Key Developmental Signaling Pathways Research Articles

STEM-14CELLULAR RESTING TRANSMEMBRANE POTENTIAL INSTRUCTS DIFFERENTIATION STATE OF GBM STEM-LIKE CELLS

INTRODUCTION: Glioblastoma (GBM) is the most common malignant brain tumor in adults with a poor prognosis despite maximal treatment. Resting membrane potential (RMP) has been shown to be an important regulator of the differentiation state of embryonic and somatic stem cells. We evaluated the relevance of RMP in GBM stem-like cells (GSC). METHODS: Whole cell patch clamp technique was used to determine the RMP. RESULTS: Undifferentiated GSC had a RMP of -5mV +/-4 (n = 33) compared to their differentiated counterparts, which had a mean RMP of -24mV +/-20 (n = 31, P < 0.001). To determine whether the cellular RMP played an instructive role in the differentiation state of GSC we used pharmacologic and genetic approaches to alter the potential. We added additional potassium chloride (KCl) to differentiated GCS in order to induce depolarization, and compared to osmotic controls using sodium chloride (NaCl). We found that GSC exposed to high KCl retained stem markers and sphere forming capacity compared to controls. We then examined whether hyperpolarization was sufficient to induce differentiation. We treated GSC with vacuolar ATPase inhibitors and salinomycin (potassium ionophore), which induced relative hyperpolarization of GSC and induction of differentiation. Transduction of GSC with inducible potassium ion channel led to hyperpolarization and differentiation. To identify potential mechanisms coupling electrical alterations to biochemical signal transduction pathways we performed extensive phospho-protein array studies comparing the undifferentiated GSC and cells hyperpolarized with omeprazole or salinomycin. Attenuation of key developmental and pro-growth signaling pathways was observed. CONCLUSIONS: Alteration of RMP may represent a new therapeutic strategy.

Hedgehog Signaling in the Maintenance of Cancer Stem Cells.

Cancer stem cells (CSCs) represent a rare population of cells with the capacity to self-renew and give rise to heterogeneous cell lineages within a tumour. Whilst the mechanisms underlying the regulation of CSCs are poorly defined, key developmental signaling pathways required for normal stem and progenitor functions have been strongly implicated. Hedgehog (Hh) signaling is an evolutionarily-conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various types of cancer and is implicated in multiple aspects of tumourigenesis, including the maintenance of CSCs. Here, we discuss the mounting evidence suggestive of Hh-driven CSCs in the context of haematological malignancies and solid tumours and the novel strategies that hold the potential to block many aspects of the transformation attributed to the CSC phenotype, including chemotherapeutic resistance, relapse and metastasis.

An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell—material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

5-HT6 Receptor: A New Player Controlling the Development of Neural Circuits.

5-HT6 receptor (5-HT6R) is a G protein-coupled receptor that has recently emerged as a new regulator of neural development. In addition to the canonical Gs adenylyl cyclase pathway, recent proteomics approaches reveal that 5-HT6R is able to engage key developmental signaling pathways controlling neuronal circuit formation, neuronal connectivity, and psychiatric-relevant behaviors. For example, at early stages of neuronal development, expression of 5-HT6R constitutively regulates the activity of the cyclin-dependent kinase (Cdk)5 and, through this mechanism, controls cellular processes involved in circuit formation, including neuronal migration and neurite outgrowth. In addition to the Cdk5 pathway, 5-HT6R modulates a variety of key developmental targets such as Fyn, Jab1, and mammalian target of rapamycin (mTOR). Engagement of developmental pathways through 5-HT6R pharmacological manipulation has led to interesting new therapeutic perspectives in the field of psychiatric-related disorders. Indeed, 5-HT6R blockade can rescue a pathological overactivation of the mTOR pathway induced by early life insults in rodents and normalizes the associated social and episodic memory deficits. Here, we review recent evidence supporting the notion that 5-HT6R is at the interface of key developmental signaling pathways and a novel actor in the orchestration of neural circuit formation.

Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

Engineered Biomaterials Control Differentiation and Proliferation of Human-Embryonic-Stem-Cell-Derived Cardiomyocytes via Timed Notch Activation

SummaryFor cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs) promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

Regulation of the seed to seedling developmental phase transition by the LAFL and VAL transcription factor networks.

