The role of lrp-2 in C. elegans vulval cell lineage polarity

We investigated the role of the low-density lipoprotein receptor lrp-2 in C. elegans vulval cell lineage polarity. We find that despite the high conservation of the Wnt signaling component, LRP5/6, in higher order organisms it appears to have evolved after the split of Nematoda due to its absence in all nematode genomes examined. C. elegans contains multiple low-density lipoprotein receptors within its genome, two of which are lrp-1 and lrp-2. Due to the position in the genome and high sequence similarity we believe that lrp-2 is the product of a recent duplication of lrp-1 within the Caenorhabditis lineage (Minor and Sternberg, 2019d). To understand the potential role of lrp-1 and lrp-2, we sought to score the vulval phenotypes of these mutants during L4 stage. lrp-1(ku156) mutants are sick and arrest during an early larval stage. For this reason we were not able to examine the role of lrp-1 in vulval formation. Despite high sequence similarity and proposed functional redundancy with lrp-1, lrp-2 mutants are remarkably wild-type. lrp-2 is expressed in the developing vulva at the same time as both cam-1 and vang-1. By examining double and triple mutant strains we find that lrp-2 is likely to function downstream of egl-20 along with transmembrane proteins cam-1 and vang-1 (Minor and Sternberg, 2019b; Minor and Sternberg, 2019c). All three genes antagonize the lin-17/Frizzled pathway by directing the aberrant localization of SYS-1 to the posterior daughter cell of P7.p, thereby leading to the posterior orientation of the P7.p lineage (Minor and Sternberg, 2019a). These observations suggest the simple model shown in Figure 1, in which LRP-2 cooperates with CAM-1 and VANG-1 to respond to EGL-20. This work provides evidence that despite lacking a true LRP5/6 ortholog, the formation of the C. elegans vulva is controlled by another member of the low-density lipoprotein superfamily, lrp-2. This data could potentially lead to insight into the evolution of both structure and function of the highly important Wnt pathway component, LRP5/6. Despite strong genetic evidence, this work does not describe the physical interaction between LRP-2 and CAM-1, VANG-1, EGL-20. Can LRP-2 bind with the other transmembrane proteins, CAM-1 and VANG-1, involved in this pathway? Can LRP-2 physically bind the Wnt ligand, EGL-20? Future work should focus on the biochemistry of this pathway. Answers to these questions could provide interesting insights into the evolution of low-density lipoprotein receptors, including LRP5/6, as well as how Wnt signaling has evolved within nematodes without the presence of one of the most important and highly conserved transmembrane proteins

The role of lrp-2 in C. elegans vulval cell lineage polarity

We investigated the role of the low-density lipoprotein receptor lrp-2 in C. elegans vulval cell lineage polarity. We find that despite the high conservation of the Wnt signaling component, LRP5/6, in higher order organisms it appears to have evolved after the split of Nematoda due to its absence in all nematode genomes examined. C. elegans contains multiple low-density lipoprotein receptors within its genome, two of which are lrp-1 and lrp-2. Due to the position in the genome and high sequence similarity we believe that lrp-2 is the product of a recent duplication of lrp-1 within the Caenorhabditis lineage (Minor and Sternberg, 2019d). To understand the potential role of lrp-1 and lrp-2, we sought to score the vulval phenotypes of these mutants during L4 stage. lrp-1(ku156) mutants are sick and arrest during an early larval stage. For this reason we were not able to examine the role of lrp-1 in vulval formation. Despite high sequence similarity and proposed functional redundancy with lrp-1, lrp-2 mutants are remarkably wild-type. lrp-2 is expressed in the developing vulva at the same time as both cam-1 and vang-1. By examining double and triple mutant strains we find that lrp-2 is likely to function downstream of egl-20 along with transmembrane proteins cam-1 and vang-1 (Minor and Sternberg, 2019b; Minor and Sternberg, 2019c). All three genes antagonize the lin-17/Frizzled pathway by directing the aberrant localization of SYS-1 to the posterior daughter cell of P7.p, thereby leading to the posterior orientation of the P7.p lineage (Minor and Sternberg, 2019a). These observations suggest the simple model shown in Figure 1, in which LRP-2 cooperates with CAM-1 and VANG-1 to respond to EGL-20. This work provides evidence that despite lacking a true LRP5/6 ortholog, the formation of the C. elegans vulva is controlled by another member of the low-density lipoprotein superfamily, lrp-2. This data could potentially lead to insight into the evolution of both structure and function of the highly important Wnt pathway component, LRP5/6. Despite strong genetic evidence, this work does not describe the physical interaction between LRP-2 and CAM-1, VANG-1, EGL-20. Can LRP-2 bind with the other transmembrane proteins, CAM-1 and VANG-1, involved in this pathway? Can LRP-2 physically bind the Wnt ligand, EGL-20? Future work should focus on the biochemistry of this pathway. Answers to these questions could provide interesting insights into the evolution of low-density lipoprotein receptors, including LRP5/6, as well as how Wnt signaling has evolved within nematodes without the presence of one of the most important and highly conserved transmembrane proteins

