TMEM198 promotes LRP6 signaling and is required for Wnt signaling upstream of the β-catenin destruction complex.
We screened a Xenopus tropicalis embryonic cDNA library and identified a novel 7-transmembrane protein, TMEM198, that specifically and strongly cooperated with the Wnt coreceptor LRP6 in activating TOP-FLASH reporter expression (A). Among many components in the canonical Wnt pathway, TMEM198 cooperated only with LRP6, and the cooperation was dose dependent (A and data not shown). In addition, TMEM198 and LRP6 cooperated to induce β-catenin accumulation in the cytosol of HEK293T cells (B), a characteristic feature of Wnt signaling activation, and to enhance the expression of direct Wnt target genes axin2 and cyclin D1 (C). These results suggest that TMEM198 is able to activate LRP6 in Wnt signaling.
Fig. 1. TMEM198 specifically promotes LRP6 signaling in the canonical Wnt/β-catenin pathway. (A) Wnt-responsive TOP-FLASH reporter assay in HEK293T cells with indicated transfections. TMEM198 plasmid DNA was used at 1 ng and 10 ng. (B) Cytosol fractions (more ...)
LRP5 is another Wnt coreceptor closely related to LRP6 in terms of structure and function (23
). However, in contrast to LRP6, LRP5 was not activated by coexpressed TMEM198, whereas Wnt3a was able to activate both coreceptors (D). More interestingly, when the intracellular domain of LRP5 was replaced with that of LRP6, the chimeric protein (LRP5-6) could be activated by TMEM198 as strongly as LRP6 (D). These results suggest that TMEM198 selectively cooperates with LRP6 but not LRP5, and the specificity relies on the intracellular domain of the LRP5/6 receptors. Corroborating this conclusion, the constitutively active form of LRP6, LRP6ΔE1-4, was further activated by TMEM198, whereas that of LRP5, LRP5 with a deletion of the N terminus (LRP5ΔN), was not activated (E). Of note, casein kinase 1γ (CK1γ), a known kinase responsible for LRP6 phosphorylation, displayed similar selectivity on full-length as well as constitutively active LRP5/6 receptors (D and E). The reason for this selectivity is unknown; however, we cannot rule out a quantitative difference between LRP5 and LRP6 in transducing Wnt signaling.
TMEM198 is predicted to be a transmembrane protein and is conserved from fruit fly to human (F); however, its orthologs have not been characterized in any organism including Drosophila
; accession number CG14234
). Thus, we cloned TMEM198
homologues from Xenopus laevis
(GeneID 447551), human (GeneID 130612), and mouse (GeneIDs 319998 and 73827) and confirmed that all retained similar activity levels in promoting LRP6 signaling (data not shown). Topology prediction using the SMART program (EMBL) suggested that TMEM198 consists of a very short extracellular domain (31 amino acids for X. tropicalis
TMEM198), seven transmembrane domains, and a cytoplasmic tail with ~110 amino acids (G). To experimentally confirm this prediction, we transplanted the signal peptide from the Kremen protein (28
) to the N terminus of TMEM198 and found that this fusion protein was as active as the wild-type in promoting LRP6 signaling (data not shown). Moreover, TMEM198 became inactive when green fluorescent protein (GFP) was fused at the N terminus while retaining full activity when GFP was fused at the C terminus (data not shown). Immunofluorescent analysis of transfected HeLa cells indicated that a large amount of TMEM198 localized intracellularly in vesicle-like structures (H), suggesting that TMEM198 may not be a typical plasma membrane protein. However, the plasma membrane-localized TMEM198 was readily detected by the cell surface biotinylation assay, confirming that at least part of the overexpressed TMEM198 proteins were at the plasma membrane (I).
