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The presence of FGF2 signaling determines whether ??-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells
Abstract
Neural stem cells proliferate and maintain multipotency when cultured in the presence of FGF2, but subsequent lineage commitment by the cells is nevertheless influenced by the exposure to FGF2. Here we show that FGF2 effects on neural stem cells are mediated, in part, by beta-catenin. Conversely, the effects of beta-catenin in neural stem cells depend in part upon whether there is concurrent fibroblast growth factor (FGF) signaling. FGF2 increases beta-catenin signaling through several different mechanisms including increased expression of beta-catenin mRNA, increased nuclear translocation of beta-catenin, increased phosphorylation of GSK-3beta, and tyrosine phosphorylation of beta-catenin. Overexpression of beta-catenin in the presence of FGF2 helps to maintain neural progenitor cells in a proliferative state. However, overexpression of beta-catenin in the absence of FGF2 enhances neuronal differentiation. Further, chromatin immunoprecipitation (ChIP) assays demonstrate that both beta-catenin and Lef1 bind directly to the neurogenin promoter, and luciferase reporter assays demonstrate that beta-catenin is directly involved in the regulation of neurogenin 1 and possibly other proneural genes when neural stem cells are cultured in the presence of FGF2. We suggest that the balance between the mitogenic effects and the proneural effects of beta-catenin is determined by the presence of FGF signaling.
... Meanwhile, numerous recent studies have demonstrated the crosstalk between FGF and Wnt/β -catenin signaling. For example, β -catenin is activated by FGF signaling through the RAS/ERK or PI3K/AKT cascade, and acts downstream of these pathways in several stem cells or progenitor cells [27][28][29][30] . In addition, FGF can activate Wnt signaling by phosphorylation of the Wnt co-receptor LRP6 31,32 or inactivation of the Wnt inhibitor Dkk1 33 . ...
... For example, in the absence of bFGF, β -catenin is only located in the membrane with E-cadherin, whereas it accumulates and is stably translocated into the nucleus to activate downstream genes in the presence of bFGF 14,27 . In the presence of bFGF, β -catenin helps neural stem cells proliferate when it is overexpressed, while enhancing neuronal differentiation in the absence of bFGF 28 . Moreover, bFGF increases β -catenin signaling in neural progenitor cells via multiple mechanisms: increasing the level of β -catenin mRNA, nuclear translocation of β -catenin, and phosphorylation of GSK-3β 47 . ...
... These results suggest that bFGF signaling promotes the proliferation of chicken PGCs via the activation of β -catenin and its downstream signaling. Many studies have shown that β -catenin is a target gene of FGF signaling through RAS/ ERK or PI3K/AKT cascades [27][28][29][30] . Therefore, it is worthwhile studying which downstream signals are involved in their crosstalk in PGCs. ...
Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3'-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes.
... instance, in vitro studies showed that WNT/β-catenin, through transcriptional regulation of N-myc and Ngn1/2, promotes the differentiation of neural progenitors (Hirabayashi et al., 2004;Israsena et al., 2004;Kuwahara et al., 2010). Also, ablation of LRP6 (low-density lipoprotein receptor-related protein 6) in mice does not affect the proliferation of NSC but decreases neuronal differentiation (Zhou et al., 2006). ...
... Overall, the WNT pathway has multiple and critical roles in neurogenesis. Some of the findings are conflicting, particularly those related to NSCs' decision to proliferate or differentiate during neuronal development (Chenn and Walsh, 2002;Viti et al., 2003;Zechner et al., 2003;Israsena et al., 2004;Yu et al., 2006;Shin et al., 2014). Several factors could account for this: First, WNTs are secreted proteins that form gradients with thresholds to be attained to function. ...