C. elegans LRP-2 functions in vulval precursor cell polarity

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. Specifically, in the absence of all Wnts, the VPCs display a randomized orientation, which is likely the default (Green et al., 2008; Minor et al. 2013). Two separate Wnts from the anchor cell, LIN-44 and MOM-2 acting through receptors LIN-17/Frizzled and LIN-18/Ryk, respectively, regulate P7.p orientation (Ferguson et al., 1987; Sternberg and Horvitz, 1988; Sawa et al., 1996; Inoue et al., 2004; Gleason et al., 2006). In the absence of these signals the orientation of the progeny of P7.p mimic those of P5.p and face toward the posterior of the worm, a phenotype referred to as posterior-reversed vulval lineage (P-Rvl). This posterior orientation is dependent on the instructive signal EGL-20, a Wnt expressed in the tail acting through CAM-1/ROR and VANG-1/Van Gogh, and is referred to as “ground polarity” (Green et al., 2008). Here we examine the role of a low-density lipoprotein receptor, lrp-2, and its role in controlling the orientation of P7.p daughter cells. To investigate this interaction double mutants were constructed with both alleles of lrp-2 and lin-17(n671) (Table 1). Much like cam-1(gm122) and vang-1(ok1142), both alleles of lrp-2 suppress the lin-17(n671) phenotype from 74 to approximately 50% P-Rvl leading us to hypothesize that lrp-2 functions in the same pathway as cam-1 and vang-1. Furthering this hypothesis we have shown that, like cam-1 and vang-1, lrp-2 controls the localization of SYS-1/b-catenin (Minor and Sternberg, 2019). To ensure that this phenotype was a result of loss of lrp-2 function as opposed to background effects we injected a fosmid (WRM0617cA02) containing the full-length sequence of lrp-2 and found that it does rescue the double mutant phenotype of lin-17(n671); lrp-2(gk272) from 55 to 73%. In order to better test this hypothesis a triple mutant was constructed between lin-17(n671), lrp-2(gk272), and cam-1(gm122) (Table 1). This triple mutant displays the same P-Rvl penetrance as both the lin-17(n671); lrp-2(gk272) and lin-17(n671); cam-1(gm122) double mutants confirming that lrp-2 functions in the same pathway as cam-1

LRP-2 controls the localization of C. elegans SYS-1/beta-catenin

The polarity of the C. elegans P7.p cell divisions is controlled by the Wnt/β-catenin asymmetry pathway (Green et al., 2008; Minor et al., 2013). This pathway includes the β-catenin-like proteins SYS-1 and WRM-1, POP-1/TCF, and the Nemo-like-kinase, LIT-1 (reviewed by Mizumoto and Sawa, 2007). The Wnt/β-catenin asymmetry pathway ensures different ratios of SYS-1 to POP-1, controlling the differential transcription of Wnt target genes between daughters of an asymmetric cell division. Because our genetic data indicate an antagonism between LRP-2 and LIN-17 similar to that between CAM-1 and VANG-1 and LIN-17 (Minor and Sternberg, 2019), we wanted to determine if LRP-2 can control the asymmetric localization of SYS-1 between the daughter cells of P7.p during anaphase of the first cell division. The initial establishment of vulval polarity can be observed through the localization of VENUS::SYS-1 (VNS::SYS-1), localized in a high (P7.pa)/low (P7.pp) pattern in the wild-type worm, reciprocal to the localization of POP-1/TCF (Phillips et al., 2007; Green et al., 2008). It was previously reported (Green et al. 2008) that VNS::SYS-1 asymmetry in P7.p daughter cells is often lost in lin-17(n671) and lin-18(e620) mutants. These mutants display two aberrant patterns of VNS::SYS-1 localization as well as the wild-type pattern, though less frequently. The two deviant localization patterns include one in which both P7.pa and P7.pp express equal amounts of VNS::SYS-1 and a reversed VNS::SYS-1 pattern in which P7.pp is enriched with VNS::SYS-1. By observing VNS::SYS-1 localization in a lin-17(n671); lrp-2(gk272) background we see that the aberrant localization of SYS-1 is suppressed to a similar degree to that of lin-17(n671); cam-1(gm122) and lin-17(n671); vang-1(ok1142). This observation confirms LRP-2 controls vulval cell polarity by antagonizing LIN-17 in a similar fashion to CAM-1 and VANG-1, and that the effect of LRP-2 is at the level of P7.p rather than its progeny

