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State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China
2Correspondence: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. E-mail: A.M., mengam{at}mail.tsinghua.edu.cnY.-G.C., ygchen{at}tsinghua.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: receptor degradation • embryonic expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. These factors are essential molecules for germ layer formation and patterning during early embryogenesis (5
, 6)
. TGF-ß elicits its signaling by binding to its type II receptor TßRII and type I receptor ALK5 (TßRI) on the cell surface. ALK5, which is activated by TßRII via phosphorylation, phosphorylates Smad2/Smad3, and promotes their complex formation with Smad4 in the cytoplasm. Then the Smad complex accumulates in the nucleus and regulates the expression of their target genes. Although this canonical signaling pathway is relatively simple, they are tightly controlled both extracellularly and intracellularly (7
, 8)
. Many receptor-binding proteins have been suggested to modulate receptor complex formation, receptor-Smad interaction, or receptor stability; SARA (9)
, Smad7 (10
, 11)
, FKBP12 (12)
and BAMBI (13)
are a few examples.
Dapper (Dpr) was first identified as a Dishevelled (Dsh)-associated antagonist of Wnt signaling in Xenopus, and it can inhibit both the canonical Wnt/ß-catenin pathway and the noncanonical Wnt/c-Jun N-terminal kinase [c-Jun NH2-terminal kinase (JNK)] pathway (14)
. Knockdown of maternal Dpr expression by antisense oligonucleotides results in loss of the notochord and head structures in Xenopus embryos, suggesting that Dpr is required for normal vertebrate development. Another Dsh-binding protein, Frodo, which shares 90% identity with Dpr at the amino acid level, was reported as an essential positive regulator of Wnt signaling in Xenopus embryogenesis (15)
. Furthermore, Dpr1, the Xenopus Dpr ortholog, and its related Dpr2 were reported or predicted in zebrafish, mouse, rat, and human (16
17
18
19)
. Later analyses suggest that Dpr family members act as either activators or inhibitors of the Wnt signaling. Zebrafish Dpr1 and Dpr2 were reported to participate in distinct Wnt-dependent developmental process –Dpr1 was shown to enhance Wnt/ß-catenin activity in zebrafish embryos, while Dpr2 is required for normal convergent extension movements that are hypomorphic for Stbm or Wnt11, a noncanonical pathway (18)
. Although zebrafish Dpr1 and Dpr2 can synergize with Dvl2 to induce TOPFlash reporter expression in HEK293T cells and to induce Wnt/ß-catenin target genes in Xenopus animal caps, both Dpr orthologs inhibit the Wnt-mediated activation of the luciferase reporter (18)
. Our recent results demonstrate that mammalian Dpr1 also antagonizes Wnt/Dsh signaling and it functions to promote Dvl2 degradation (20)
. In synergy with Dsh, Frodo induces the secondary axis formation, and inhibition of Frodo expression interferes with XWnt8- and XDsh-induced axial development. It was also showed that Frodo associates with the transcription repressor TCF3 and synergizes with Dpr in inducing head formation (21)
. Therefore, Frodo was believed to transduce Wnt signaling in ß-catenin-dependent and -independent manners. Furthermore, zebrafish Dpr2 is reported to inhibit TGF-ß/Nodal signaling by promoting lysosomal degradation of their type I receptors and thereby modulates Nodal signaling during mesoderm induction (22
, 23)
. Thus, Dpr2 may be able to regulate TGF-ß/Nodal signals as well as noncanonical Wnt signals through different mechanisms.
Albeit sharing a low overall homology, Dpr proteins are conserved significantly in four discrete domains: an NH2-terminal leucine-zipper domain, two serine-rich domains (one right after the leucine-zipper domain and the other in the C-terminal region), and a domain containing a PDZ-binding motif in the very C-terminus (14
, 16
, 18)
. The PDZ-binding motif was reported to be involved in Dishevelled binding whereas the function of the other two domains still needs to be defined. A recent report demonstrated that the middle region of Xenopus Frodo can interact with XDbf4, a cell cycle and DNA replication related protein, to down-regulate Wnt signal (24)
.