In parallel, TMEM198 was identified in a genome-wide siRNA screen for genes required for Wnt/β-catenin signaling in HEK293T cells (A) (14
). To further address the involvement of TMEM198 in canonical Wnt signaling, we designed two specific siRNAs to knock down TMEM198 in HEK293T cells (B). As shown in C, Wnt3a signaling was significantly reduced when TMEM198 siRNAs were applied, while β-catenin signaling was largely unaffected. Signaling induced by Dvl2 was also markedly downregulated by TMEM198 siRNA (C), consistent with the model of Dvl involvement in promoting LRP6 activation and signaling via LRP6 (5
). The siRNA effect was specific because the activity was rescued by cotransfected X. tropicalis TMEM198
(XtTMEM198) (D). Further confirming specificity, TMEM198 siRNA affected Wnt3a-induced TOP-FLASH expression but had no effect on the expression of the CAGA-luciferase reporter induced by constitutively active TGFβ type I receptor (CA-TGFβRI) (E). Epistatically, TMEM198 was required for β-catenin accumulation induced by Wnt3a but not LiCl, a potent GSK3 inhibitor (F), suggesting that TMEM198 functions upstream of β-catenin accumulation. Moreover, the TMEM198/LRP6 signal was completely blocked with cotransfection of Axin, the scaffold protein of the β-catenin degradation complex (data not shown). Taken together, these results indicate that TMEM198 is able to activate LRP6 and is required for canonical Wnt signaling upstream of the β-catenin destruction complex.
Fig. 2. TMEM198 is required for canonical Wnt signaling upstream of the β-catenin destruction complex. (A) Wnt-responsive reporter assay in HEK293T cells. LOC130612 (human TMEM198) was identified in a genome-wide siRNA screen for genes required for Wnt/β-catenin (more ...) TMEM198 associates with LRP6 and promotes phosphorylation.
Next, we addressed whether TMEM198 was able to interact with LRP6. In coimmunoprecipitation (co-IP) assays with transfected HEK293T cells, TMEM198 associated with LRP6 (A). Since an antibody recognizing endogenous TMEM198 protein was lacking, we transfected FLAG-TMEM198 into HEK293T cells and detected coimmunoprecipitated endogenous LRP6 (B). The interaction is likely mediated by the transmembrane domains because the TMEM198 protein lacking the cytoplasmic portion (TMEM198-ΔC) was also coimmunoprecipitated with LRP6 (data not shown) and LRP6ΔE1-4, an extracellular truncated form of LRP6 (C). The interaction between overexpressed TMEM198 and LRP5 was also detected (data not shown) although the latter was not activated.
Fig. 3. TMEM198 associates with LRP6 and promotes phosphorylation. (A) Western blots of immunoprecipitates (IP) or initial lysates from HEK293T cells transfected as indicated. TMEM198-M2-FLAG is a mutant form of TMEM198. Note that the precipitated LRP6 from a (more ...)
Activation of LRP6 in canonical Wnt signaling requires phosphorylation; therefore, the status of LRP6 phosphorylation at three key residues was monitored using phospho-specific antibodies. As expected, LRP6 phosphorylation at Thr-1479, Ser-1490, and Thr-1493 was dramatically increased when TMEM198 was coexpressed (D and E). As a positive control, CK1γ induced massive phosphorylation at Thr-1479, whereas GSK3β did not induce phosphorylation (D). A mutant form of TMEM198 (TMEM198-M2) was also tested in this assay and showed no effect (E). Consistently, this mutant had greatly reduced activity in promoting LRP6 signaling (F). TMEM198-M2 was expressed at an equal level (A and E) and distributed in a similar pattern in HeLa cells as the wild type (data not shown) and retained binding to LRP6 (A). As another control of the seven-transmembrane protein, Frizzled 7 was cotransfected with LRP6, and no enhancement of LRP6 phosphorylation was observed (data not shown), suggesting that TMEM198 specifically promoted LRP6 phosphorylation. Further demonstrating the specificity and consistent with the results of the activity assay (D) were our findings that TMEM198 did not enhance Thr-1493 phosphorylation of LRP5 while overexpression of GSK3/Axin did enhance phosphorylation (G). Together, these results indicate that TMEM198 specifically activates LRP6 by promoting phosphorylation.
TMEM198 facilitates casein kinase 1 family members in phosphorylating LRP6.