LRP-2 likely acts downstream of EGL-20/Wnt

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. egl-20/Wnt is expressed in the tail (Whangbo and Kenyon, 2000) and forms a posterior-to-anterior concentration gradient (Coudreuse et al., 2006). It has previously been shown that EGL-20 acts instructively during vulva development by imparting directional information, as opposed to being permissive, where it would only be required for polarization (Green et al., 2008; Minor et al., 2013). By moving the source of egl-20 expression from the posterior of the worm to the anchor cell, the axis of symmetry of the developing vulva, we can reorient the daughter cells of P5.p and P7.p toward the center in a wild-type configuration. Expression of egl-20 from the center of the axis of symmetry partially suppresses the lin-17(n671) phenotype (Green et al., 2008; Table 1). To test whether LRP-2 acts downstream of EGL-20, we ectopically expressed egl-20 from the anchor cell in a lin-17(n671); lrp-2(gk272) double mutant background and compared it to a lin-17(n671); lrp-2(+) strain. If LRP-2 acts downstream of EGL-20, then anteriorly-expressed EGL-20 will not be able to suppress the lin-17 phenotype, with is the result observed (Table 1). Thus, like CAM-1 and VANG-1, LRP-2 likely acts downstream of EGL-20

C. elegans LRP-2 functions in vulval precursor cell polarity

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. Specifically, in the absence of all Wnts, the VPCs display a randomized orientation, which is likely the default (Green et al., 2008; Minor et al. 2013). Two separate Wnts from the anchor cell, LIN-44 and MOM-2 acting through receptors LIN-17/Frizzled and LIN-18/Ryk, respectively, regulate P7.p orientation (Ferguson et al., 1987; Sternberg and Horvitz, 1988; Sawa et al., 1996; Inoue et al., 2004; Gleason et al., 2006). In the absence of these signals the orientation of the progeny of P7.p mimic those of P5.p and face toward the posterior of the worm, a phenotype referred to as posterior-reversed vulval lineage (P-Rvl). This posterior orientation is dependent on the instructive signal EGL-20, a Wnt expressed in the tail acting through CAM-1/ROR and VANG-1/Van Gogh, and is referred to as “ground polarity” (Green et al., 2008). Here we examine the role of a low-density lipoprotein receptor, lrp-2, and its role in controlling the orientation of P7.p daughter cells. To investigate this interaction double mutants were constructed with both alleles of lrp-2 and lin-17(n671) (Table 1). Much like cam-1(gm122) and vang-1(ok1142), both alleles of lrp-2 suppress the lin-17(n671) phenotype from 74 to approximately 50% P-Rvl leading us to hypothesize that lrp-2 functions in the same pathway as cam-1 and vang-1. Furthering this hypothesis we have shown that, like cam-1 and vang-1, lrp-2 controls the localization of SYS-1/b-catenin (Minor and Sternberg, 2019). To ensure that this phenotype was a result of loss of lrp-2 function as opposed to background effects we injected a fosmid (WRM0617cA02) containing the full-length sequence of lrp-2 and found that it does rescue the double mutant phenotype of lin-17(n671); lrp-2(gk272) from 55 to 73%. In order to better test this hypothesis a triple mutant was constructed between lin-17(n671), lrp-2(gk272), and cam-1(gm122) (Table 1). This triple mutant displays the same P-Rvl penetrance as both the lin-17(n671); lrp-2(gk272) and lin-17(n671); cam-1(gm122) double mutants confirming that lrp-2 functions in the same pathway as cam-1