As Dpr proteins have been shown to be implicated in regulating early embryogenesis via different mechanisms in zebrafish and Xenopus, it is important to understand the function of mammalian Dpr proteins. To elucidate the function of Dpr2 in mammals, we cloned mouse Dpr2 (mDpr2). Here, we provide evidence that unlike Dpr1 that modulates Wnt signaling, mDpr2 negatively regulates TGF-ß signaling and promotes TGF-ß receptor degradation in lysosomes. Overexpression of mDpr2 in the zebrafish embryos inhibits mesoderm tissue development. These data indicate that the conserved function of Dpr2 in fish and mouse. We also find that mDpr2 is expressed ubiquitously at early stages and specifically in the somites, the neural tube, ear and gut during development of mouse embryos, suggesting roles in development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture, transfection, luciferase assay, immunoprecipitation, immunoblotting and immunofluorescence were performed as described previously (20
, 22)
.
RNA interference
pSUPER.retro (OligoEngine) was used for the expression of siRNA. The target sequence of mDpr2 is 5'-GGACAGCCTCAAGCAACAT-3', corresponding to nt 1143–1161 of mDpr2 (Dpr2-siRNA1). This sequence is conserved in the human Dpr2 gene. Another siRNA construct Dpr2-siRNA2, targeting the sequence of mDpr2 5'-GATCCGAAGGTTTCAGCCA-3' (nt 1301
1320 of mDpr2) also has effect on the expression of both human and mouse Dpr2. A non-specific siRNA expression vector, (NS-RNAi) (5'-AGCGGACTAAGTCCATTGC-3'), was constructed as a negative control. These sequences were all analyzed by a basic local alignment search tool (BLAST) search of the GenBank database to avoid similar sequences found in the human genome. Oligonucleotides were synthesized (Bioasin, Shanghai, China) and inserted into the pSR vector in the BglII and HindIII sites.
Whole-mount in situ hybridization (ISH) of mouse embryos
The plasmid pBluscriptIIKS-mDpr2 was linearized with XhoI as the template. Digoxigenin-uridine triphosphate (UTP) labeled antisense RNA probe was generated by in vitro transcription. The whole-mount RNA ISH was performed essentially using the protocol described previously (25)
.
Microinjection of zebrafish embryos
Plasmid pXT7-mDpr2 was linearized with BamHI and used as template to in vitro synthesize mDpr2 mRNA using T7 Cap-Scribe kit (Roche). The same plasmid was linearized with HindIII, which cut at the other end of mDpr2 cDNA, and used as template to synthesize mDpr2 antisense RNA (cDpr2 RNA) using Sp6 Cap-Scribe kit (Roche). cDpr2 RNA has a sequence complementary to mDpr2 mRNA and was used as an injection control. Zebrafish embryos at the one-cell stage were microinjected with 100pg of mDpr2 mRNA, or 100 pg of GFP mRNA or 120 pg of cDpr2 RNA as controls. The amount of sqt mRNA was 0.5 pg per embryo.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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18
19)
, and zebrafish Dpr2 was shown to modulate noncanonical Wnt signaling (18)
or TGF-ß/Nodal signaling (22)
. To investigate whether mDpr2 functions as an antagonist of TGF-ß signaling, the transcriptional activity of TGF-ß was examined with two Smad-responsive reporters: ARE-luciferase (26)
and CAGA-luciferase (27)
. As shown in Fig. 1
A, TGF-ß1 stimulated the expression of ARE-luciferase in human hepatoma Hep3B cells, and exogenous expression of mDpr2 attenuated TGF-ß activity. Similarly, mDpr2 also interfered with the expression of ARE-luciferase induced by constitutively active TGF-ß type I receptor ALK5 or activin/Nodal type I receptor ALK4 (Fig. 1B
). Furthermore, mDpr2 inhibits active ALK5-induced CAGA-luciferase expression in a dose-dependent manner (Fig. 1C
). Similar inhibitory effect of mDpr2 on TGF-ß-induced expression of CAGA-luciferase was also observed in HepG2 (data not shown).