Neurogenesis and tumorigenesis share signaling molecules/pathways involved in cell proliferation, differentiation, migration, and death. Self- renewal of neural stem cells is a tightly regulated process that secures the accuracy of cell division and eliminates cells that undergo mitotic errors. Abnormalities in the molecular mechanisms controlling this process can trigger aneuploidy and genome instability, leading to neoplastic transformation. Mutations that affect cell adhesion, polarity, or migration enhance the invasive potential and favor the progression of tumors. Here, we review recent evidence of the WNT pathway’s involvement in both neurogenesis and tumorigenesis and discuss the experimental progress on therapeutic opportunities targeting components of this pathway.
... Hence, it is generally thought that the primary role of canonical WNT signalling is to promote NPC self-renewal, a phenomenon that is also observed in other parts of the developing nervous system and is mediated by the transcriptional activity of bcatenin (Zechner et al, 2003;Gulacsi & Anderson, 2008;Draganova et al, 2015). However, in vitro studies have demonstrated that WNT/b-catenin, through transcriptional regulation of N-myc and the neurogenic transcription factors Ngn1/2, can also promote differentiation of NPCs (Hirabayashi et al, 2004;Israsena et al, 2004;Kuwahara et al, 2010). Wnt7a and Wnt7b promote NPC proliferation (Viti et al, 2003;Qu et al, 2013). ...
... This is particularly evident in b-catenin mutant mice, which display increased AP cell cycle exit and premature neurogenesis (Mutch et al, 2010;Draganova et al, 2015). Although some studies have shown that b-catenin can also promote neuronal differentiation of NPCs (Hirabayashi et al, 2004;Israsena et al, 2004;Kuwahara et al, 2010), these studies were mostly performed in vitro, and are not supported by in vivo evidence. More recently, electroporation of Wnt3a into the neocortex of developing mouse embryos increased both, proliferation of APs and differentiation of BPs, seemingly resolving the controversy and leading the authors to conclude that canonical WNT can indeed promote both processes in a cell type-specific manner (Munji et al, 2011). ...
The role of WNT/β-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/β-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.
... HCV infection upregulates the expression of microRNA-155 (miR-155), which directly restrains APC expression, one of the major negative regulators in the destruction complex to regulate cytoplasmic β-catenin levels [45]. Additionally, HCV infection increases epidermal growth factor receptor (EGFR) and fibroblast growth factor (FGF) signaling, both of which lead to the release of β-catenin from the β-catenin/E-cadherin complexes as a result of tyrosine phosphorylation of β-catenin at residue Y654 and the inactivation of GSK3β through stimulation of PI3K/Akt and Ras/Raf/MEK/ERK cascades [46,47]. ...
... Overexpression of LEF-1 and β-catenin downstream c-Myc and cyclin D1 [24][25][26] other Insertion of HBV gene into a LINE1 element produces an oncogenic HBV-LINE1 chimeric transcript, inducing the nuclear localization of β-catenin. [18,27] HCV core protein Elevated expression levels of Wnt ligands, FZD, and LRP5/6 receptors [33,34] Downregulated transcription of Wnt antagonists SFRP2 and DKK1 [35,36] Hypermethylation at the CDH1 promoter leading to the reduction of E-cadherin and dissociation of the β-catenin/E-cadherin complexes at the cell-cell adhesion sites [37] NS5A Combination and stabilization of β-catenin protein [39] Stimulation of PI3K/Akt pathway to further inactivate GSK3β [40][41][42] E2 Activation of SHP-2, promoting tyrosine dephosphorylation of parafibromin to bind and stabilize β-catenin in the nucleus [43,44] others Upregulation of miR-155 to restrain APC expression [45] Activation of EGFR and FGF signaling, leading to tyrosine phosphorylation of β-catenin at residue Y654 and its release from the β-catenin/E-cadherin complexes, as well as inactivation of GSK3β through PI3K/Akt and Ras/Raf/MEK/ERK cascades [46,47] ...