In the seed, a fundamental transition between embryo and vegetative phases of plant development is coordinated by the interaction between the AFL and VAL sub-clades of the plant specific B3 domain transcription factor family. The AFL B3 factors together with LEC1-type HAP3 transcription factors promote embryo maturation; whereas the VAL B3 factors repress the LEC1/AFL (LAFL) network during seed germination. Recent advances reveal that genes in key developmental programs and hormone signaling pathways are downstream targets of the LAFL network highlighting the central role of the LAFL network in integration of intrinsic developmental and hormonal signals during plant development. The VAL B3 proteins are proposed to mediate repression by recruiting a histone deacetylase complex (HDAC) to LAFL genes that contain the Sph/RY cis-element recognized by AFL and VAL B3-DNA-binding domains. In addition to VAL B3 factors, epigenetic mechanisms are implicated in maintaining repression of LAFL network during vegetative development. WIREs Dev Biol 2014, 3:135–145. doi: 10.1002/wdev.126

Drosophila SETs Its Sights on Cancer: Trr/MLL3/4 COMPASS-Like Complexes in Development and Disease

Drosophila SETs Its Sights on Cancer: Trr/MLL3/4 COMPASS-Like Complexes in Development and Disease

Cutaneous wound healing: recruiting developmental pathways for regeneration

Following a skin injury, the damaged tissue is repaired through the coordinated biological actions that constitute the cutaneous healing response. In mammals, repaired skin is not identical to intact uninjured skin, however, and this disparity may be caused by differences in the mechanisms that regulate postnatal cutaneous wound repair compared to embryonic skin development. Improving our understanding of the molecular pathways that are involved in these processes is essential to generate new therapies for wound healing complications. Here we focus on the roles of several key developmental signaling pathways (Wnt/β-catenin, TGF-β, Hedgehog, Notch) in mammalian cutaneous wound repair, and compare this to their function in skin development. We discuss the varying responses to cutaneous injury across the taxa, ranging from complete regeneration to scar tissue formation. Finally, we outline how research into the role of developmental pathways during skin repair has contributed to current wound therapies, and holds potential for the development of more effective treatments.

Activation of Wnt/β-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of ex vivo cerebellar slice culture and in vivo genetic manipulation to dysregulate its activity during late embryonic development. Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis.

CREB signalling in neural stem/progenitor cells: Recent developments and the implications for brain tumour biology

This paper discusses the evidence for the role of CREB in neural stem/progenitor cell (NSPC) function and oncogenesis and how these functions may be important for the development and growth of brain tumours. The cyclic-AMP response element binding (CREB) protein has many roles in neurons, ranging from neuronal survival to higher order brain functions such as memory and drug addiction behaviours. Recent studies have revealed that CREB also has a role in NSPC survival, differentiation and proliferation. Recent work has shown that over-expression of CREB in transgenic animals can impart oncogenic properties on cells in various tissues and that aberrant CREB expression is associated with tumours in patients. It is the central position of CREB, downstream of key developmental and growth signalling pathways, which give CREB the ability to influence a spectrum of cell activities, such as cell survival, growth and differentiation in both normal and cancer cells.

Medulloblastoma stem cells: where development and cancer cross pathways

Brain tumors are the leading cause of childhood cancer mortality, with medulloblastoma (MB) representing the most frequent malignant tumor. The recent molecular classification of MB has reconceptualized the heterogeneity that exists within pathological subtypes by giving context to the role of key developmental signaling pathways in MB pathogenesis. The identification of cancer stem cell (CSC) populations, termed brain tumor-initiating cells (BTICs), in MB has provided novel cellular targets for the study of these aberrantly activated signaling pathways, namely, Sonic hedgehog (Shh) and Wingless (Wnt), along with the identification of novel BTIC self-renewal pathways. In this review, we discuss recent evidence for the presence of a MB stem cell that drives tumorigenesis in this malignant childhood tumor. We focus on evidence from cerebellar development, the recent identification of BTICs, the presence of activated developmental signaling pathways in MB, the role of epigenetic stem cell regulatory mechanisms, and how these developmental and epigenetic pathways may be targeted for novel therapeutic options.