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To investigate the effect of endogenous Dpr2 on TGF-ß signaling, RNA interference was performed to knock down endogenous Dpr2 expression in Hep3B cells, which express Dpr2 as detected by RT-polymerase chain reaction (RT-PCR) (data not shown). Dpr2-siRNA1, which targets a DNA sequence conserved in both mouse and human Dpr2 (see Materials and Methods) and effectively decreased the Dpr2 mRNA level in Hep3B cells and exogenously expressed mDpr2 expression (data not shown), enhanced TGF-ß-induced expression of CAGA-luciferase, while a non-specific siRNA (NS-siRNA) had no effect (Fig. 1D
). Dpr2-siRNA2, which targeted on mDpr2 on another region and deceased mDpr2 expression, also increased TGF-ß-induced expression of CAGA-luciferase (Fig. 1E,F
). Furthermore, both Dpr2-siRNA1 and Dpr2-siRNA2 could partially reverse the inhibitory effect on TGF-ß activity by Dpr2 overexpression, and it alone enhanced the reporter expression, possibly by eliminating Dpr2 inhibition on the basal TGF-ß activity. To demonstrate the specificity of Dpr2-siRNA1, we generated mDpr2(Mut) construct that encoded the wild-type (WT) mDpr2 protein but was resistant to Dpr2-siRNA1 (Fig. 1E
). This mutant behaved as the WT mDpr2 in inhibiting TGF-ß-induced reporter expression, and Dpr2-siRNA2 was able to rescue the inhibition by mDpr2(Mut) while Dpr2-siRNA1 had no effect (Fig. 1E
).
Mouse Dpr2 in zebrafish embryos mimics zebrafish Dpr2 activity
Our previous observation indicates that zebrafish Dpr2 regulates mesoderm formation by influencing Nodal signaling in fish embryos (22)
. To test whether the function of mouse Dpr2 is evolutionally conserved, we investigated the activity of mDpr2 in the zebrafish embryos. Injection of one-cell zebrafish embryos with 100 pg mDpr2 mRNA resulted in partial or complete fusion of eyes in 13.7% or 12.7% of embryos (n=284, pooled from 4 experiments), respectively, of 24 h post-fertilization (hpf) (Fig. 2
A). In contrast, none of embryos injected with 120 pg of Dpr2 antisense RNA (cDpr2) (n=70), which has a sequence complementary to mDpr2 mRNA, showed fusion of eyes. The phenotype of fused eyes resembles that caused by loss-of-function mutation of one-eye pinhead (oep), which encodes a coreceptor required for Nodal signaling (28
, 29)
. Consistently, overexpression of mDpr2 reduced the expression of no tail (ntl), a mesoderm marker, in the presumptive dorsal blastodermal margin at the shield stage (Fig. 2B
), mimicking ntl reduction in sqt;cyc double mutants (30)
or in embryos injected with the Nodal antagonist antivin/lefty1 mRNA (31)
. mDpr2 injection also led to decrease of the expression of goosecoid (gsc), a shield-specific marker that requires Nodal signals for expression (29
, 30)
. Furthermore, expansion of gsc expression was induced by overexpression of sqt (a zebrafish Nodal-related gene), and this effect was inhibited to a certain degree by coinjection of mDpr2 mRNA (Fig. 2C
) while it was not affected by coinjection of GFP mRNA (data not shown). Taken together, these data suggest that mDpr2 also functions as an antagonist of Nodal signaling.
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Mouse Dpr2 inhibits TGF-ß signaling through TGF-ß receptors
Our previous study suggested that zebrafish Dpr2 inhibits TGF-ß/Nodal signaling by associating with and targeting their type I receptors for degradation. To investigate whether mouse Dpr2 also interacts with the TGF-ß type I receptor ALK5, we transfected HEK293T cells with Myc-tagged mDpr2 and hemagglutinin (HA)-tagged WT or active form of ALK5. ALK5-mDpr2 complexes were detected by anti-HA immunoprecipitation and anti-Myc immunoblotting. As shown in Fig. 3
A, mDpr2 bound to both WT and active ALK5 and exhibited a higher affinity to active ALK5. When coexpressed with mDpr2, a low protein level of ALK5 was noticed (Fig. 3A
, lower panel), implying that mDpr2 might regulate receptor protein stability. To test this possibility, HEK293T cells were cotransfected with mDpr2 and active ALK5 and then treated with different protein degradation inhibitors. Lysosomal inhibitors such as bafilomycine A, NH4Cl and chloroquine blocked mDpr2-induced degradation of ALK5, whereas the proteosome inhibitor MG132 had no effect (Fig. 3B
). Consistent with previous report (32)
, MG132 was effective in preventing Smad7 degradation via the ubiquitination-proteosome pathway mediated by the ubiquitin E3 ligase Smurf1 (Fig. 3C
). These results suggest that mDpr2 interferes with TGF-ß by directly binding to and targeting the receptors for lysosomal inhibitor-sensitive degradation.
