Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2^LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2^LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2^LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.     

The E6 oncoprotein from HPV16 enhances the canonical Wnt/β-catenin pathway in skin epidermis in vivo

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2(+/LacZ) mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin-responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2(+/LacZ). Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.     

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.     

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2(Tg)). The predicted protein encoded by Rspo2(Tg) neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2(Tg/Tg) mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2(Tg/Tg) lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2(Tg) and Lrp6(-) alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.     

Wnt5a regulates Shh and Fgf10 signaling during lung development

The role of WNT signaling and its interactions with other morphogenetic pathways were investigated during lung development. Previously, we showed that targeted disruption of Wnt5a results in over-branching of the epithelium and thickening of the interstitium in embryonic lungs. In this study, we generated and characterized transgenic mice with lung-specific over-expression of Wnt5a from the SpC promoter. Over-expression of Wnt5a interfered with normal epithelial-mesenchymal interactions resulting in reduced epithelial branching and dilated distal airways. During early lung development, over-expression of Wnt5a in the epithelium resulted in increased Fgf10 in the mesenchyme and decreased Shh in the epithelium. Both levels and distribution of SHH receptor, Ptc were reduced in SpC-Wnt5a transgenic lungs and were reciprocally correlated to changes of Fgf10 in the mesenchyme, suggesting that SHH signaling is decreased by over-expression of Wnt5a. Cultured mesenchyme-free epithelial explants from SpC-Wnt5a transgenic lungs responded abnormally to recombinant FGF10 supplied uniformly in the Matrigel with dilated branch tips that mimic the in vivo phenotype. In contrast, chemotaxis of transgenic epithelial explants towards a directional FGF10 source was inhibited. These suggest that over-expression of Wnt5a disrupts epithelial-response to FGF10. In conclusion, Wnt5a regulates SHH and FGF10 signaling during lung development.     

The role of Axin2 in calvarial morphogenesis and craniosynostosis

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of beta-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of beta-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis.     

A unique cell population in the mouse olfactory bulb displays nuclear beta-catenin signaling during development and olfactory sensory neuron regeneration

Olfactory sensory neurons (OSNs) in the nose form precise connections with neurons in the brain. However, mechanisms that account for the formation of such precise neuronal connections are incompletely understood. Recent studies implicate the function of Wnt growth factors in the formation of neuronal connections. To assess the role of Wnt signaling in the olfactory system, we examined the expression of beta-galactosidase (beta-gal) in the TOPGAL mouse, a transgenic strain in which beta-gal expression reports the activation of the canonical Wnt signaling pathway. In the olfactory epithelium, no beta-gal expression was observed at any developmental stages. In the olfactory bulb (OB), beta-gal expression was observed in a population of cells located at the interface of the olfactory nerve layer and the glomerular layer. The beta-gal expression was developmentally regulated with the peak expression occurring at late embryonic and early postnatal stages and a greatly reduced expression in adulthood. Further, forced OSN regeneration and subsequent reinnervation of the OB led to a reactivation of beta-gal expression in mature animals. The temporal coincidence between the peak of beta-gal expression and formation of OSN connections, together with the spatial localization of these cells, suggests a potential role of these cells and canonical Wnt signaling in the formation of OSN connections in the OB during development and regeneration.     

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (β-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2^lacZ reporter mice, we find Wnt/β-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/β-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.     

Brachy-syndactyly caused by loss of Sfrp2 function

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.     

Fuz regulates craniofacial development through tissue specific responses to signaling factors

