The function and evolution of Wnt genes in arthropods

Wnt signalling is required for a wide range of developmental processes, from cleavage to patterning and cell migration. There are 13 subfamilies of Wnt ligand genes and this diverse repertoire appeared very early in metazoan evolution.In this review, we first summarise the known Wnt gene repertoire in various arthropods. Insects appear to have lost several Wnt subfamilies, either generally, such as Wnt3, or in lineage specific patterns, for example, the loss of Wnt7 in Anopheles. In Drosophila and Acyrthosiphon, only seven and six Wnt subfamilies are represented, respectively; however, the finding of nine Wnt genes in Tribolium suggests that arthropods had a larger repertoire ancestrally.We then discuss what is currently known about the expression and developmental function of Wnt ligands in Drosophila and other insects in comparison to other arthropods, such as the spiders Achaearanea and Cupiennius. We conclude that studies of Wnt genes have given us much insight into the developmental roles of some of these ligands. However, given the frequent loss of Wnt genes in insects and the derived development of Drosophila, further studies of these important genes are required in a broader range of arthropods to fully understand their developmental function and evolution.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Expression of the decapentaplegic ortholog in embryos of the onychophoran Euperipatoides rowelli

The gene decapentaplegic (dpp) and its homologs are essential for establishing the dorsoventral body axis in arthropods and vertebrates. However, the expression of dpp is not uniform among different arthropod groups. While this gene is expressed along the dorsal body region in insects, its expression occurs in a mesenchymal group of cells called cumulus in the early spider embryo. A cumulus-like structure has also been reported from centipedes, suggesting that it might be either an ancestral feature of arthropods or a derived feature (=synapomorphy) uniting the chelicerates and myriapods. To decide between these two alternatives, we analysed the expression patterns of a dpp ortholog in a representative of one of the closest arthropod relatives, the onychophoran Euperipatoides rowelli. Our data revealed unique expression patterns in the early mesoderm anlagen of the antennal segment and in the dorsal and ventral extra-embryonic tissue, suggesting a divergent role of dpp in these tissues in Onychophora. In contrast, the expression of dpp in the dorsal limb portions resembles that in arthropods, except that it occurs in the mesoderm rather than in the ectoderm of the onychophoran limbs. A careful inspection of embryos of E. rowelli revealed no cumulus-like accumulation of dpp expressing cells at any developmental stage, suggesting that this feature is either a derived feature of chelicerates or a synapomorphy uniting the chelicerates and myriapods.     

Roles of Wnt8a during formation and patterning of the mouse inner ear

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle.     

Embryonic expression patterns of Wnt genes in the RTA-clade spider Cupiennius salei

Spiders represent widely used model organisms for chelicerate and even arthropod development and evolution. Wnt genes are important and evolutionary conserved factors that control and regulate numerous developmental processes. Recent studies comprehensively investigated the complement and expression of spider Wnt genes revealing conserved as well as diverged aspects of their expression and thus (likely) function among different groups of spiders representing Mygalomorphae (tarantulas), and both main groups of Araneae (true spiders) (Haplogynae/Synspermiata and Entelegynae). The allegedly most modern/derived group of entelegyne spiders is represented by the RTA-clade of which no comprehensive data on Wnt expression were available prior to this study. Here, we investigated the embryonic expression of all Wnt genes of the RTA-clade spider Cupiennius salei. We found that most of the Wnt expression patterns are conserved between Cupiennius and other spiders, especially more basally branching species. Surprisingly, most differences in Wnt gene expression are seen in the common model spider Parasteatoda tepidariorum (a non-RTA clade entelegyne species). These results show that data and conclusions drawn from research on one member of a group of animals (or any other organism) cannot necessarily be extrapolated to the group as a whole, and instead highlight the need for comprehensive taxon sampling.     

Wnt signaling regulates the proliferation potential and lineage commitment of human umbilical cord derived mesenchymal stem cells

Relatively less is known about the interactions that tightly regulate the mesenchymal stem cells (MSCs) to maintain their pluripotency. Recent studies reports that Wnt proteins might play an important role in governing the MSC cell fate. In this study, we tested the hypothesis that Wnt proteins differentially regulate in vitro differentiation of human umbilical cord derived MSCs. Stromal cells from human umbilical cord (hUCMSCs) were isolated and treated with Wnt inhibitor/activator. FACS analysis of hUCMSCs for CD29, CD90, CD73, CD44, CD45 marker expression and gene expression of Wnt target genes and lineage specific genes were performed after Lithium Chloride (LiCl) and Quercetin treatment for 6 days. The cultured primary hUCMSCs demonstrated elevated MSC surface marker expression with clonogenic properties and differentiation potentials towards osteogenic, adipogenic and chondrogenic lineages. Downregulation in the expression of Wnt with Quercetin treatment was noted. LiCl treatment increased cellular proliferation but did not influence differentiation suggesting that the cells retain pluripotency whereas Quercetin treatment downregulated stemness markers, Wnt target gene expression and promoted osteogenesis as demonstrated by FACS analysis, calcium estimation and gene expression studies. Shift of differentiation potential after the inhibition of Wnt signaling by Quercetin was evident from the gene expression data and elevated calcium production, driving MSCs towards probable osteogenic lineage. The findings in particular are likely to open an interesting avenue of biomedical research, summarizing the impact of Wnt signaling on lineage commitment of MSCs.