Differential Deployment of REST and CoREST Promotes Glial Subtype Specification and Oligodendrocyte Lineage Maturation

BackgroundThe repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master transcriptional regulator that binds to numerous genomic RE1 sites where it acts as a molecular scaffold for dynamic recruitment of modulatory and epigenetic cofactors, including corepressor for element-1-silencing transcription factor (CoREST). CoREST also acts as a hub for various cofactors that play important roles in epigenetic remodeling and transcriptional regulation. While REST can recruit CoREST to its macromolecular complex, CoREST complexes also function at genomic sites independently of REST. REST and CoREST perform a broad array of context-specific functions, which include repression of neuronal differentiation genes in neural stem cells (NSCs) and other non-neuronal cells as well as promotion of neurogenesis. Despite their involvement in multiple aspects of neuronal development, REST and CoREST are not believed to have any direct modulatory roles in glial cell maturation.Methodology/Principal FindingsWe challenged this view by performing the first study of REST and CoREST in NSC-mediated glial lineage specification and differentiation. Utilizing ChIP on chip (ChIP-chip) assays, we identified distinct but overlapping developmental stage-specific profiles for REST and CoREST target genes during astrocyte (AS) and oligodendrocyte (OL) lineage specification and OL lineage maturation and myelination, including many genes not previously implicated in glial cell biology or linked to REST and CoREST regulation. Amongst these factors are those implicated in macroglial (AS and OL) cell identity, maturation, and maintenance, such as members of key developmental signaling pathways and combinatorial transcription factor codes.Conclusions/SignificanceOur results imply that REST and CoREST modulate not only neuronal but also glial lineage elaboration. These factors may therefore mediate critical developmental processes including the coupling of neurogenesis and gliogenesis and neuronal-glial interactions that underlie synaptic and neural network plasticity and homeostasis in health and in specific neurological disease states.

Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1

Ovol2 belongs to the Ovo family of evolutionarily conserved zinc finger transcription factors that act downstream of key developmental signaling pathways including Wg/Wnt and BMP/TGF-beta. We previously reported Ovol2 expression in the basal layer of epidermis, where epidermal stem/progenitor cells reside. In this work, we use HaCaT human keratinocytes to investigate the cellular and molecular functions of Ovol2. We show that depletion of Ovol2 leads to transient cell expansion but a loss of cells with long term proliferation potential. Mathematical modeling and experimental findings suggest that both faster cycling and precocious withdrawal from the cell cycle underlie this phenotype. Ovol2 depletion also accelerates extracellular signal-induced terminal differentiation in two- and three-dimensional culture models. By chromatin immunoprecipitation, luciferase reporter, and functional rescue assays, we demonstrate that Ovol2 directly represses two critical downstream targets, c-Myc and Notch1, thereby suppressing keratinocyte transient proliferation and terminal differentiation, respectively. These findings shed light on how an epidermal cell maintains a proliferation-competent and differentiation-resistant state.

Prdm1a is necessary for posterior pharyngeal arch development in zebrafish

Multiple tissue interactions and signaling within the pharyngeal arches are required for development of the craniofacial skeleton. Here, we focus on the role of the transcription factor prdm1a in the differentiation of the posterior skeleton. prdm1a is expressed in the presumptive pharyngeal arch region and later in an endodermal pouch, the otic vesicle, and pharyngeal teeth. prdm1a mutants display a reduction in pharyngeal arch markers, a loss of posterior ceratobranchial cartilages, and a reduction in most neural crest-derived dermal bones. This is likely caused by a decrease in the number of proliferating cells but not an increase in cell death. Finally, a reduction in two key developmental signaling pathways, Fgf and retinoic acid, alters prdm1a expression, suggesting that prdm1a expression is mediated by these signaling pathways to pattern the posterior craniofacial skeleton. Together, these results indicate an essential role for prdm1a in the development of the zebrafish craniofacial skeleton.

Ovol1 represses its own transcription by competing with transcription activator c-Myb and by recruiting histone deacetylase activity

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-β/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter.

The mouse Ovol2 gene is required for cranial neural tube development

The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-β. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early–mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis.

Functions of heparan sulfate proteoglycans in cell signaling during development

Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan (GAG) chains. In vitro studies have demonstrated the roles of these molecules in many cellular functions, and recent in vivo studies have begun to clarify their essential functions in development. In particular, HSPGs play crucial roles in regulating key developmental signaling pathways, such as the Wnt, Hedgehog, transforming growth factor-beta, and fibroblast growth factor pathways. This review highlights recent findings regarding the functions of HSPGs in these signaling pathways during development.