Nemo-like kinase 1 (Nlk1) and paraxial protocadherin (PAPC) cooperatively control Xenopus gastrulation through regulation of Wnt/planar cell polarity (PCP) signaling.

The Wnt/planar cell polarity (PCP) pathway directs cell migration during vertebrate gastrulation and is essential for proper embryonic development. Paraxial protocadherin (PAPC, Gene Symbol pcdh8.2) is an important activator of Wnt/PCP signaling during Xenopus gastrulation, but how PAPC activity is controlled is incompletely understood. Here we show that Nemo-like kinase 1 (Nlk1), an atypical mitogen-activated protein (MAP) kinase, physically associates with the C-terminus of PAPC. This interaction mutually stabilizes both proteins by inhibiting polyubiquitination. The Nlk1 mediated stabilization of PAPC is essential for Wnt/PCP signaling, tissue separation and gastrulation movements. We identified two conserved putative phosphorylation sites in the PAPC C-terminus that are critical for Nlk1 mediated PAPC stabilization and Wnt/PCP regulation. Intriguingly, the kinase activity of Nlk1 itself was not essential for its cooperation with PAPC, suggesting an indirect regulation for example by impeding a different kinase that promotes protein degradation. Overall these results outline a novel, kinase independent role of Nlk1, wherein Nlk1 regulates PAPC stabilization and thereby controls gastrulation movements and Wnt/PCP signaling during development.

Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.

Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity.

Mutation or knock-down of 17ß-hydroxysteroid dehydrogenase type 10 cause loss of MRPP1 and impaired processing of mitochondrial heavy strand transcripts.

17ß-Hydroxysteroid dehydrogenase type 10 (HSD10) is multifunctional protein coded by the X-chromosomal HSD17B10 gene. Mutations in this gene cause HSD10 disease characterized by progressive neurological abnormalities and cardiomyopathy. Disease progression and severity of symptoms is unrelated to the protein's dehydrogenase activity. Recently, it was shown that HSD10 is an essential component of mitochondrial Ribonuclease P (RNase P), an enzyme required for mitochondrial tRNA processing, but little is known about the role of HSD10 in RNase P function. RNase P consists of three different proteins MRPP1, MRPP2 (HSD10) and MRPP3, each of which is essential for RNase P function. Here, we show that HSD10 protein levels are significantly reduced in fibroblasts from patients carrying the HSD17B10 mutation p.R130C. A reduction in HSD10 levels was accompanied by a reduction in MRPP1 protein but not MRPP3 protein. In HSD10 knock-down cells, MRPP1 protein content was also reduced, indicating that HSD10 is important for the maintenance of normal MRPP1 protein levels. Ectopic expression of HSD10 partially restored RNA processing in HSD10 knock-down cells and fibroblasts, and also expression of MRPP1 protein was restored to values comparable to controls. In both, patient fibroblasts and HSD10 knock-down cells, there was evidence of impaired processing of precursor tRNA transcripts of the mitochondrial heavy strand but not the light strand compared with controls. Our findings indicate that HSD10 is important for the maintenance of the MRPP1-HSD10 subcomplex of RNase P and that loss of HSD10 causes impaired mitochondrial precursor transcript processing which may explain mitochondrial dysfunction observed in HSD10 disease.

Lack of phosphomannomutase 2 affects Xenopus laevis morphogenesis and the non-canonical Wnt5a/Ror2 signalling.

Reduced phosphomannomutase 2 activity in man leads to hypoglycosylation of glycoconjugates causing PMM2-CDG, the most common type of congenital disorders of glycosylation. Here we show that an antisense morpholino-mediated knockdown of the Xenopus laevis phosphomannomutase 2 gene provoked a general underglycosylation in frog embryos, which led to an altered phenotype and reduced glycosylation of Wnt5a as member of the non-canonical Wnt signalling. Loss of function experiments in hemi-sectioned embryos proved that due to the phosphomannomutase 2 knockdown expression of the Wnt5a/Ror2 target gene paraxial protocadherin was significantly decreased. Regarding the expression of paraxial protocadherin, a gain of function could only be achieved by injections of wnt5a and ror2 in dorsal neighbouring blastomeres, while a parallel injection of phosphomannomutase 2 morpholino led to a significant reduced level of expression. Our data show for the first time that a knockdown of phosphomannomutase 2 influences in vivo the non-canonical Wnt signalling during early embryogenesis.

IGF antagonizes the Wnt/ß-Catenin pathway and promotes differentiation of extra-embryonic endoderm.