Because there is no predicted kinase domain within TMEM198, we speculate that TMEM198 cannot phosphorylate LRP6 directly. As shown in the in vitro
kinase assay, unlike CK1ε, TMEM198 was not able to phosphorylate purified LRP6-C, the cytoplasmic domain of LRP6 (A). This failure to phosphorylate the cytoplasmic domain confirmed that TMEM198 per se
is not a kinase; however, it may assist kinases. As casein kinase 1 (CK1) family members and GSK3 have been implicated in LRP6 phosphorylation (17
), we verified the potential interaction between TMEM198 and casein kinase 1 or GSK3β. As shown in B, all four CK1s (CK1α/ε/δ/γ) were coimmunoprecipitated with TMEM198, whereas GSK3β was not coimmunoprecipitated (data not shown). In the Wnt-responsive reporter assay, LRP6/TMEM198 signaling was inhibited by dominant negative CK1γ ([DN-CK1γ] which specifically blocks CK1γ activity [17
]) and dominant negative CK1δ ([DN-CK1δ] which blocks the activity of both CK1ε and CK1δ [58
]) (C). The inhibition was specific because dominant negative CK1α ([DN-CK1α] which blocks CK1α activity [58
]) had no effect (C). Moreover, DN-CK1γ and DN-CK1δ, but not DN-CK1α, significantly blocked TMEM198-induced LRP6 phosphorylation (D). These results indicate that casein kinase 1 family members are required for TMEM198-induced LRP6 phosphorylation and activation.
Fig. 4. Casein kinase 1 is involved in TMEM198 stimulated LRP6 phosphorylation. (A) Western blots and Coomassie blue staining of SDS-PAGE samples after the in vitro kinase assay. Coomassie blue staining indicated MBP-LRP6-C protein. Tp-1493 staining indicated (more ...)
CK1 overexpression is able to induce LRP6 phosphorylation. Therefore, we addressed whether endogenous TMEM198 was involved in this process. As shown in E, knockdown of TMEM198 using siRNA significantly reduced Wnt signaling activated by LRP6/CK1γ or LRP6/CK1ε. Consistently, LRP6 phosphorylation induced by either CK1γ or CK1ε was also reduced with TMEM198 depletion (F and G). Taken together, these results suggest that TMEM198 and casein kinases are mutually dependent for LRP6 phosphorylation and activation.
TMEM198 is a multitransmembrane protein, and the C terminus likely extends toward the cytosol while the casein kinases are either membrane associated (CK1γ, via lipid modification) or cytosolic. The topology suggested that TMEM198 might interact with CK1s via its C-terminal domain (C domain). Indeed, TMEM198 with a deletion of the C terminus (TMEM198ΔC) exhibited greatly reduced interaction with all CK1s (A and data not shown) as well as significantly decreased Wnt promoting activity (B). This result suggested that the TMEM198 C-terminal domain was crucial for CK1 binding. Similarly, TMEM198-M2, a mutant with greatly reduced activities in Wnt signaling and in promoting LRP6 phosphorylation (E and F), bound undetectable CK1ε or CK1γ in the co-IP assay (C). Furthermore, although either TMEM198-M2 or TMEM198ΔC retained some activity, TMEM198-M2ΔC was completely inactive (B), suggesting that the CK1 binding capacity is indispensable for promoting LRP6 signaling. Direct interactions between the C domains of TMEM198 (GST-TMEM198-C purified from E. coli) and CK1ε that was either in vitro translated (D) or overexpressed in HEK293T cells (data not shown) were detected in GST pulldown experiments. These results suggest that the TMEM198 cytoplasmic domain interacts with CK1 directly.
Fig. 5. TMEM198 recruits casein kinase via the cytoplasmic domain. (A) Western blots of immunoprecipitates (IP) or initial lysates from HEK293T cells transfected as indicated. The asterisk indicates a nonspecific band. (B) Wnt-responsive reporter assay in HEK293T (more ...)
The above results suggested that TMEM198 likely activated LRP6 via recruitment of casein kinase 1 family members. Therefore, we investigated whether TMEM198 was able to assist CK1 with phosphorylating LRP6. When cotransfected, minimal doses of TMEM198 and CK1ε indeed synergistically promoted LRP6 phosphorylation (E). To demonstrate a cofactor feature of TMEM198 for CK-mediated LRP6 phosphorylation, we fused the LRP6 cytoplasmic domain (LRP6-C) with the TMEM198 C domain (TMEM198-C) and tested whether this fusion protein was an improved substrate for casein kinases in transfected cells. As shown in F, the fusion protein (LRP6-C-TMEM198-C) was, indeed, more strongly phosphorylated than LRP6-C. This result strongly suggests that TMEM198 is able to facilitate CK1-mediated LRP6 phosphorylation. Taken together, these results suggest that TMEM198 promotes LRP6 phosphorylation and activation partially via recruiting and facilitating CK1.
TMEM198 promotes LRP6 aggregation.
