Limits...
A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

Hartin SN, Hudson ML, Yingling C, Ackley BD - PLoS ONE (2015)

Bottom Line: We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood.We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3.We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, United States of America.

ABSTRACT

Background: The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.

Results: We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.

Conclusions: PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

No MeSH data available.


Related in: MedlinePlus

sdn-1 mutations enhance lin-44 gastrulation defects.A: In C. elegans, gastrulation is initiated by the inward migration of the endodermal precursor cells Ea and Ep (black asterisks). In sdn-1 and ptp-3 mutant animals, the cells ingress, become completely surrounded by neighboring cells, then divide laterally (white asterisks). Note that Ea and Ep are completely internalized prior to the lateral cell division. In lin-44 and sdn-1 mutants, Ea/Ep ingression is often asynchronous. In addition, some lin-44 embryos show a more severe phenotype, where the Ea/Ep cells completely fail to ingress. The subsequent lateral cell division positions two of the daughter cells onto the surface of the embryo, generating a “Gut on the exterior”, or Gex phenotype [77]. Similar but more penetrant defects are observed in lin-44; sdn-1 double mutants. B: Summary of Ea and Ep cell ingression behavior by genotype. The lin-44; sdn-1 double mutants exhibit a higher rate of Ea and Ep ingression defects than either single mutant. C: Relative timing of developmental milestones as a function of genotype. Note the lin-44; sdn-1 double mutants have a significantly longer period in which the gastrulation cleft is open.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4418752&req=5

pone.0121397.g005: sdn-1 mutations enhance lin-44 gastrulation defects.A: In C. elegans, gastrulation is initiated by the inward migration of the endodermal precursor cells Ea and Ep (black asterisks). In sdn-1 and ptp-3 mutant animals, the cells ingress, become completely surrounded by neighboring cells, then divide laterally (white asterisks). Note that Ea and Ep are completely internalized prior to the lateral cell division. In lin-44 and sdn-1 mutants, Ea/Ep ingression is often asynchronous. In addition, some lin-44 embryos show a more severe phenotype, where the Ea/Ep cells completely fail to ingress. The subsequent lateral cell division positions two of the daughter cells onto the surface of the embryo, generating a “Gut on the exterior”, or Gex phenotype [77]. Similar but more penetrant defects are observed in lin-44; sdn-1 double mutants. B: Summary of Ea and Ep cell ingression behavior by genotype. The lin-44; sdn-1 double mutants exhibit a higher rate of Ea and Ep ingression defects than either single mutant. C: Relative timing of developmental milestones as a function of genotype. Note the lin-44; sdn-1 double mutants have a significantly longer period in which the gastrulation cleft is open.

Mentions: Using time-lapse video microscopy, we found highly penetrant defects in the migration of endodermal precursor cells Ea and Ep, in lin-44; sdn-1 double mutants at the 24-cell stage of development (Fig 5). In wild type animals, gastrulation begins when Ea and Ep rotate and then ingress from the surface of the embryo to the center [51]. In lin-44; sdn-1 double mutants, 48% of embryos (11/24) showed defective Ea/Ep ingression, compared to 21% of lin-44 (5/23) and 20% of sdn-1 (3/15) single mutant embryos. As a comparison, we also examined ptp-3; sdn-1 embryos for Ea/Ep ingression failure. 29% (2/7) embryos showed defects in this process. In one embryo, the Ea/Ep cells completely failed to ingress, while in another, Ep ingressed before Ea. These data are not significantly different from sdn-1 mutants alone, suggesting that ptp-3 has no obvious role in early gastrulation. These defects in Ea/Ep ingression often resulted in endodermal cells appearing on the embryo’s surface later in development (Gut on the exterior, or Gex phenotype), with catastrophic consequences for subsequent epithelial cell migrations (Fig 5). Further analysis of our time-lapse data revealed that 15% (3/20) of lin-44 embryos showed defects in neuroblast migration as manifested by an increase in gastrulation cleft duration and failure of epithelial cells to enclose the embryo (Fig 3A and Fig 5). This is likely due to mis-positioned gut cells inhibiting or blocking epithelial cell migrations, or causing defects in overall embryonic organization. While the lin-44; sdn-1 Ea/Ep ingression phenotypes appear to be additive when compared to each single mutant, the increase in embryonic lethality is clearly synergistic (Table 1). As such, it appears that small defects in Ea/Ep ingression early in development lead to severe consequences in later developmental events. Together these results indicate that both lin-44 and sdn-1 contribute to the normal migration of Ea and Ep cells at the onset of gastrulation, and that ptp-3 has no obvious role in this process. In addition, both lin-44 and sdn-1 may also be involved in controlling neuroblast migration and gastrulation cleft closure later in embryogenesis, although in the case of lin-44, we cannot rule out that defects at later time points are a consequence of earlier defects in Ea/Ep migration.