Mouse F9 teratocarcinoma cells are an established model for the differentiation of extra-embryonic endoderm (ExEn). Primitive endoderm, parietal and visceral endoderm can be generated by stimulation of F9 cells with retinoic acid and dibutyryl cyclic adenosine monophosphate. Here we show that Wnt/ß-Catenin signaling is down-regulated during ExEn differentiation in F9 cells and that the inhibition of the Wnt pathway promotes differentiation of the three extra-embryonic endoderm lineages. Wnt inhibition is achieved through the IGF pathway, which is up-regulated during differentiation. IGF signaling antagonizes the Wnt pathway by stimulating transcription of axin2 and by stabilizing Axin1 protein. Both Axin1 and Axin2 are components of the ß-Catenin destruction complex and act as intra-cellular inhibitors of the Wnt/ß-Catenin pathway. The data presented reveal a mechanism which restricts pluripotency of undifferentiated cells and directs them toward extra-embryonic lineages.

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Xenopus paraxial protocadherin inhibits Wnt/ß-catenin signalling via casein kinase 2ß.

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/ß-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2ß), which is part of the CK2 holoenzyme. The CK2α/ß complex stimulates Wnt/ß-catenin signalling, and the physical interaction of CK2ß with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.

Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia.

The heart's rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 (-/-) hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 (-/-) hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.

A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling.

BACKGROUND: Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/ß-catenin signalling, and also interacts with those Wnt ligands that act on ß-catenin-independent Wnt pathways. FINDINGS: Here we show that Xenopus Fz4-v1 can activate and inhibit the ß-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/ß-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced ß-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin. CONCLUSIONS: For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators.

Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart.

Heart formation requires a highly balanced network of transcriptional activation of genes. The homeodomain transcription factor, Shox2, is essential for the formation of the sinoatrial valves and for the development of the pacemaking system. The elucidation of molecular mechanisms underlying the development of pacemaker tissue has gained clinical interest as defects in its patterning can be related to atrial arrhythmias. We have analyzed putative targets of Shox2 and identified the Bmp4 gene as a direct target. Shox2 interacts directly with the Bmp4 promoter in chromatin immunoprecipitation assays and activates transcription in luciferase-reporter assays. In addition, ectopic expression of Shox2 in Xenopus embryos stimulates transcription of the Bmp4 gene, and silencing of Shox2 in cardiomyocytes leads to a reduction in the expression of Bmp4. In Tbx5(del/+) mice, a model for Holt-Oram syndrome, and Shox2(-/-) mice, we show that the T-box transcription factor Tbx5 is a regulator of Shox2 expression in the inflow tract and that Bmp4 is regulated by Shox2 in this compartment of the embryonic heart. In addition, we could show that Tbx5 acts cooperatively with Nkx2.5 to regulate the expression of Shox2 and Bmp4. This work establishes a link between Tbx5, Shox2 and Bmp4 in the pacemaker region of the developing heart and thus contributes to the unraveling of the intricate interplay between the heart-specific transcriptional machinery and developmental signaling pathways.

xGit2 and xRhoGAP 11A regulate convergent extension and tissue separation in Xenopus gastrulation.

In a microarray-based screen for genes that are involved in tissue separation downstream of Paraxial Protocadherin (PAPC) and Frizzled-7 (Fz7)-mediated signaling we identified xGit2 and xRhoGAP 11A, two GTPase-activating proteins (GAP) for small GTPases. xGit2 and xRhoGAP 11A are expressed in the dorsal ectoderm, and their transcription is downregulated in the involuting dorsal mesoderm by PAPC and Fz7. Overexpression of xGit2 and xRhoGAP 11A inhibits Rho activity and impairs convergent extension movements as well as tissue separation behaviour. We propose that Rho activity in the involuting mesoderm is enhanced through inhibition of xGit2 and xRhoGAP 11A transcription by PAPC and Fz7. By this mechanism xRhoGAP 11A and xGit2 are restricted to the dorsal ectoderm, while Rho signaling is inhibited.

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior­posterior patterning and left­right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Molecular basis of morphogenesis during vertebrate gastrulation.

Gastrulation is a crucial step in early embryogenesis. During gastrulation, a set of morphogenetic processes takes place leading to the establishment of the basic body plan and formation of primary germ layers. A rich body of knowledge about these morphogenetic processes has been accumulated over decades. The understanding of the molecular mechanism that controls the complex cell movement and inductive processes during gastrulation remains a challenge. Substantial progress has been made recently to identify and characterize pathways and molecules implicated in the modulation of morphogenesis during vertebrate gastrulation. Here, we summarize recent findings in the analysis of signaling pathways implicated in gastrulation movements, with the aim to generalize the basic molecular principles of vertebrate morphogenesis.