TMEM198 promoted LRP6 phosphorylation at all three amino acid sites tested, Thr-1479, Ser-1490, and Thr-1493. Among them, Thr-1479 and Thr-1493 are typical casein kinase targets while Ser-1490 could be phosphorylated by GSK3, GRK5, or Pftk1 (9
). Our results indicated that TMEM198 does not interact with GSK3β or Pftk1 (data not shown), suggesting that the effect on Ser-1490 by TMEM198 is secondary. One explanation could be that the phosphorylation on Thr-1479 enhanced that on Ser-1490, and this possibility is supported by a previous report (56
). Another possibility is that TMEM198 affects LRP6 phosphorylation globally and provides a microenvironment that facilitates phosphorylation.
Dishevelled (Dvl)-mediated LRP6 clustering or aggregation has been proposed as a key step in LRP6 phosphorylation and activation, and upon Wnt ligand stimulation the LRP6 signalosome is induced (5
). We therefore addressed whether TMEM198 was able to affect the subcellular distribution of LRP6. As shown in A, LRP6 was detected at the cell membrane and colocalized with cotransfected Frizzled 5 (90% of cotransfected cells). In contrast, when TMEM198 was cotransfected, LRP6 formed cytosolic punctate structures together with TMEM198 (95% of cotransfected cells), suggesting the formation of signalosome-like structures (A). To rule out unspecific aggregation with overexpressed transmembrane proteins, we coexpressed TMEM198 together with the TGFβ type I receptor (TGFβRI) and observed no colocalization although TMEM198 itself was punctate (100% of cotransfected cells) (A). The LRP6 signalosome has been shown to consist of caveolin-containing acidic vesicles with an unclear identity (5
) but which are thought to correspond to multivesicular bodies (MVBs) (46
). We therefore performed immunofluorescence analysis and observed that TMEM198/LRP6 structures were partially colocalized with caveolin but not with clathrin or EEA (an early endosome marker) (data not shown). To further confirm that TMEM198 was able to promote LRP6 aggregation, we performed sucrose sedimentation experiments using LRP6- or LRP6/TMEM198-transfected HEK293T cells. As shown in B, significantly more total LRP6 as well as phosphorylated LRP6 was detected in high-molecular-weight (HMW) fractions when TMEM198 was coexpressed. Importantly, when we separately harvested the low-molecular-weight (LMW) and HMW fractions and performed immunoprecipitation against FLAG-TMEM198, much more LRP6 was coprecipitated from the HMW fractions although less LRP6 was contained in the HMW fractions before precipitation (C). Similarly, in the HMW precipitates, phospho-LRP6 was enriched (C). These results suggest that TMEM198 is likely capable of promoting LRP6 aggregation, thus facilitating LRP6 phosphorylation.
Fig. 6. TMEM198 promotes LRP6 aggregation and is required for the spontaneous aggregation and activation of LRP6ΔE1-4. (A) Confocal microscopy images of HeLa cells transfected as indicated. LRP6-GFP (green) was cotransfected with Mesd and Fz5 (red) or (more ...)
It has been proposed that LRP6ΔE1-4, the constitutively active form, signals independently of Dvl because of spontaneous self-aggregation (5
). However, how this ligand- and Dvl-independent self-aggregation occurs was unknown. We found that TMEM198 was able to associate with LRP6ΔE1-4 (C) to further enhance its activity (E) and was required for its activation in the Wnt-responsive reporter assay (D). Moreover, in sucrose sedimentation experiments, a significant portion of LRP6ΔE1-4 protein was detected in the HMW fractions (E), as previously reported (5
). However, in TMEM198 knockdown cells (F) this HMW distribution was slightly but consistently reduced (E). These results suggest that TMEM198 is probably involved in self-aggregation and spontaneous activation of LRP6ΔE1-4.