     

Study on the function of Helicoverpa armigera Wnt1 gene using CRISPR/Cas9 system

The Wnt signaling pathway, as a highly conserved signaling pathway in evolution, plays an important role in many biological processes. The research of Wnt signaling pathway through gene editing has been implemented in a variety of organisms. Among the various genome editing tools available for functional genomic research, CRISPR is popular because of its ease of use and versatility. Here, we use the CRISPR/Cas9 system to knock out the HaWnt1 gene of the important agricultural pest Helicoverpa armigera to explore the impacts on embryo development. Direct injection of Cas9 protein and Wnt1-specific single guide RNA (sgRNA) into H. armigera embryos successfully induced Wnt1 gene deletion mutants, which showed high lethality, abnormal segmentation, defected appendages and defected pigmentation. qRT-PCR analysis revealed that the deletion of Wnt1 gene affected the expression of several genes, which were closely related to the growth and development of insects. Our results indicate that HaWnt1 signaling pathway is essential for embryonic development of H. armigera. The study of the function of HaWnt1 gene not only lays the foundation for the study of the somatic development pattern of H. armigera, but also provides a candidate gene for genetic control of H. armigera.     

Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation

Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.     

Divergent Wnt8a Gene Expression in Teleosts

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.     

Wnt signaling in heart valve development and osteogenic gene induction

Wnt signaling mediated by β-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of β-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.     

Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus

Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. We have identified a second Xenopus Wnt11-Related gene (Wnt11-R) that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube.     

The IGSF1, Wnt5a,FGF14, and ITPR1 Gene Expression and Prognosis Hallmark of Prostate Cancer

Background:Prostate cancer is considered as the second leading cause of cancer related death in men worldwide and the third frequent cancer among Iranian men. Despite the use of PSA as the only biomarker for early diagnosis of prostate cancer, its application in clinical settings is under debate. Therefore, the introduction of new molecular markers for early detection of prostate cancer is needed. Methods:In the present study we intended to evaluate the expression of IGSF1, Wnt5a, FGF14, and ITPR1 in prostate cancer specimens by real time PCR. Biopsy samples of 40 prostate cancer cases and 41 healthy Iranian men were compared to determine the relative gene expression of IGSF1, Wnt5a, FGF14, and ITPR1 by real time PCR. Results:Our results showed that Wnt5a, FGF14, and IGSF1 were significantly overexpressed in the prostate cancer patients while the mean relative expression of ITPR1 showed a significant decrease in PCa samples compared to healthy controls. Conclusion:According to results of the present study, the combination panel of IGSF1, Wnt5a, FGF14, and ITPR1 genes could be considered as potential genetic markers for prostate cancer diagnosis. However further studies on larger populations and investigating the clinicopathological relevance of these genes is needed.Key Words: FGF14, IGSF1, ITPR1, Prostate Cancer, Wnt5a     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Sfrp1a and Sfrp5 function as positive regulators of Wnt and BMP signaling during early retinal development

Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-β catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.     

Prognostic role of Wnt and Fzd gene families in acute myeloid leukaemia

Wnt-Fzd signalling pathway plays a critical role in acute myeloid leukaemia (AML) progression and oncogenicity. There is no study to investigate the prognostic value of Wnt and Fzd gene families in AML. Our study screened 84 AML patients receiving chemotherapy only and 71 also undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT) from the Cancer Genome Atlas (TCGA) database. We found that some Wnt and Fzd genes had significant positive correlations. The expression levels of Fzd gene family were independent of survival in AML patients. In the chemotherapy group, AML patients with high Wnt2B or Wnt11 expression had significantly shorter event-free survival (EFS) and overall survival (OS); high Wnt10A expressers had significantly longer OS than the low expressers (all P < .05), whereas, in the allo-HSCT group, the expression levels of Wnt gene family were independent of survival. We further found that high expression of Wnt10A and Wnt11 had independent prognostic value, and the patients with high Wnt10A and low Wnt11 expression had the longest EFS and OS in the chemotherapy group. Pathway enrichment analysis showed that genes related to Wnt10A, Wnt11 and Wnt 2B were mainly enriched in ‘cell morphogenesis involved in differentiation’, ‘haematopoietic cell lineage’, ‘platelet activation, signalling and aggregation’ and ‘mitochondrial RNA metabolic process’ signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo-HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.     

Selective Modulation of Wnt Ligands and Their Receptors in Adipose Tissue by Chronic Hyperadiponectinemia

Background

Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue.

Materials and Methods

We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells.

Results

The Wnt5b, Wnt6, Frizzled 6 (Fzd6), and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6) were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII) and phosphorylated Jun N-terminal kinase (p-JNK) were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin.

Conclusion

Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors accompanied by the inhibition of the Wnt/Ca²⁺ and JNK signaling pathways, which may be involved in the altered adipocyte cellularity, endogenous adiponectin production, and anti-inflammatory action induced by hyperadiponectinemia.