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Identification of functional domains of mDpr2
To map the functional domains of mDpr2 important for its inhibitory effect on TGF-ß signaling, mDpr2 deletion mutants (Fig. 4
A) were transfected into HepG2 cells together with CAGA-luciferase. As shown in Fig. 4B
, the N-terminal region containing the first 281 amino acids of mDpr2 was sufficient to inhibit active ALK5-mediated expression of this reporter, while the C-terminal region mutants had no effect (Fig. 4C
). This result is consistent with the results obtained with zebrafish Dpr2 whose functional domain was also mapped to its N terminus (22)
.
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Distinct function of mDpr1 and mDpr2 in regulating Wnt and TGF-ß signaling
Previous studies suggested distinct functions of Dpr1 and Dpr2: Dpr1 of zebrafish and Xenopus negatively regulates canonical and noncanonical Wnt signaling (14
, 20)
, while zebrafish Dpr2 interferes with noncanonical Wnt signaling (18)
or TGF-ß/Nodal signaling (22)
. To investigate whether the distinct functions between Dpr1 and Dpr2 are conserved in mammalian orthologs, we examined the effect of mDpr2 in the canonical and noncanonical Wnt signaling pathways. A ß-catenin-responsive reporter, LEF-luciferase was cotransfected into HEK293T cells together with Wnt1 in the presence or absence of human Dpr1 (hDpr1), mouse Dpr1 (mDpr1), or mDpr2, and the reporter expression was analyzed by measuring luciferase activity. Whereas both hDpr1 and mDpr1 had no influence on ALK5-stimulated expression of CAGA-luciferase (Fig. 5
A), it remarkably inhibited the Wnt1-induced expression of LEF-luciferase (Fig. 5B
). Similar results were obtained with the Wnt/ß-catenin-responsive reporter TopFlash in HeLa cells, and Dpr expression had no effect on the Wnt-nonresponsive reporter FopFlash (Fig. 5C
). Furthermore, mDpr2 had little effect on Wnt/ß-catenin signaling.
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Zebrafish Dpr2 was suggested to be required for noncanonical Wnt signaling in regulating normal convergence extension movements in embryos (18)
, and Xenopus Dpr has been suggest to inhibit the JNK-mediated noncanonical Wnt signaling pathways (14)
. We then examined whether mDpr2 has any effect on JNK activity. As JNK reporters could be used as reporters for noncanonical Wnt signaling pathways in cell culture, Stratagene’s (La Jolla, CA, USA) PathDetect c-Jun trans-Reporting System, which is activated by MEKK and JNK. Human Dpr1 and mDpr1, but not mDpr2, interfered with the reporter expression in a dose-dependent manner in HeLa (Fig. 5D
) and in HEK293 cells (and data not shown). When they were expressed at high levels (100 ng DNA), mDpr2 also exhibited a limited inhibitory effect on the reporter expression (Fig. 5D
). These results implicate that the JNK-mediated noncanonical Wnt signaling is more sensitive to the inhibitory effect of Dpr1, although Dpr2 may function as a negative regulator of noncanonical Wnt signaling pathways when it is highly expressed.
Expression pattern of Dpr2 in mouse embryos
To help understand in vivo functions of mDpr2, we examined its dynamic expression during embryogenesis. mDpr2 transcripts were detected in embryos at 3 d post-coitum (dpc) by RT-PCR and Northern blotting (data not shown). Whole-mount ISH revealed that mDpr2 was expressed throughout embryonic tissues around the onset of gastrulation (Fig. 6
A, C, E). A cross section of a 7.5-dpc embryo indicated that mDpr2 transcripts were present in embryonic endoderm, mesoderm, and ectoderm. Specific expression of mDpr2 was detected at the onset of segmentation. At 8.0 dpc, mDpr2 transcripts are distributed in domains lateral to the node, which represent the presomitic mesoderm, but not within the node (Fig. 6F, G
). At 8.5 dpc, the expression occurs at a high level in the newly formed somites (Fig. 6H, I
) and in the neural plate and the gut tube (Fig. 6J
). At 10.0 dpc and 10.5 dpc, mDpr2 is expressed in the somites with a higher level posteriorly as well as in the roof plate of the neural tube, otic vesicle, and gut (Fig. 6L
–O). The expression pattern during segmentation is correlated with that during gastrulation. The expression pattern of mDpr2 implies that it might be involved in early development of mouse embryos.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In this study, we identified mouse Dpr2 and found that it also negatively regulates TGF-ß signaling in cultured cells. We further showed that, like zebrafish Dpr2, mDpr2 associated with TGF-ß type I receptor ALK5 and induced ALK5 degradation in a lysosomal inhibitor-sensitive mechanism. Our biochemical data strongly suggest that the function of Dpr2 proteins in inhibiting TGF-ß signal transduction is well conserved during evolution from fish to mammals.