Previous studies revealed that Frizzled and Dishevelled proteins are required for Wnt-dependent LRP6 phosphorylation (5
). We next investigated whether they were involved in TMEM198-induced LRP6 phosphorylation and activation. In HEK293T cells, Fz2/Fz4/Fz5 (Fz2/4/5) and Dvl2/Dvl3 (Dvl2/3) are expressed at detectable levels, and previous reports have confirmed that siRNA-mediated knockdown of these genes efficiently blocks Wnt signaling or LRP6 receptor activation (38
). We therefore used these siRNA combinations (A and C) and found that knockdown of either Fz or Dvl proteins did not significantly affect TMEM198-induced LRP6 phosphorylation (B and D). Moreover, TMEM198/LRP6-induced β-catenin accumulation was not affected by Dvl siRNAs (E). These results suggest that TMEM198 promotes LRP6 phosphorylation probably independent or downstream of Dvl-mediated receptor aggregation. However, we noted that when Fz genes were knocked down with siRNAs, TMEM198/LRP6-induced β-catenin accumulation was slightly reduced (F). An explanation for this observation is that besides leading to LRP6 phosphorylation, Fz may have another activity required for β-catenin stabilization.
Fig. 7. TMEM198 promotes LRP6 phosphorylation independent of Fz and Dvl. (A to D) RT-PCR result shows that in specific-siRNA-expressing HEK293T cells, which were used in the experiments shown in panels B and F, Fz2, Fz4 and Fz5 mRNA levels were significantly (more ...) TMEM198 activates LRP6 in Xenopus embryos and is involved in neural patterning.
Canonical Wnt signaling plays a vital role during early embryonic development (26
). To address TMEM198 function in Xenopus
embryos, we analyzed its expression pattern by RT-PCR and in situ
hybridization. By RT-PCR, tmem198
mRNA was detected constantly from the two-cell stage to tadpole embryos (data not shown). By in situ
mRNA was detected in cleavage embryos with enrichment in the animal blastomeres, indicative of maternal distribution (A and data not shown). At stage 10+, tmem198
mRNA was detected mainly in the organizer region, just above the dorsal blastopore lip (B). Its expression extended toward the ventral side along the blastopore formation (C). During gastrulation and neuralization, the expression domain of tmem198
extended anteriorly and covered the entire neural plate as well as the underlying mesodermal cells (D and E and data not shown). At stage 14, a gradual enhanced expression was detected toward the posterior pore (D). At the late neural stage, tmem198
was expressed in the neural tube, brain subdomains, branch arches, and quite strongly in the eye regions (F).
Fig. 8. TMEM198 activates LRP6 in Xenopus embryos. (A to F) Whole-mount in situ hybridization of tmem198 mRNA in developing X. laevis embryos. (A) Stage 6.5, animal view. (B) Stage 10+, dorsal-vegetal view. (C) Stage 11.5, vegetal view with dorsal up. (D) Stage (more ...)
embryos, TMEM198 cooperated with LRP6 in activating Wnt-responsive reporter gene expression (data not shown) and inducing the expression of siamois
) and Xnr3
, two direct Wnt target genes, in animal caps (G). In addition, tmem198
mRNA injection into animal blastomeres inhibited head formation (data not shown), a hallmark of overactivated Wnt signaling (11
), and a suboptimal dose of TMEM198 cooperated with LRP6 in the same assay (n
= 37 embryos; 92%) (H). These results suggest that TMEM198 is able to activate LRP6-mediated Wnt signaling in Xenopus
Two independent morpholino antisense oligonucleotides (MO) were both effective in blocking mRNA translation of Xenopus laevis
TMEM198 (A) and were used in combination to assess whether TMEM198 is necessary for the normal development of Xenopus
embryos. Embryos injected with TMEM198-MOs developed largely unaffected at the gastrula and early neurula stages. However, at the tadpole stage, morphants were often less pigmented (n
= 94; 67%) with ventrally bent tails (n
= 94; 47%) (B). Anteriorly, these embryos developed smaller eyes (often only the dorsal half of the retina was retained) and forebrain structures (n
= 94; 62%) than the control-MO-injected embryos (B). The overall phenotype resembled that of the LRP6 morphants (22
) as well as embryos that overexpressed Dkk1, a Wnt antagonist (21
). TMEM198 morphants were specific because in X. tropicalis tmem198
mRNA-coinjected embryos, the defects in pigmentation, tail, and anterior structures were reduced to 11%, 9%, and 13%, respectively (B lower panel) (n
= 45). Loss of pigmentation is an indication of defective neural crest formation, a process regulated by Wnt/β-catenin signaling (42
). We therefore investigated the role of tmem198
in neural crest development. Control- or TMEM198-MO, together with LacZ
mRNA as a lineage tracer, was injected into one of the two blastomeres in two-cell-stage embryos, and at the early neurula stage, the expression of neural crest markers was examined using in situ
hybridization. In the TMEM198-MO-injected side, expression levels of Sox10
= 46; 96%), Sox9
= 28; 50%), FoxD3a
= 25; 60%), Slug
= 20; 40%), and Twist
= 26; 63%) were markedly reduced (C), confirming that TMEM198 is required for neural crest formation. In contrast, the pan-neural marker Sox3
was not affected (data not shown), suggesting that the deficient formation of neural crests was not due to the defective neural induction. The downregulation of neural crest marker expression by TMEM198-MO was specific because the expression of Sox10
was rescued in 63% of embryos coinjected with X. tropicalis tmem198
mRNA, which is refractory to MO targeting X. laevis tmem198
= 28) (D).