Bone morphogenetic protein-4 and Noggin signaling regulates pigment cell distribution in the axolotl trunk.

Wild-type (dark) and white mutant axolotl (Ambystoma mexicanum) embryos were used to investigate the role of the secreted growth factor bone morphogenetic protein-4 (BMP-4) and its antagonist, Noggin, in dorso-lateral trunk neural crest (NC) migration. Implantation of a BMP-4-coated microbead caused a melanophore-free zone around the bead, reduction of the dorsal fin above the bead, and disappearance of myotome tissue. We established a novel method that allows controlled induction of protein synthesis and release. Xenopus animal cap (XAC) cells injected with heat shock-inducible constructs for BMP-4 and Noggin were implanted into axolotl embryos and protein expression was induced at defined time points. With this approach, we could demonstrate for the first time that Noggin can stimulate melanophore migration in the white mutant. We further showed that implantation of BMP-4 expressing XAC cells alters pigment cell distribution without affecting muscle and dorsal fin development.

Wnt-frizzled interactions in Xenopus.

The Wnt signaling cascades are regulatory modules which are involved in embryonic patterning, cell differentiation, morphogenesis, and diseases. The Wnt pathways are activated when secreted Wnt ligands interact with 7-trans-membrane receptors of the Frizzled (Fz) family. Specific readouts are determined by the ligand/receptor combinations and the cellular context. Here we describe two methods for the analysis of Wnt/Frizzled interactions in Xenopus embryos. Physical interaction of ligand and receptor are demonstrated by co-immunoprecipitation assays. The activation of Wnt targets in Xenopus animal cap tissue provides a versatile test system for activating and inhibitory components of the Wnt/beta -catenin pathway.

An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish.

BACKGROUND: In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization. RESULTS: To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, we tested different vasa homologs for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish. CONCLUSIONS: Our results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline.

Frizzled7 dictates embryonic morphogenesis: implications for colorectal cancer progression.

Recent insights from diverse fields of basic and clinical research reveal that the biological processes that govern embryonic development and organogenesis are also commonly involved in the pathologies that arise in that organ or tissue in the adult. This striking parallel between embryonic development and pathology is exemplified by Wnt signalling in the intestinal tract. Wnt signalling is critical throughout embryonic development of the mammalian gut. Moreover, competent Wnt signalling is essential for the homeostatic control of the adult intestinal epithelium. On the other hand, aberrant Wnt signalling in the adult intestine leads to cancer and other pathologies. This critical role of the Wnt pathway in gut development and homeostasis is conserved through evolution, emphasizing the importance of this pathway in this tissue. Interestingly, expression of the Wnt receptor FZD7 in gut tissue is also conserved through evolution, suggesting that this receptor may be integral to the important role assigned to Wnt signalling in gut tissues.

Developmental expression of Shisa-2 in Xenopus laevis.

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.

Expression of the ALK1 family of type I BMP/ADMP receptors during gastrula stages in Xenopus embryos.

Multiple members of the transforming growth factor beta (TGFß) family of secreted factors play central inductive and patterning roles during embryogenesis. During gastrulation in vertebrates, the bone morphogenetic protein (BMP) sub-family is linked to formation of the embryonic organizer, Spemann's organizer in Xenopus, and dorsal-ventral mesoderm patterning. Our knowledge regarding the BMP receptors mediating this signaling is still very incomplete. The BMPR1A (ALK3) and BMPR1B (ALK6) receptors are known to mediate the BMP4 signal. These receptors belong to the ALK1 subfamily of type I receptors that also includes ACVR1 (ALK2), and ACVRL1 (ALK1). We studied by qPCR and in situ hybridization the spatio-temporal expression patterns of ALK2 and ALK1 and compared them to ALK3 and ALK6, and to the main BMPs expressed during gastrulation, i.e., BMP4, BMP7, BMP2, and ADMP, in an attempt to establish a link between ligands and receptors. There is extensive overlap between BMP4, and ALk3 and Alk6 expression, supporting their functional interaction. Robust Alk6 expression was observed from mid-gastrula. Animal region expression of both receptors shows co-expression with BMP4 and BMP7. Alk2 transcripts were detected within the organizer, overlapping with its proposed ligand, ADMP, suggesting a probable function within the organizer. Alk1 is very weakly expressed during gastrula, but its transcripts were localized to the lateral marginal zone flanking the organizer domain. No receptor closely matched the maternal BMP2 expression, although Alk2, Alk3, and Alk6, have transcripts of maternal origin. Our analysis shows that the BMP ligands and their receptors exhibit dynamic expression patterns during gastrula stages.