Zebrafish Dpr2 is specifically expressed in mesoderm precursors at the onset of gastrulation, and in dorsal neural tube, lateral mesoderm, and tailbud during segmentation (18
, 19
, 22)
. Unlike zebrafish Dpr2, mDpr2 is expressed in all three germ layers during early gastrulation, suggesting an evolutionary divergence. After somitogenesis starts, mDpr2 is expressed in developing somites and the roof plate, which is similar to dpr2 expression pattern in zebrafish embryos. In mouse, Nodal gene is expressed throughout the epiblast before gastrulation and is essential for mesoderm induction (33
, 34)
. The mDpr2 expression domains appear to overlap the Nodal domain. This raises a possibility that Dpr2 functions to antagonize mesoderm induction activity of Nodal signaling during mouse embryo development. In fact, overexpression of mDpr2 in zebrafish embryos can inhibit the formation of dorsal mesoderm and induce the cyclopia phenotype, which suggests that mDpr2 has a biological activity similar to zebrafish Dpr2.
We previously found that morpholino knockdown of zebrafish dpr2 causes expansion of dorsal mesoderm (22)
. In this study, we attempted to rescue the effect of dpr2 knockdown by overexpressing mDpr2, but coinjection of dpr2-MO with 100 pg of mDpr2 mRNA still led to expansion of gsc at the shield stage and of ntl at the 5-somite stage. Although overexpression of mDpr2 resulted decreased expression of both ntl, a mesoderm marker, and gsc, a shield-specific marker, mDpr2 had stronger effect on ntl expression. Together, these data suggest that mDpr2 and fish Dpr2 may have different levels of activity in regulating different signaling pathways, or mDpr2 overexpression may affect additional signaling pathways besides nodal signaling. Definite roles of Dpr2 in development of mouse embryos need to be elucidated through knockout approach.
Dpr1 and Dpr2 share several conserved domains from fish to human (16
, 18
, 19)
. However, increasing evidence suggests that they act as the modulators for different signaling pathways and their functions may be context-dependent. Indeed, Dpr1 orthologs have been reported to inhibit Wnt signaling or to enhance Wnt signaling (14
, 18
, 19
, 21)
. Waxman et al. suggested that Dpr1 and Dpr2 participate in different Wnt-dependent developmental processes in zebrafish embryos: Dpr1 is involved in the canonical Wnt/ß-catenin pathway and functionally interacts with Dishevelled while Dpr2 participates in the noncanonical Wnt/Ca2+-PCP pathway (18)
. Our previous results indicated that zebrafish Dpr2 modulates Nodal signaling in induction of mesoderm formation, at least in part by controlling the cell surface protein level of TGF-ß/Nodal type I receptors (22)
. Here we further provide evidence that mDpr2 antagonizes the transcriptional activity of TGF-ß similarly by interacting and promoting type I receptor degradation. In contrast, Dpr1 inhibits the function of Wnt1 in activating the expression of the ß-catenin-responsive LEF-luciferase reporter, consistent with the observation that mDpr1 can promote Dishevelled degradation (20)
. In agreement with that zebrafish Dpr2 has no effect on Wnt1-induced LEF-luciferase expression (22)
, mDpr2 has minor effect on LEF-luciferase expression. Interestingly, we found that both mDpr1 and mDpr2 could attenuate the expression of a JNK-responsive reporter albeit mDpr1 was more effective, supporting the early observations that Xenopus Dpr1 and zebrafish Dpr2 modulate noncanonical Wnt pathways (14
, 18)
. However, different from the report of Waxman et al. that zebrafish Dpr2 is essential for the noncanonical Wnt/Ca2+-PCP pathway (18)
, our data suggest that mDpr2 inhibits the Wnt/JNK pathway. Therefore, Dpr proteins may function to regulate distinct signaling pathways in context-dependent manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 14, 2006. Accepted for publication October 25, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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