Isolation of Two Novel WNT Genes, WNT14 and WNT15, One of Which (WNT15) Is Closely Linked to WNT3 on Human Chromosome 17q21

TheWntgene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of theWntgene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of theWntgene family that also includes theWnt9sequences found in jawless and cartilaginous fishes. AWnt14cDNA was also isolated from chicken and a partialWnt15sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.     

WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells

Human WNT10A and WNT6 were cloned and characterized. WNT10A encoded a 417-amino-acid polypeptide with WNT core domain, and WNT6 encoded a 365-amino-acid polypeptide with N-terminal signal peptide, WNT core domain, and RGD motif. WNT10A and WNT6 genes were clustered in the head-to-tail manner with an interval less than 7.0 kb in human chromosome 2q35 region. Among human WNT family, WNT10A was most homologous to WNT10B (59.2% amino-acid identity), and WNT6 was most homologous to WNT1 (47.4% amino-acid identity). WNT10B and WNT1 genes were also clustered in human chromosome 12q13 region. Two WNT gene clusters in human chromosome 2q35 and 12q13 regions might be generated due to duplication of ancestral gene cluster. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in fetal kidney, placenta, adult spleen and kidney. The 2.0-kb WNT6 mRNA was coexpressed with WNT10A in placenta and adult spleen. WNT10A and WNT6 were strongly coexpressed in SW480 (colorectal cancer). In addition to SW480, WNT10A was strongly expressed in HL-60 (promyelocytic leukemia) and Raji (Burkitt's lymphoma), and WNT6 in HeLa S3 (cervical cancer). Overexpression WNT10A and WNT6 might play key roles in human carcinogenesis through activation of WNT-beta-catenin-TCF signaling pathway, just like Wnt10b and Wnt1.     

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b . We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl ( Ambystoma mexicanum ), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.     

Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. We show that expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, we find that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis.     

Mouse Wnt9b transforming activity,tissue-specific expression,and evolution

The members of the Wnt family of secreted factors have oncogenic potential and important roles as developmental regulators. We report an analysis of mouse Wnt9b (also called Wnt15 and Wnt14b), including its cDNA sequence, chromosomal mapping, epithelial cell transforming activity, adult and embryonic tissue expression patterns, and evolution. We also deduced the full-length amino acid sequence of its close relative, Wnt9a (also called Wnt14), from unannotated genomic DNA sequences in GenBank. Full-length comparisons among Wnt amino acid sequences provide evidence that Wnt9b and Wnt9a are close paralogs of each other and are orthologs of Wnt9 genes from shark and hagfish. Mapping Wnt9b to The Jackson Laboratory BSS interspecific backcross panel places it at 63.0 cM on chromosome 11. Sequence comparisons of two pairs of linked Wnt genes (the Wnt9a-Wnt3a pair and the Wnt9b-Wnt3 pair) suggest that they arose from the relatively recent duplication of a single ancestral Wnt gene pair, confirming the close paralogous relationship of Wnt9a and Wnt9b. Wnt9b expression is primarily restricted to the kidney in the adult mouse, with lower levels detected in the preputial gland, liver, and mammary gland. Testing of staged whole mouse embryos from 9.5 to 17.5 days of gestation showed expression at all stages with a peak at day 10.5. In situ hybridization analysis showed expression in most but not all tissues of the 16.5-day embryo. No significant elevation of Wnt9b expression was detected in 29 mouse mammary tumor virus-induced tumors. Overexpression of Wnt9b in C57MG mammary epithelial cells caused small transformed foci in cell monolayers and a moderate morphological transformation in pooled colonies compared with Wnt1.     

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/beta-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.     

BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells

WNT and bone morphogenetic protein (BMP) signaling are known to stimulate hemogenesis from pluripotent embryonic stem (ES) cells. However, osteochondrogenic mesoderm was generated effectively when BMP signaling is kept to a low level, while WNT signaling was strongly activated. When mesoderm specification from ES cells was exogenous factor dependent, WNT3a addition supported the generation of cardiomyogenic cells expressing lateral plate/extraembryonic mesoderm genes, and this process involved endogenous BMP activities. Exogenous BMP4 showed a similar effect that depended on endogenous WNT activities. However, neither factor induced robust chondrogenic activity. In support, ES cell differentiation in the presence of either WNT3a or BMP4 was associated with elevated levels of both Bmp and Wnt mRNAs, which appeared to provide sufficient levels of active BMPs and WNTs to promote the nonchondrogenic mesoderm specification. The osteochondrogenic mesoderm expressed PDGFRα, which also expressed genes that mark somite and rostral presomitic mesoderm. A strong WNT signaling was required for generating the mesodermal progeny, while approximately 50- to 100-fold lower concentration of WNT3a was sufficient for specifying axial mes(end)oderm. Thus, depending on the dose and cofactor (BMP), WNT signaling stimulates the generation of different biological activities and specification of different types of mesodermal progeny from ES cells.     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

Differential transformation of mammary epithelial cells by Wnt genes.

The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent.     

