R-Spondins in Cutaneous Biology: Nails and Cancer

The R-spondins are a relatively recently identified family of secreted proteins linked to the Wnt signalling pathway. Recently, human phenotypes have been associated with mutations in two human R-spondins. Germline RSPO4 and RSPO1 mutations result in recessive anonychia (absence of all fingernails and toenails) and a recessive syndrome with XX-male sex reversal, PPK and a predisposition to SCC, respectively. This review discusses the key roles R-spondins play in embryogenesis, adult tissue maintenance and skin carcinogenesis.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

Structures of Wnt-Antagonist ZNRF3 and Its Complex with R-Spondin 1 and Implications for Signaling

Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 – R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.     

A novel Wnt5a‐Frizzled4 signaling pathway mediates activity‐independent dendrite morphogenesis via the distal PDZ motif of Frizzled 4

The morphology of the dendritic tree is critical to neuronal function and neural circuit wiring. Several Wnt family members have been demonstrated to play important roles in dendrite development. However, the Wnt receptors responsible for mediating this process remain largely elusive. Using primary hippocampal neuronal cultures as a model system, we report that Frizzled4 (Fzd4), a member of the Fzd family of Wnt receptors, specifically signals downstream of Wnt5a to promote dendrite branching and growth. Interestingly, the less conserved distal PDZ binding motif of Fzd4, and not its conserved proximal Dvl‐interacting PDZ motif, is required for mediating this effect. We further showed that Dvl signaled parallel to and independent of Fzd4 in promoting dendrite growth. Unlike most previously described pathways, Wnt5a/Fzd4 signaling promoted dendrite development in an activity‐independent and autocrine fashion. Together, these results provide the first identification of a Wnt receptor for regulating dendrite development in the mammalian system, and demonstrate a novel function of the distal PDZ motif of Fzd4 in dendrite morphogenesis, thereby expanding our knowledge of the complex roles of Wnt signaling in neural development. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 805℃822, 2015     

Wnt4 expression in the differentiating gonad of the frog Rana rugosa

Wnt4, a member of the Wnt family, is known to influence the sex-determination cascade. In mice having a targeted deletion of Wnt4, masculinization occurs in XX pups. Therefore, in addition to Sry, Wnt4 is also involved in sex determination in mice. In humans, a chromosomal duplication of the WNT4 causes feminization of XY-individuals. Thus, for better understanding of the mechanism of sex determination in vertebrates, it is necessary to examine the expression of Wnt4 at early gonadal development stages in non-mammalians. We first isolated the Wnt4 cDNA from the tetsis of the frog Rana rugosa. R. rugosa Wnt4 had a high similarity (>86%) at the amino acid level with zebra fish, chicken, mouse, and human Wnt4s. We next employed RT-PCR analysis to examine whether Wnt4 was expressed in a sexually dimorphic fashion at early stages of gonadal development in R. rugosa. Wnt4 was transcribed first in the embryos at the late gastrula stage, and its expression was maintained until the indifferent gonad differentiated into a testis or an ovary. Wnt4 expression in the differentiating gonad appeared in a non-sexually dimorphic pattern. The results, taken together, suggest that Wnt4 is highly conserved through evolution, and that its expression in the indifferent gonad takes place with no sexual dimorphism. Thus, Wnt4 is not a key factor to initiate the development of a testis or an ovary from the indifferent gonads in R. rugosa. However, this gene probably forms part of a gonadal-development pathway in this species.     

Wnt signaling: the good and the bad

Since the first Wnt gene was identified in 1982, the functions and mechanisms of Wnt signaling have been extensively studied. Wnt signaling is conserved from invertebrates to vertebrates and regulates early embryonic development as well as the homeostasis of adult tissues. In addition, both embryonic stem cells and adult stem cells are regulated by Wnt signaling. Deregulation of Wnt signaling is associated with many human diseases, particularly cancers. In this review, we will discuss in detail the functions of many components involved in the Wnt signal transduction pathway. Then, we will explore what is known about the role of Wnt signaling in stem cells and cancers.     

Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.     

Negative Feedback Regulation of Wnt4 Signaling by EAF1 and EAF2/U19

Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.     

Uterine Msx-1 and Wnt4 signaling becomes aberrant in mice with the loss of leukemia inhibitory factor or Hoxa-10: evidence for a novel cytokine-homeobox-Wnt signaling in implantation

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.     

Wnt signaling components in the chicken intestinal tract

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. Here, we offer an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut. This paper describes the first comprehensive characterization of the dynamic expression of Wnt signaling molecules during gut development. These data form the basis for future studies to determine the role of Wnt signaling in the developing gastrointestinal tract.     

Total anonychia congenita: a rare heterogeneic disorder

Total anonychia congenita is a very rare disorder in which all the finger nails and the toenails are absent without significant bone anomalies. This condition is reported to have an autosomal dominant inheritance pattern. There are a variety of syndromes or conditions associated with anonychia congenita. In this study we present a monozygotic twin with isolated anonychia congenita totals. Interestingly there is no family history of anonychia and this condition seems to be caused by a sporadic mutation.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal

Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1-/-, Tcf3(flox), and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.     

A Naturally Occurring Lgr4 Splice Variant Encodes a Soluble Antagonist Useful for Demonstrating the Gonadal Roles of Lgr4 in Mammals

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.     

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development.     

A heat shock protein and Wnt signaling crosstalk during axial patterning and stem cell proliferation

Both Wnt signaling and heat shock proteins play important roles in development and disease. As such, they have been widely, though separately, studied. Here we show a link between a heat shock protein and Wnt signaling in a member of the basal phylum, Cnidaria. A heat shock at late gastrulation in the clonal marine hydrozoan, Hydractinia, interferes with axis development, specifically inhibiting head development, while aboral structures remain unaffected. The heat treatment upregulated Hsc71, a constitutive Hsp70 related gene, followed by a transient upregulation, and long-term downregulation, of Wnt signaling components. Downregulating Hsc71 by RNAi in heat-shocked animals rescued these defects, resulting in normal head development. Transgenic animals, ectopically expressing Hsc71, had similar developmental abnormalities as heat-shocked animals in terms of both morphology and Wnt3 expression. We also found that Hsc71 is upregulated in response to ectopic Wnt activation, but only in the context of stem cell proliferation and not in head development. Hsc71's normal expression is consistent with a conserved role in mitosis and apoptosis inhibition. Our results demonstrate a hitherto unknown crosstalk between heat shock proteins and Wnt/β-catenin signaling. This link likely has important implications in understanding normal development, congenital defects and cancer biology.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.