Liver cancer is among the leading global healthcare issues associated with high morbidity and mortality. Liver cancer consists of hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), hepatoblastoma (HB), and several other rare tumors. Progression has been witnessed in understanding the interactions between etiological as well as environmental factors and the host in the development of liver cancers. However, the pathogenesis remains poorly understood, hampering the design of rational strategies aiding in preventing liver cancers. Accumulating evidence demonstrates that aberrant activation of the Wnt/β-catenin signaling pathway plays an important role in the initiation and progression of HCC, CCA, and HB. Targeting Wnt/β-catenin signaling potentiates a novel avenue for liver cancer treatment, which may benefit from the development of numerous small-molecule inhibitors and biologic agents in this field. In this review, we discuss the interaction between various etiological factors and components of Wnt/β-catenin signaling early in the precancerous lesion and the acquired mechanisms to further enhance Wnt/β-catenin signaling to promote robust cancer formation at later stages. Additionally, we shed light on current relevant inhibitors tested in liver cancers and provide future perspectives for preclinical and clinical liver cancer studies.
... Many studies also point to somatic stem cells as major targets of FGF signaling in both tissue homeostasis and repair (25). FGFs appear to promote self-renewing proliferation and inhibit cellular senescence, further increasing the reliability of these factors (26)(27)(28). ...
There is a great need for studying mechanisms of hair follicle cycles and designing small molecular drugs, biologics, formulation, and surgical treatment for androgenic alopecia (AGA, a common form of hair recession). While minoxidil has served as a solution in the past, it is a vasodilator and can react heavily with alcohol. Furthermore, minoxidil must be used at night, as it requires 6 hours for the product to settle and perform its function. Therefore, alternative approaches are needed to treat AGA. Numerous clinical trials are studying the use of fibroblast growth factors (FGFs) for tissue regeneration applications. Additionally, FGFs have been proven to stimulate stem cell growth and tissue regeneration. In the present study, we tested the effects of four FGFs, namely FGF1 (aFGF), FGF2 (bFGF), FGF10 (KGF-2), and FGF21, as compared to the gold standard, minoxidil, on isolated primary mouse hair follicle dermal papilla (DP) cells. We also used plain cell culture media (without growth factors or serum) as a negative control. Cell proliferation and viability were measured using both a biochemical assay and fluorescent microscope examinations. Our results indicate that several FGFs can promote DP cell proliferation. Interestingly, our results show that minoxidil promotes DP cell proliferation in the absence of a vascular system. In addition, DP cells cultured in FGFs exhibited a more organized cytoskeleton as compared to the ones cultured with minoxidil, suggesting the potential advantages of FGFs in general cellular health. In that regard, we hypothesize that FGFs can stimulate hair growth and treat AGA.
... Israsena et al. report that FGF2 helps to maintain neural progenitor cells in a proliferative state [21]. On the other hand, NSCs isolated from neonatal rats, passaged as neurospheres and plated as single cells in media composed of 2% N2 and 5% FBS, differentiate equally into GFAP+ astrocytes and TUBB3+ neurons [22]. ...
In vitro cell culture is a routinely used method which is also applied for in vitro modeling of various neurological diseases. On the other hand, media used for cell culture are often not strictly standardized between laboratories, which hinders the comparison of the obtained results. Here, we compared the effects of homocysteine (Hcy), a molecule involved in neurodegeneration, on immature cells of the nervous system cultivated in basal medium or media supplemented by either fetal bovine serum or basic fibroblast growth factor. The number of cells in basal media supplemented with basic fibroblast growth factor (bFGF) was 2.5 times higher in comparison to the number of cells in basal media supplemented with fetal bovine serum (FBS). We also found that the neuron-specific β-3-tubulin protein expression dose dependently decreased with increasing Hcy exposure. Interestingly, bFGF exerts a protective effect on β-3-tubulin protein expression at a concentration of 1000 µM Hcy compared to FBS-treated neural stem cells on Day 7. Supplementation with bFGF increased SOX2 protein expression two-fold compared to FBS supplementation. GFAP protein expression increased five-fold on Day 3 in FBS-treated neural stem cells, whereas on Day 7, bFGF increased GFAP expression two-fold compared to FBS-treated neural stem cells. Here, we have clearly shown that the selection of culturing media significantly influences various cellular parameters, which, in turn, can lead to different conclusions in experiments based on in vitro models of pathological conditions.
