus