Calcium signalling and cell-fate choice in B cells

Alterations in the cytosolic concentration of calcium ions (Ca2+) transmit information that is crucial for the development and function of B cells. Cytosolic Ca2+ concentration is determined by a balance of active transport and gradient-driven Ca2+ fluxes, both of which are subject to the influence of multiple receptors and environmental sensing pathways. Recent advances in genomics have allowed for the compilation of an increasingly comprehensive list of Ca2+ transporters and channels expressed by B cells. The increasing understanding of the function and regulation of these proteins has begun to shift the frontier of Ca2+ physiology in B cells from molecular analysis to determining how diverse inputs to cytosolic Ca2+ concentration are integrated in specific immunological contexts.     

Wnt2b/beta-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye

Wnt signaling orchestrates multiple aspects of central nervous system development, including cell proliferation and cell fate choices. In this study, we used gene transfer to activate or inhibit canonical Wnt signaling in vivo in the developing eye. We found that the expression of Wnt2b or constitutively active (CA) beta-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina was sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, led to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. We propose that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris. Because wingless (wg) plays a similar role in the induction of peripheral eye tissues of Drosophila, these findings indicate a possible conservation of the process that patterns the photoreceptive and support structures of the eye.     

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.     

Wnt5 signaling in vertebrate pancreas development

Background  

Signaling by the Wnt family of secreted glycoproteins through their receptors, the frizzled (Fz) family of seven-pass transmembrane proteins, is critical for numerous cell fate and tissue polarity decisions during development.     

The orientation of cell division influences cell-fate choice in the developing mammalian retina

Asymmetric segregation of cell-fate determinants during cell division plays an important part in generating cell diversity in invertebrates. We showed previously that cells in the neonatal rat retina divide at various orientations and that some dividing cells asymmetrically distribute the cell-fate determinant Numb to the two daughter cells. Here, we test the possibility that such asymmetric divisions contribute to retinal cell diversification. We have used long-term videomicroscopy of green-fluorescent-protein (GFP)-labeled retinal explants from neonatal rats to visualize the plane of cell division and follow the differentiation of the daughter cells. We found that cells that divided with a horizontal mitotic spindle, where both daughter cells should inherit Numb, tended to produce daughters that became the same cell type, whereas cells that divided with a vertical mitotic spindle, where only one daughter cell should inherit Numb, tended to produce daughters that became different. Moreover, overexpression of Numb in the dividing cells promoted the development of photoreceptor cells at the expense of interneurons and Müller glial cells. These findings indicate that the plane of cell division influences cell-fate choice in the neonatal rat retina and support the hypothesis that the asymmetric segregation of Numb normally influences some of these choices.     

Wnt signaling mediates regional specification in the vertebrate face

At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.     

The canonical Wnt pathway in embryonic axis polarity

The canonical Wnt pathway plays crucial roles in multiple developmental processes, including in axis specification. Throughout the animal kingdom, this pathway has been reported to drive patterning of axes as different as the animal-vegetal axis in echinoderms to the dorsal-ventral axis in vertebrates. Intriguingly enough, this pathway appears structurally and functionally well conserved during evolution. However, differences between these phyla are observed that explain how a same pathway can mediate establishment of two such apparently distinct axes. This review compares the axis specification processes used in two evolutionarily distant embryos, the sea urchin and Xenopus.     

Wnt4 participates in the formation of vertebrate neuromuscular junction

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4−/− NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.     

The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.     

Wnt11 promotes cardiomyocyte development by caspase-mediated suppression of canonical Wnt signals

Specification and early patterning of the vertebrate heart are dependent on both canonical and noncanonical wingless (Wnt) signal pathways. However, the impact of each Wnt pathway on the later stages of myocardial development and differentiation remains controversial. Here, we report that the components of each Wnt signal conduit are expressed in the developing and postnatal heart, yet canonical/β-catenin activity is restricted to nonmyocardial regions. Subsequently, we observed that noncanonical Wnt (Wnt11) enhanced myocyte differentiation while preventing stabilization of the β-catenin protein, suggesting active repression of canonical Wnt signals. Wnt11 stimulation was synonymous with activation of a caspase 3 signal cascade, while inhibition of caspase activity led to accumulation of β-catenin and a dramatic reduction in myocyte differentiation. Taken together, these results suggest that noncanonical Wnt signals promote myocyte maturation through caspase-mediated inhibition of β-catenin activity.     

Common activation of canonical Wnt signaling in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.     

Histology of the heterostracan dermal skeleton: Insight into the origin of the vertebrate mineralised skeleton

Living vertebrates are divided into those that possess a fully formed and fully mineralised skeleton (gnathostomes) versus those that possess only unmineralised cartilaginous rudiments (cyclostomes). As such, extinct phylogenetic intermediates of these living lineages afford unique insights into the evolutionary assembly of the vertebrate mineralised skeleton and its canonical tissue types. Extinct jawless and jawed fishes assigned to the gnathostome stem evidence the piecemeal assembly of skeletal systems, revealing that the dermal skeleton is the earliest manifestation of a homologous mineralised skeleton. Yet the nature of the primitive dermal skeleton, itself, is poorly understood. This is principally because previous histological studies of early vertebrates lacked a phylogenetic framework required to derive evolutionary hypotheses. Nowhere is this more apparent than within Heterostraci, a diverse clade of primitive jawless vertebrates. To this end, we surveyed the dermal skeletal histology of heterostracans, inferred the plesiomorphic heterostracan skeleton and, through histological comparison to other skeletonising vertebrate clades, deduced the ancestral nature of the vertebrate dermal skeleton. Heterostracans primitively possess a four‐layered skeleton, comprising a superficial layer of odontodes composed of dentine and enameloid; a compact layer of acellular parallel‐fibred bone containing a network of vascular canals that supply the pulp canals (L1); a trabecular layer consisting of intersecting radial walls composed of acellular parallel‐fibred bone, showing osteon‐like development (L2); and a basal layer of isopedin (L3). A three layered skeleton, equivalent to the superficial layer L2 and L3 and composed of enameloid, dentine and acellular bone, is possessed by the ancestor of heterostracans + jawed vertebrates. We conclude that an osteogenic component is plesiomorphic with respect to the vertebrate dermal skeleton. Consequently, we interpret the dermal skeleton of denticles in chondrichthyans and jawless thelodonts as independently and secondarily simplified. J. Morphol. 276:657–680, 2015. © 2015 The Authors Journal of Morphology Published by Wiley Periodicals, Inc.     

SALL4 is directly activated by TCF/LEF in the canonical Wnt signaling pathway

The SALL4 promoter has not yet been characterized. Animal studies showed that SALL4 is downstream of and interacts with TBX5 during limb and heart development, but a direct regulation of SALL4 by TBX5 has not been demonstrated. For other SAL genes, regulation within the Shh, Wnt, and Fgf pathways has been reported. Chicken csal1 expression can be activated by a combination of Fgf4 and Wnt3a or Wnt7a. Murine Sall1 enhances, but Xenopus Xsal2 represses, the canonical Wnt signaling. Here we describe the cloning and functional analysis of the SALL4 promoter. Within a minimal promoter region of 31bp, we identified a consensus TCF/LEF-binding site.The SALL4 promoter was strongly activated not only by LEF1 but also by TCF4E. Mutation of the TCF/LEF-binding site resulted in decreased promoter activation. Our results demonstrate for the first time the direct regulation of a SALL gene by the canonical Wnt signaling pathway.     

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.     

Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.