... Together, these mechanisms enact a range of FGF-dependent cellular responses, enabling dual and context-dependent FGF roles in the progression from radial glial to intermediate progenitors. [66][67][68][69][70][71] For instance, in the mouse cortex, triple deletion of FGFR1/2/3 results in increased cell specification from radial glial progenitors, resulting in progenitor cell depletion and small cortex development. 67 Conversely, in the diencephalon, fgf8a mutant zebrafish fail to initiate or maintain dbx1b expression in dorsal habenular progenitors. ...
Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.
... Besides, many cellular processes, controlling cell proliferation, differentiation and migration, neural patterning and organogenesis, are also regulated by Wnt proteins during embryonic development [86]. Wnt signaling stimulates the Neurog2 expression and is a direct regulator of Atoh1 and Neurog1 [116][117][118][119]. Ectopic expression of Neurog1/2 is induced by components of Wnt signaling in the ventral telencephalon [116,117,119,120]. Wnt signaling regulates the Neurog2 expression only during the early cortical neurogenesis, thereby temporarily restricting the regulation of Neurog2 by Wnt [117,121]. ...
The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the circuits can cause neurodegenerative diseases such as Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Treatment of such neurodegenerative diseases caused by neuronal loss includes some strategies of cell therapy employing stem cells (such as neural progenitor cells (NPCs)) and gene therapy through cell fate conversion. In this report, we review how bHLH acts as a regulator in neuronal differentiation, reprogramming, and cell fate determination. Moreover, several different researchers are conducting studies to determine the importance of bHLH factors to direct neuronal and glial cell fate specification and differentiation. Therefore, we also investigated the limitations and future directions of conversion or transdifferentiation using bHLH factors.
... The in vitro treatment of rodent NPCs with varying doses of IL6 [87], IL1B [88], and palmitate [89,90] inhibits neurogenesis at higher doses, likely via STAT3 activation [87,90]. In addition to these, an array of other factors including hormones (leptin and insulin) [38], β-catenin, and growth factors (IGF1, FGF) influence neurogenesis by promoting NPC proliferation and determining neuronal versus glial fate [91][92][93]. ...
Maternal obesity results in programmed offspring hyperphagia and obesity. The increased offspring food intake is due in part to the preferential differentiation of hypothalamic neuroprogenitor cells (NPCs) to orexigenic (AgRP) vs. anorexigenic (POMC) neurons. The altered neurogenesis may involve hypothalamic bHLH (basic helix–loop–helix) neuroregulatory factors (Hes1, Mash1, and Ngn3). Whilst the underlying mechanism remains unclear, it is known that mitochondrial function is critical for neurogenesis and is impacted by proinflammatory cytokines such as TNFα. Obesity is associated with the activation of inflammation and oxidative stress pathways. In obese pregnancies, increased levels of TNFα are seen in maternal and cord blood, indicating increased fetal exposure. As TNFα influences neurogenesis and mitochondrial function, we tested the effects of TNFα and reactive oxidative species (ROS) hydrogen peroxide (H2O2) on hypothalamic NPC cultures from newborn mice. TNFα treatment impaired NPC mitochondrial function, increased ROS production and NPC proliferation, and decreased the protein expression of proneurogenic Mash1/Ngn3. Consistent with this, AgRP protein expression was increased and POMC was decreased. Notably, treatment with H2O2 produced similar effects as TNFα and also reduced the protein expression of antioxidant SIRT1. The inhibition of STAT3/NFκB prevented the effects of TNFα, suggesting that TNFα mediates its effects on NPCs via mitochondrial-induced oxidative stress that involves both signaling pathways.
... The fate of stem cells is regulated by a variety of signaling pathways, including Wnt, Hippo, MAPK pathways, etc. The balance of NSCs is regulated by WNT, Notch, FGF, and BMP signaling cascades (14,15). In this study, we explored which signaling pathway is affected by SPIO and SMF treatment. ...