A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

Hartin SN, Hudson ML, Yingling C, Ackley BD - PLoS ONE (2015)

sdn-1 mutations enhance lin-44 gastrulation defects.A: In C. elegans, gastrulation is initiated by the inward migration of the endodermal precursor cells Ea and Ep (black asterisks). In sdn-1 and ptp-3 mutant animals, the cells ingress, become completely surrounded by neighboring cells, then divide laterally (white asterisks). Note that Ea and Ep are completely internalized prior to the lateral cell division. In lin-44 and sdn-1 mutants, Ea/Ep ingression is often asynchronous. In addition, some lin-44 embryos show a more severe phenotype, where the Ea/Ep cells completely fail to ingress. The subsequent lateral cell division positions two of the daughter cells onto the surface of the embryo, generating a “Gut on the exterior”, or Gex phenotype [77]. Similar but more penetrant defects are observed in lin-44; sdn-1 double mutants. B: Summary of Ea and Ep cell ingression behavior by genotype. The lin-44; sdn-1 double mutants exhibit a higher rate of Ea and Ep ingression defects than either single mutant. C: Relative timing of developmental milestones as a function of genotype. Note the lin-44; sdn-1 double mutants have a significantly longer period in which the gastrulation cleft is open.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418752&req=5

pone.0121397.g005: sdn-1 mutations enhance lin-44 gastrulation defects.A: In C. elegans, gastrulation is initiated by the inward migration of the endodermal precursor cells Ea and Ep (black asterisks). In sdn-1 and ptp-3 mutant animals, the cells ingress, become completely surrounded by neighboring cells, then divide laterally (white asterisks). Note that Ea and Ep are completely internalized prior to the lateral cell division. In lin-44 and sdn-1 mutants, Ea/Ep ingression is often asynchronous. In addition, some lin-44 embryos show a more severe phenotype, where the Ea/Ep cells completely fail to ingress. The subsequent lateral cell division positions two of the daughter cells onto the surface of the embryo, generating a “Gut on the exterior”, or Gex phenotype [77]. Similar but more penetrant defects are observed in lin-44; sdn-1 double mutants. B: Summary of Ea and Ep cell ingression behavior by genotype. The lin-44; sdn-1 double mutants exhibit a higher rate of Ea and Ep ingression defects than either single mutant. C: Relative timing of developmental milestones as a function of genotype. Note the lin-44; sdn-1 double mutants have a significantly longer period in which the gastrulation cleft is open.
Mentions: Using time-lapse video microscopy, we found highly penetrant defects in the migration of endodermal precursor cells Ea and Ep, in lin-44; sdn-1 double mutants at the 24-cell stage of development (Fig 5). In wild type animals, gastrulation begins when Ea and Ep rotate and then ingress from the surface of the embryo to the center [51]. In lin-44; sdn-1 double mutants, 48% of embryos (11/24) showed defective Ea/Ep ingression, compared to 21% of lin-44 (5/23) and 20% of sdn-1 (3/15) single mutant embryos. As a comparison, we also examined ptp-3; sdn-1 embryos for Ea/Ep ingression failure. 29% (2/7) embryos showed defects in this process. In one embryo, the Ea/Ep cells completely failed to ingress, while in another, Ep ingressed before Ea. These data are not significantly different from sdn-1 mutants alone, suggesting that ptp-3 has no obvious role in early gastrulation. These defects in Ea/Ep ingression often resulted in endodermal cells appearing on the embryo’s surface later in development (Gut on the exterior, or Gex phenotype), with catastrophic consequences for subsequent epithelial cell migrations (Fig 5). Further analysis of our time-lapse data revealed that 15% (3/20) of lin-44 embryos showed defects in neuroblast migration as manifested by an increase in gastrulation cleft duration and failure of epithelial cells to enclose the embryo (Fig 3A and Fig 5). This is likely due to mis-positioned gut cells inhibiting or blocking epithelial cell migrations, or causing defects in overall embryonic organization. While the lin-44; sdn-1 Ea/Ep ingression phenotypes appear to be additive when compared to each single mutant, the increase in embryonic lethality is clearly synergistic (Table 1). As such, it appears that small defects in Ea/Ep ingression early in development lead to severe consequences in later developmental events. Together these results indicate that both lin-44 and sdn-1 contribute to the normal migration of Ea and Ep cells at the onset of gastrulation, and that ptp-3 has no obvious role in this process. In addition, both lin-44 and sdn-1 may also be involved in controlling neuroblast migration and gastrulation cleft closure later in embryogenesis, although in the case of lin-44, we cannot rule out that defects at later time points are a consequence of earlier defects in Ea/Ep migration.

Bottom Line: We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood.We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3.We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, United States of America.

ABSTRACT

Background: The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.

Results: We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.

Conclusions: PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

No MeSH data available.


Related in: MedlinePlus