Fig. 9. TMEM198 functions in neural crest development. (A) Western blot of total cell lysate from Xenopus embryos injected with indicated RNA or morpholino oligonucleotides (MO). Four nanograms of X. laevis TMEM198-EGFP mRNA (XlTMEM198-EGFP) and 40 ng of control (more ...)
Wnt/β-catenin signaling is involved in antero-posterior (A-P) body axis determination of both vertebrates and invertebrates (18
). To uncover a role of TMEM198 in Wnt-mediated A-P patterning, we used Xenopus
animal caps that were neuralized with injected Noggin and further posteriorized with coinjected Xenopus
Wnt8. In this experiment, neuralized animal cap cells were transformed with Wnt/β-catenin signaling into posterior type neural tissues as indicated by the expression of engrailed 2
), a midbrain-hindbrain-boundary (MHB) marker, and HoxB9
, a spinal cord marker (A). The neural crest marker gene Slug
was also induced, which confirmed Wnt signaling involvement during neural crest induction (A). As expected, depletion of Wnt coreceptor LRP6 using a specific MO abolished the induction of all three genes by Wnt. TMEM198-MO also blocked the activity of Wnt in inducing the expression of En2
, and Slug
, indicating that TMEM198 is required downstream of Wnt ligand in this experimental setting (A). Coinjection of X. tropicalis tmem198
mRNA rescued En2
expression, suggesting that the effect of TMEM198-MO was specific (B). To further confirm that TMEM198 acted through the β-catenin-dependent Wnt signaling, we monitored the expression of a Wnt-responsive reporter gene in these animal caps and found that the reporter was consistently downregulated by TMEM198-MO (C). Moreover, the blocking effect of TMEM198-MO on Wnt-induced En2
expression was reversed by coinjection of β-catenin S37A, a constitutively active form (D). These results indicate that TMEM198 is required for Wnt/β-catenin-mediated posteriorization of neural tissues.
Fig. 10. TMEM198 is required for Wnt-mediated antero-posterior patterning in Xenopus embryos. (A and B) Four-cell-stage embryos were injected animally in each blastomere with 100 pg of noggin and 50 pg of Wnt8 mRNA or 10 ng of control-MO (Ctrl-MO), 5 ng of LRP6-MO, (more ...)
Using in situ
hybridization in whole embryos, we next verified the expression of Otx2
, and HoxB9
, which are often used to demarcate different compartments of the central nervous system along the A-P axis. Surprisingly, none except En2
was consistently downregulated by TMEM198-MO injection, suggesting a rather specific role of TMEM198 in the A-P patterning of the central nervous system (E and data not shown). En2
has been demonstrated as a direct target of Wnt/β-catenin signaling (30
), and its expression in the MHB was induced/maintained directly by Wnt1 (31
) and further upstream by fibroblast growth factor 8 (FGF8) (24
). We observed that the expression of neither wnt1
was altered in TMEM198-MO-injected embryos (E), suggesting that TMEM198 likely functions downstream of Wnt1. We also verified the expression of Otx2
, two transcription factors that antagonistically regulate formation of the MHB and wnt1
expression. Our results revealed that the expression levels of these two genes were also largely unaffected (data not shown), indicating that the specification of the MHB was not defective and suggesting again that TMEM198 was required more downstream for Wnt1 signaling. In line with this conclusion, we were able to demonstrate that in Noggin-neutralized animal caps, TMEM198 and LRP6 together could mimic the activity of Wnt8 to activate the expression of En2
(F). These results suggest that TMEM198 is required for Wnt-mediated induction of En2
expression in Xenopus