Background: Due to the excellent reliable traceability and superparamagnetic properties, superparamagnetic iron oxide nanoparticles (SPIOs) are widely used for the applications in the field of biomedicine, including tissue engineering and regenerative medicine. However, the regulation of SPIOs on the gene expressions in the stem cells is not clear.
Methods: In this study, by RNA-Seq analysis, we analyzed the gene expression pattern in the neural stem cells (NSCs) treated with SPIOs in the presence or absence of static magnetic field (SMF).
Results: It was found that SPIOs with SMF regulated more gene expression in NSCs, while most of these genes have been previously reported to play a crucial role in NSCs fate decision.
Conclusions: Our findings reveal the ability of SPIOs and SMF in the regulation of gene expression in NSCs, which may provide an experimental basis for its applications.
... FGF2 is known to play an important role in neurogenesis and its temporospatial expression corresponds to areas of neurogenesis in the developing CNS [52,53]. During development, and in vitro, FGF2 is a major NPC mitogen that stimulates neural progenitor cell proliferation and regulates neural cell differentiation [54][55][56]. This is underscored by the observation that FGF2 deficient mice display impaired neuronal proliferation and differentiation [57,58]. ...
Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphami-dase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.
... For example, Wnt-mediated upregulation of Empty Spiracles Homeobox 2 (Emx2) expression promotes stem cell proliferation , while Wnt-mediated upregulation of Paired Box 6 (Pax6), Ngn1/Ngn2, and N-Myc expression stimulates neuronal differentiation (Gunhaga et al., 2003;Hirabayashi et al., 2004;Kuwahara et al., 2010). Interestingly, the regulation of this switch in Wnt function may be mediated by Fibroblast Growth Factor 2 (FGF2) presence (which promotes proliferation) or absence (which promotes differentiation) (Israsena et al., 2004). In the chick developing brain, N-Myc represses Notch signaling to promote neuronal differentiation, indicating that Wnt additionally promotes neurogenesis through N-Myc-mediated Notch repression (Zinin et al., 2014). ...
Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that post-translationally modifies proteins via poly (ADP-ribosylation) (PARylation). PARP1 serves various functions, including DNA damage repair, regulation of cell death pathways, chromatin modification, RNA processing, and transcriptional regulation. Accordingly, mutations in Parp1 or Adprhl2 (encoding the protein ADP-ribosylhydrolase 3, which removes PAR polymers) cause intellectual disability, ataxia, episodic psychosis, neurodegeneration, and developmental delay. Altered PARP1 expression is also associated with numerous neurodegenerative and neuroimmune disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, rheumatoid arthritis, major depressive disorder, and epilepsy. Despite ubiquitous expression and an apparent connection with brain disorders, PARP1's role in neurodevelopment has not been widely studied. Our lab has recently uncovered a novel interaction between PARP1 and the receptor tyrosine kinase ErbB4, which binds its ligand NRG1 to mediate numerous functions during neurodevelopment, including radial migration of excitatory neurons, tangential migration of inhibitory neurons, synaptogenesis, and differentiation. Additionally, ErbB4 has multiple splice forms that confer different signaling modalities. Specifically, the ErbB4-juxtamembrane (JM)-a isoform is cleavable via the enzymes tumor necrosis factor-alpha (TACE) and presenilin/gamma-secretase. Upon NRG1 binding and ErbB4-JMa cleavage, the ErbB4 intracellular domain (E4ICD) is released, which regulates transcription through direct promoter binding. Previous findings have shown that E4ICD complexes with co-factors to repress gliogenesis during early development. Due to PARP1's prominent roles in chromatin modification and transcriptional control, this begs the question as to whether PARP1 is likewise regulating glial gene expression via E4ICD. The aims of this dissertation are two-fold: 1) investigate the role of PARP1 in regulating astrocytic gene expression via E4ICD and 2) further characterize the effect of PARP1 loss on brain development. To explore the role of PARP1-E4ICD in the regulation of astrogenesis, I utilized mouse primary embryonic neural precursor cell (NPC) cultures and transgenic mice with