LRP-2 controls the localization of C. elegans SYS-1/beta-catenin

The polarity of the C. elegans P7.p cell divisions is controlled by the Wnt/β-catenin asymmetry pathway (Green et al., 2008; Minor et al., 2013). This pathway includes the β-catenin-like proteins SYS-1 and WRM-1, POP-1/TCF, and the Nemo-like-kinase, LIT-1 (reviewed by Mizumoto and Sawa, 2007). The Wnt/β-catenin asymmetry pathway ensures different ratios of SYS-1 to POP-1, controlling the differential transcription of Wnt target genes between daughters of an asymmetric cell division. Because our genetic data indicate an antagonism between LRP-2 and LIN-17 similar to that between CAM-1 and VANG-1 and LIN-17 (Minor and Sternberg, 2019), we wanted to determine if LRP-2 can control the asymmetric localization of SYS-1 between the daughter cells of P7.p during anaphase of the first cell division. The initial establishment of vulval polarity can be observed through the localization of VENUS::SYS-1 (VNS::SYS-1), localized in a high (P7.pa)/low (P7.pp) pattern in the wild-type worm, reciprocal to the localization of POP-1/TCF (Phillips et al., 2007; Green et al., 2008). It was previously reported (Green et al. 2008) that VNS::SYS-1 asymmetry in P7.p daughter cells is often lost in lin-17(n671) and lin-18(e620) mutants. These mutants display two aberrant patterns of VNS::SYS-1 localization as well as the wild-type pattern, though less frequently. The two deviant localization patterns include one in which both P7.pa and P7.pp express equal amounts of VNS::SYS-1 and a reversed VNS::SYS-1 pattern in which P7.pp is enriched with VNS::SYS-1. By observing VNS::SYS-1 localization in a lin-17(n671); lrp-2(gk272) background we see that the aberrant localization of SYS-1 is suppressed to a similar degree to that of lin-17(n671); cam-1(gm122) and lin-17(n671); vang-1(ok1142). This observation confirms LRP-2 controls vulval cell polarity by antagonizing LIN-17 in a similar fashion to CAM-1 and VANG-1, and that the effect of LRP-2 is at the level of P7.p rather than its progeny

Low density lipoprotein receptors LRP-1 and LRP-2 in C. elegans

The regulation of vulval cell lineage polarity is controlled by Wnt signaling. Previously known components involved in the regulation of vulval cell lineage polarity include LIN-17, LIN-18, CAM-1, and VANG-1 (Inoue et al., 2004; Gleason et al., 2006; Green et al., 2008). A directed bioinformatics screen of known Wnt pathway components was performed to find additional genes involved in directing vulval orientation. A BLAST was run using other known Wnt receptors and it was determined that C. elegans does not contain a true ortholog of Drosophila LRP5/6 (Arrow) (He et al., 2004; Eisenmann, 2005), but does have multiple low-density lipoprotein receptors, including LRP-1 and LRP-2 (Figure 1). Like other low-density lipoprotein receptors, both LRP-1 and LRP-2 contain many LDLR Domain Class A and Class B repeats, EGF-like domains, and a transmembrane domain. However, having approximately three times as many amino acids, LRP-1 and LRP-2 are more similar to megalin than LRP5/6 (Yochem et al., 1999). The absence of LRP5/6 within C. elegans but presence in flies and all other higher order organisms suggests that the gene encoding LRP5/6 arose after nematodes, potentially from either LRP1 or LRP2/megalin, as both receptors contain the entire extracellular portion of LRP5/6 in a single contiguous sequence block (Figure 1). Our examination of the protein sequence of LRP-1 and LRP-2 indicates that most nematodes have at least two copies of LRP-like proteins with C. elegans LRP-1 and LRP-2 being highly similar possibly due to a recent duplication and divergence (Figure 2). Comparing the sequences across Caenorhabditis we find that LRP-1 proteins cluster together and LRP-2 proteins also form their own cluster. Based on location in the genome and sequence similarity from protein alignment, we believe that Caenorhabditis lrp-2 is a recent duplication and divergence of lrp-1 (Figure 2)

Origin and Evolution of Dishevelled

Dishevelled (Dsh or Dvl) is an important signaling protein, playing a key role in Wnt signaling and relaying cellular information for several developmental pathways. Dsh is highly conserved among metazoans and has expanded into a multigene family in most bilaterian lineages, including vertebrates, planarians, and nematodes. These orthologs, where explored, are known to have considerable overlap in function, but evidence for functional specialization continues to mount. We performed a comparative analysis of Dsh across animals to explore protein architecture and identify conserved and divergent features that could provide insight into functional specialization with an emphasis on invertebrates, especially nematodes. We find evidence of dynamic evolution of Dsh, particularly among nematodes, with taxa varying in ortholog number from one to three. We identify a new domain specific to some nematode lineages and find an unexpected nuclear localization signal conserved in many Dsh orthologs. Our findings raise questions of protein evolution in general and provide clues as to how animals have dealt with the complex intricacies of having a protein, such as Dsh, act as a central messenger hub connected to many different and vitally important pathways. We discuss our findings in the context of functional specialization and bring many testable hypotheses to light

FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans