β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme.     

Notch signalling pathway in tooth development and adult dental cells

Notch signalling is a highly conserved intercellular signal transfer mechanism that includes canonical and non-canonical pathways. It regulates differentiation and proliferation of stem/progenitor cells by means of para-inducing effects. Expression and activation of Notch signalling factors (receptors and ligands) are critical not only for development of the dental germ but also for regeneration of injured tissue associated with mature teeth. Notch signalling plays key roles in differentiation of odontoblasts and osteoblasts, calcification of tooth hard tissue, formation of cusp patterns and generation of tooth roots. After tooth eruption, Notch signalling can also be triggered in dental stem cells of the pulp, where it induces them to differentiate into odontoblasts, thus generating fresh dentine tissue. Other signalling pathways, such as TGFβ, NF-κB, Wnt, Fgf and Shh also interact with Notch signalling during tooth development.     

The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways

The members of the transforming growth factor beta (TGFβ) family of cytokines, including bone morphogenetic proteins (BMP), play fundamental roles in development and tissue homeostasis. Hence, aberrant TGFβ/BMP signalling is associated with several human diseases such as fibrosis, bone and immune disorders, cancer progression and metastasis. Consequently, targeting TGFβ signalling for intervention potentially offers therapeutic opportunities against these diseases. Many investigations have focussed on understanding the molecular mechanisms underpinning the regulation of TGFβ signalling. One of the key areas has been to investigate the regulation of the protein components of the TGFβ/BMP signal transduction pathways by ubiquitylation and deubiquitylation. In the last 15 years, extensive research has led to the discovery and characterisation of several E3 ubiquitin ligases that influence the TGFβ pathway. However, the research on DUBs regulating the TGFβ pathway has received prominence only recently and is still an emerging field. This review will provide a concise summary of our current understanding of how DUBs regulate TGFβ signalling.     

Bmp,Fgf and Wnt signalling in programmed cell death and chondrogenesis during vertebrate limb development: the role of Dickkopf-1

Dickkopf-1 (Dkk-1) is a potent head inducer in Xenopus. This effect can be attributed to its capability to specifically inhibit Wnt/beta-catenin signalling. Recent data point to a crucial role for Dkk-1 in the control of programmed cell death during vertebrate limb development. In this paper, we present a comparative expression analysis of Dkk-1, Bmp-4 and Sox-9 as well as data on the regulation of Dkk-1 by Wnt. Finally, we summarize the current knowledge of its potential function in the developing limb and present a model how the interplay of the Bmp, Fgf and Wnt signalling pathways might differentially regulate programmed cell death versus chondrogenic differentiation in limb mesodermal cells.     

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP''s and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

miRNAs and morphogen gradients

Morphogens induce biological diversity by operating in a dose-dependent manner. Here we review recent evidences indicating that microRNAs (miRNAs) are ideally suited to serve the morphogen cause. miRNAs regulate the establishment of morphogen gradients, including TGFβ, Wnt and other growth factors by acting on their secretion, distribution and clearance. miRNA are also critical in receiving cells, establishing context-dependency and threshold responses. Moreover, miRNAs contributes to gene networks that transform the graded activity of a morphogen into robust cell fate decisions. Finally, we discuss in the perspective section the implication of the new ceRNA hypothesis for morphogen biology.     

Antagonistic crosstalk of Wnt/β-catenin/Bmp signaling within the Apical Ectodermal Ridge (AER) regulates interdigit formation

Digit and interdigit (D/ID) development is one of the important research fields in molecular developmental biology. Interdigital cell death (ICD) is a morphogenetic event which has been considered as an essential process for D/ID formation. Although some growth factors including Bmp and Fgf signaling can modulate ICD, growth factor crosstalk regulating ICD is poorly understood. Wnt canonical pathway and Bmp signal crosstalk has been considered as the essential growth factor crosstalk in organogenesis. To elucidate the crosstalk to regulate the D/ID formation, we analyzed conditional mutant mice with limb bud ectoderm expressing constitutively activated β-catenin signaling. We showed that modulation of Wnt/β-catenin signal in the limb ectoderm including the AER regulates ID apoptosis. We also demonstrated that Wnt/β-catenin signaling in the ectoderm can positively regulate Fgf8 possibly antagonizing the epithelial derived Bmp signaling. Human birth defects for digit abnormalities have been known to be affected by multiple parameters. Elucidation of the potential mechanisms underlying such D/ID development is an urgent medical issue to be solved. This work would be one of the first studies showing essential growth factor cascades in the D/ID formation.     

Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling

In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.     

A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.     

Altered signal transduction in Folr1-/- mouse embryo fibroblasts

Mice lacking the gene for Folr1 (folic acid receptor 1) have an NTD (neural tube defect) that is rescued by maternal folate supplementation. Primary cultures of MEFs (mouse embryonic fibroblasts) were established from these embryos and the effect on various signalling pathways examined. TGFβ1 (transforming growth factor β1) inhibited the proliferation of wild-type and Folr1-/- MEFs, and folate restriction, either in growth medium or through folate uptake, led to further inhibition of growth. This effect may be Smad-independent because reporter assays using the Smad-dependent reporter, p3TP-lux, revealed attenuation of TGFβ1/Smad signalling in Folr1-/- MEFs. Signalling through the canonical Wnt pathway, measured by Wnt-3a stimulated expression of the target gene, Axin2, demonstrated increased activity in Folr1-/- MEFs. Only minor changes in the expression of a panel of TGFβ (transforming growth factor β) and Wnt pathway-associated genes were revealed when Folr1-/- MEFs were compared with wild-type cells. These results demonstrate that under conditions of reduced folate (Folr-/-) signalling, pathways crucial for proper development of the neural tube are significantly altered.     

Dynamic Assignment and Maintenance of Positional Identity in the Ventral Neural Tube by the Morphogen Sonic Hedgehog

Morphogens are secreted signalling molecules that act in a graded manner to control the pattern of cellular differentiation in developing tissues. An example is Sonic hedgehog (Shh), which acts in several developing vertebrate tissues, including the central nervous system, to provide positional information during embryonic patterning. Here we address how Shh signalling assigns the positional identities of distinct neuronal subtype progenitors throughout the ventral neural tube. Assays of intracellular signal transduction and gene expression indicate that the duration as well as level of signalling is critical for morphogen interpretation. Progenitors of the ventral neuronal subtypes are established sequentially, with progressively more ventral identities requiring correspondingly higher levels and longer periods of Shh signalling. Moreover, cells remain sensitive to changes in Shh signalling for an extended time, reverting to antecedent identities if signalling levels fall below a threshold. Thus, the duration of signalling is important not only for the assignment but also for the refinement and maintenance of positional identity. Together the data suggest a dynamic model for ventral neural tube patterning in which positional information corresponds to the time integral of Shh signalling. This suggests an alternative to conventional models of morphogen action that rely solely on the level of signalling.     

WNTs in synapse formation and neuronal circuitry

Wnt proteins play important roles in wiring neural circuits. Wnts regulate many aspects of neural circuit generation through their receptors and distinct signalling pathways. In this review, we discuss recent findings on the functions of Wnts in various aspects of neural circuit formation, including neuronal polarity, axon guidance, synapse formation, and synaptic plasticity in vertebrate and invertebrate nervous systems.