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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

The dual loss of ptp-3 and sdn-1 results in synergistic defects during embryogenesis.A: Using 4D time-lapse microscopy we monitored embryogenesis in N2 wild-type, ptp-3, sdn-1, lin-44 single mutants, and lin-44; sdn-1 and ptp-3; sdn-1 double mutants. The gastrulation clefts (outlined regions) present in the single mutants are more likely to close during development than the clefts in the double mutants. The arrowhead indicates cells that have extruded from the internal region of the embryo through the open gastrulation cleft (panel 418 minutes). B: Terminal fates of the embryos plotted by genotype.
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pone.0121397.g003: The dual loss of ptp-3 and sdn-1 results in synergistic defects during embryogenesis.A: Using 4D time-lapse microscopy we monitored embryogenesis in N2 wild-type, ptp-3, sdn-1, lin-44 single mutants, and lin-44; sdn-1 and ptp-3; sdn-1 double mutants. The gastrulation clefts (outlined regions) present in the single mutants are more likely to close during development than the clefts in the double mutants. The arrowhead indicates cells that have extruded from the internal region of the embryo through the open gastrulation cleft (panel 418 minutes). B: Terminal fates of the embryos plotted by genotype.

Mentions: To better understand what might be contributing to ptp-3; sdn-1 lethality during embryogenesis, we used 4D time-lapse microscopy to observe cell division and migration during the first 10 hours of embryonic development. The first cell migration event that occurs during C. elegans development is the onset of gastrulation, where the gut precursor cells, Ea and Ep, ingress into the center of the embryo [55]. In wild-type embryos, Ea and Ep ingress in concert, side-by-side, with the space vacated by their ingression filled by movements from six surrounding cells [56]. The next major developmental event occurs when cells of the endodermal and mesodermal lineages begin to ingress at the posterior end, leaving a transient gastrulation cleft on the ventral surface (Fig 3) [57]. Onset of cleft opening was phenotypically normal in the embryos observed (n = 10) although one embryo showed gastrulation cleft opening at the anterior end. However, the relative timing of gastrulation cleft opening was significantly delayed in ptp-3 embryos when compared to Ea/Ep ingression and comma stage. The gastrulation cleft is flanked by neuroblasts; in wild-type embryos, these normally migrate towards the midline of the ventral surface, closing the cleft in about 55 minutes [53]. These subsequently form a substrate for epithelial cell migration, which intercalate and extend from the dorsal and lateral surfaces to enclose the embryo during epiboly.


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)

The dual loss of ptp-3 and sdn-1 results in synergistic defects during embryogenesis.A: Using 4D time-lapse microscopy we monitored embryogenesis in N2 wild-type, ptp-3, sdn-1, lin-44 single mutants, and lin-44; sdn-1 and ptp-3; sdn-1 double mutants. The gastrulation clefts (outlined regions) present in the single mutants are more likely to close during development than the clefts in the double mutants. The arrowhead indicates cells that have extruded from the internal region of the embryo through the open gastrulation cleft (panel 418 minutes). B: Terminal fates of the embryos plotted by genotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121397.g003: The dual loss of ptp-3 and sdn-1 results in synergistic defects during embryogenesis.A: Using 4D time-lapse microscopy we monitored embryogenesis in N2 wild-type, ptp-3, sdn-1, lin-44 single mutants, and lin-44; sdn-1 and ptp-3; sdn-1 double mutants. The gastrulation clefts (outlined regions) present in the single mutants are more likely to close during development than the clefts in the double mutants. The arrowhead indicates cells that have extruded from the internal region of the embryo through the open gastrulation cleft (panel 418 minutes). B: Terminal fates of the embryos plotted by genotype.
Mentions: To better understand what might be contributing to ptp-3; sdn-1 lethality during embryogenesis, we used 4D time-lapse microscopy to observe cell division and migration during the first 10 hours of embryonic development. The first cell migration event that occurs during C. elegans development is the onset of gastrulation, where the gut precursor cells, Ea and Ep, ingress into the center of the embryo [55]. In wild-type embryos, Ea and Ep ingress in concert, side-by-side, with the space vacated by their ingression filled by movements from six surrounding cells [56]. The next major developmental event occurs when cells of the endodermal and mesodermal lineages begin to ingress at the posterior end, leaving a transient gastrulation cleft on the ventral surface (Fig 3) [57]. Onset of cleft opening was phenotypically normal in the embryos observed (n = 10) although one embryo showed gastrulation cleft opening at the anterior end. However, the relative timing of gastrulation cleft opening was significantly delayed in ptp-3 embryos when compared to Ea/Ep ingression and comma stage. The gastrulation cleft is flanked by neuroblasts; in wild-type embryos, these normally migrate towards the midline of the ventral surface, closing the cleft in about 55 minutes [53]. These subsequently form a substrate for epithelial cell migration, which intercalate and extend from the dorsal and lateral surfaces to enclose the embryo during epiboly.

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