WNT5A Encodes Two Isoforms with Distinct Functions in Cancers

WNT5A, a member of the WNT family of secreted lipid-modified glycoproteins, is a critical regulator of a host of developmental processes, including limb formation, lung morphogenesis, intestinal elongation and mammary gland development. Altered WNT5A expression has been associated with a number of cancers. Interestingly, in certain types of cancers, such as hematological malignancies and colorectal carcinoma, WNT5A is inactivated and exerts a tumor suppressive function, while in other cancers, such as melanoma and gastric carcinoma, WNT5A is overexpressed and promotes tumor progression. The mechanism by which WNT5A achieves these distinct activities in cancers is poorly understood. Here, we provide evidence that the WNT5A gene produces two protein isoforms, WNT5A-long (WNT5A-L) and WNT5A-short (WNT5A-S). Amino-terminal sequencing and a WNT5A-L specific antibody demonstrate that the mature and secreted isoforms are distinct, with WNT5A-L carrying an additional 18 N-terminal amino acids. Biochemical analysis indicates that both purified proteins are similar with respect to their stability, hydrophobicity and WNT/β-catenin signaling activity. Nonetheless, modulation of these two WNT5A isoforms, either through ectopic expression or knockdown, demonstrates that they exert distinct activities in cancer cell lines: while WNT5A-L inhibits proliferation of tumor cell lines, WNT5A-S promotes their growth. Finally, we show that expression of these two WNT5A isoforms is altered in breast and cervix carcinomas, as well as in the most aggressive neuroblastoma tumors. In these cancers, WNT5A-L is frequently down-regulated, whereas WNT5A-S is found overexpressed in a significant fraction of tumors. Altogether, our study provides evidence that the distinct activities of WNT5A in cancer can be attributed to the production of two WNT5A isoforms.     

A local Wnt-3a signal is required for development of the mammalian hippocampus

The mechanisms that regulate patterning and growth of the developing cerebral cortex remain unclear. Suggesting a role for Wnt signaling in these processes, multiple Wnt genes are expressed in selective patterns in the embryonic cortex. We have examined the role of Wnt-3a signaling at the caudomedial margin of the developing cerebral cortex, the site of hippocampal development. We show that Wnt-3a acts locally to regulate the expansion of the caudomedial cortex, from which the hippocampus develops. In mice lacking Wnt-3a, caudomedial cortical progenitor cells appear to be specified normally, but then underproliferate. By mid-gestation, the hippocampus is missing or represented by tiny populations of residual hippocampal cells. Thus, Wnt-3a signaling is crucial for the normal growth of the hippocampus. We suggest that the coordination of growth with patterning may be a general role for Wnts during vertebrate development.     

<Emphasis Type="Italic">Wnt5</Emphasis>a plays a crucial role in determining tooth size during murine tooth development

We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14–17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial–mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.     

Wnt16 Is Associated with Age-Related Bone Loss and Estrogen Withdrawal in Murine Bone

Genome Wide Association Studies suggest that Wnt16 is an important contributor to the mechanisms controlling bone mineral density, cortical thickness, bone strength and ultimately fracture risk. Wnt16 acts on osteoblasts and osteoclasts and, in cortical bone, is predominantly derived from osteoblasts. This led us to hypothesize that low bone mass would be associated with low levels of Wnt16 expression and that Wnt16 expression would be increased by anabolic factors, including mechanical loading. We therefore investigated Wnt16 expression in the context of ageing, mechanical loading and unloading, estrogen deficiency and replacement, and estrogen receptor α (ERα) depletion. Quantitative real time PCR showed that Wnt16 mRNA expression was lower in cortical bone and marrow of aged compared to young female mice. Neither increased nor decreased (by disuse) mechanical loading altered Wnt16 expression in young female mice, although Wnt16 expression was decreased following ovariectomy. Both 17β-estradiol and the Selective Estrogen Receptor Modulator Tamoxifen increased Wnt16 expression relative to ovariectomy. Wnt16 and ERβ expression were increased in female ERα^-/- mice when compared to Wild Type. We also addressed potential effects of gender on Wnt16 expression and while the expression was lower in the cortical bone of aged males as in females, it was higher in male bone marrow of aged mice compared to young. In the kidney, which we used as a non-bone reference tissue, Wnt16 expression was unaffected by age in either males or females. In summary, age, and its associated bone loss, is associated with low levels of Wnt16 expression whereas bone loss associated with disuse has no effect on Wnt16 expression. In the artificially loaded mouse tibia we observed no loading-related up-regulation of Wnt16 expression but provide evidence that its expression is influenced by estrogen receptor signaling. These findings suggest that while Wnt16 is not an obligatory contributor to regulation of bone mass per se, it potentially plays a role in influencing pathways associated with regulation of bone mass during ageing and estrogen withdrawal.     

Essential roles of mesenchyme-derived beta-catenin in mouse Müllerian duct morphogenesis

Members of the Wnt family of genes such as Wnt4, Wnt5a, and Wnt7a have been implicated in the formation and morphogenesis of the Müllerian duct into various parts of the female reproductive tract. These WNT ligands elicit their action via either the canonical WNT/beta-catenin or the non-canonical WNT/calcium pathway and could possibly function redundantly in Müllerian duct differentiation. By using the Müllerian duct-specific anti-Müllerian hormone receptor 2 cre (Amhr2-cre) mouse line, we established a conditional knockout model that removed beta-catenin specifically in the mesenchyme of the Müllerian duct. At birth, loss of beta-catenin in the Müllerian duct mesenchyme disrupted the normal coiling of the oviduct in the knockout embryo, resembling the phenotype of the Wnt7a knockout. The overall development of the female reproductive tract was stunted at birth with a decrease in proliferation in the mesenchyme and epithelium. We also discovered that Wnt5a and Wnt7a expression remained normal, excluding the possibility that the phenotypes resulted from a loss of these WNT ligands. We examined the expression of Frizzled (Fzd), the receptors for WNT, and found that Fzd1 is one receptor present in the Müllerian duct mesenchyme and could be the putative receptor for beta-catenin activation in the Müllerian duct. In summary, our findings suggest that mesenchymal beta-catenin is a downstream effector of Wnt7a that mediates the patterning of the oviduct and proper differentiation of the uterus.     

Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice

The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.     

Evidence for Involvement of Wnt Signalling in Body Polarities,Cell Proliferation,and the Neuro-Sensory System in an Adult Ctenophore

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.