The planar cell polarity effector gene Fuz regulates ciliogenesis and Fuz loss of function studies reveal an array of embryonic phenotypes. However, cilia defects can affect many signaling pathways and, in humans, cilia defects underlie several craniofacial anomalies. To address this, we analyzed the craniofacial phenotype and signaling responses of the Fuz(-/-) mice. We demonstrate a unique role for Fuz in regulating both Hedgehog (Hh) and Wnt/β-catenin signaling during craniofacial development. Fuz expression first appears in the dorsal tissues and later in ventral tissues and craniofacial regions during embryonic development coincident with cilia development. The Fuz(-/-) mice exhibit severe craniofacial deformities including anophthalmia, agenesis of the tongue and incisors, a hypoplastic mandible, cleft palate, ossification/skeletal defects and hyperplastic malformed Meckel's cartilage. Hh signaling is down-regulated in the Fuz null mice, while canonical Wnt signaling is up-regulated revealing the antagonistic relationship of these two pathways. Meckel's cartilage is expanded in the Fuz(-/-) mice due to increased cell proliferation associated with the up-regulation of Wnt canonical target genes and decreased non-canonical pathway genes. Interestingly, cilia development was decreased in the mandible mesenchyme of Fuz null mice, suggesting that cilia may antagonize Wnt signaling in this tissue. Furthermore, expression of Fuz decreased expression of Wnt pathway genes as well as a Wnt-dependent reporter. Finally, chromatin IP experiments demonstrate that β-catenin/TCF-binding directly regulates Fuz expression. These data demonstrate a new model for coordination of Hh and Wnt signaling and reveal a Fuz-dependent negative feedback loop controlling Wnt/β-catenin signaling.     

Investigating Tonic Wnt Signaling Throughout the Adult CNS and in the Hippocampal Neurogenic Niche of BatGal and Ins-TopGal Mice

Wnt/β-catenin signaling has a well-established role in the development of the central nervous system (CNS), and recent evidence is extending this role to include the regulation of adult hippocampal function, including neurogenesis within the dentate gyrus. While the neuroanatomical expression pattern of many canonical Wnt signaling components have been investigated, the sites of signal integration and functional downstream β-catenin activation remain comparatively less characterized in the adult CNS. Using two independent transgenic β-catenin-activated LacZ reporter mouse lines (BatGal and ins-TopGal), we demonstrate that Wnt/β-catenin signaling is active in discrete regions of the adult mouse CNS. Intriguingly, BatGal mice exhibit a broad pattern of reporter expression in the CNS, while expression in ins-TopGal mice is more restricted. Further investigation of these two lines reveals temporal differences in β-catenin-activated reporter expression during neurogenesis within the adult hippocampus. Ins-TopGal mice display peaks of Wnt/β-catenin-activated reporter expression during early and later stages of neurogenesis suggesting Wnt/β-catenin signaling plays an important role during both progenitor cell amplification as well as neuronal maturation, integration, and/or maintenance; however, results from BatGal mice are not as convincing. Thus our data using ins-TopGal mice are consistent with the idea that Wnt signaling plays diverse roles during adult hippocampal neurogenesis and support the idea that multiple transgenic reporter lines must be rigorously compared during scientific investigations.     

LEF1 identifies androgen-independent epithelium in the developing prostate

Lymphoid enhancer-binding factor (LEF)1 is a major mediator and a target in canonical Wnt/β-catenin pathway. Interactions between the androgen receptor (AR) and canonical Wnt pathways have been implicated in the development of the genitourinary organs. Here, we investigated the localization and role of LEF1-positive cells during development of the prostate gland in human and in the murine model. We show that during human prostate development, LEF1 is restricted to the basal epithelial layer of the urogenital sinus. During mouse development, Lef1 is also present in the urogenital mesenchyme in addition to the basal epithelial layer of the urogenital sinus. In the course of elongation and branching of the prostatic ducts, Lef1 is localized to the proliferating epithelium at the distal tips of the buds. Notably, during branching morphogenesis, domains of Lef1 and AR are mutually exclusive. We further employed the TOPGAL reporter strain to examine the dynamics of Wnt signaling in the context of prostate regression upon a 7-d treatment with a competitive AR inhibitor, bicalutamide. We found that Wnt/Lef1-positive basal cells are not dependent upon androgen for survival. Furthermore, upon bicalutamide treatment, Wnt/Lef1-positive basal progenitors repopulated the luminal compartment. We conclude that Wnt/Lef1 activity identifies an androgen-independent population of prostate progenitors, which is important for embryonic development and organ maintenance and regeneration in the adult.