a germline knockout of PARP1, ErbB4, or ErbB4-JMa. I found that NRG1-mediated repression of GFAP expression upon FGF removal from NPC cultures was dependent upon the presence of PARP1, ErbB4, and ErbB4-JMa. Additionally, I showed that PARP1 KO and ErbB4 KO mice overexpress GFAP at birth, indicating the importance of both proteins in vivo. To investigate the effect of PARP1 loss on neurodevelopment more broadly, I analyzed the brain and cortical size of PARP1 KO mice at birth, finding a reduction in brain weight relative to body size, which is associated with a thinner cortex and a reduced cortical surface area. Furthermore, I discovered that PARP1 loss alters early-born neuron migration and increases the density of deeper-layer neurons. To investigate changes in gene expression associated with these findings, I performed RNA-sequencing of the embryonic PARP1 KO cortex. I found that PARP1 loss increases the expression of genes involved in neuronal migration and adhesion, including Reln, which encodes the glycoprotein Reelin. Accordingly, my findings indicate that PARP1 loss increases the abundance of Reelin-expressing cells in the developing (E15.5) and adolescent (P5) mouse brain. I further demonstrated that PARP1 loss, inhibition, or acute knockdown increases Cajal-Retzius cell abundance in vitro, suggesting PARP1 regulates Cajal-Retzius cell development via a cell-autonomous mechanism. Finally, atomic force microscopy showed that NPCs isolated from the PARP1 KO cortex adhere more strongly to the cell adhesion molecule N-cadherin, likely due to excess Reelin. Overall, these findings demonstrate that PARP1 regulates astrogenesis, Cajal-Retzius cell development, and cell adhesion in the developing brain.
... Hence, signaling pathways control neuron fate and regulation, and this activity is achieved through their regulatory interactions to decrease or enhance an activity. The Wnt/β-catenin and FGF pathways regulate not only neuronal stem cell differentiation but also proliferation, and it is known that the crosstalk and the balance between them induce the growth and the development of adult brain, and the appropriate segregation of neuronal populations [50,51]. Here, upon TCQA treatment, Wnt-and FGF-related genes were upregulated in seven days treated hAECs compared with D0 control. ...
Over the past years, Human Amnion Epithelial Cells (hAECs), a placental stem cell, are gaining higher attention from the scientific community as they showed several advantages over other types of stem cells, including availability, easy accessibility, reduced rejection rate, non-tumorigenicity, and minimal legal constraint. Recently, natural compounds are used to stimulate stem cell differentiation and proliferation and to enhance their disease-treating potential. A polyphenolic compound 3,4,5-Tri- O -Caffeoylquinic Acid (TCQA) has been previously reported to induce human neural stem cell differentiation and may affect melanocyte stem cell differentiation as well. In this study, TCQA was tested on 3D cultured hAECs after seven days of treatment, and then, microarray gene expression profiling was conducted of TCQA-treated and untreated control cells on day 0 and day 7. Analyses revealed that TCQA treatment significantly enriched pigment and neural cells sets; besides, genes linked with neurogenesis, oxidation–reduction process, epidermal development, and metabolism were positively regulated. Interestingly, TCQA stimulated cell cycle arrest-related pathways and differentiation signaling. On the other hand, TCQA decreased interleukins and cytokines expression and this due to its anti-inflammatory properties as a polyphenolic compound. Results were validated to highlight the main activities of TCQA on hAECs, including differentiation, cell cycle arrest, and anti-inflammatory. This study highlights the important role of hAECs in regenerative medicine and the use of natural compounds to regulate their fate.
... Hence, signaling pathways control neuron fate and regulation, and this activity is achieved through their regulatory interactions to decrease or enhance an activity. The Wnt/β-catenin and FGF pathways regulate not only neuronal stem cell differentiation but also proliferation, and it is known that the crosstalk and the balance between them induce the growth and the development of adult brain, and the appropriate segregation of neuronal populations [50,51]. Here, upon TCQA treatment, Wnt-and FGF-related genes were upregulated in seven days treated hAECs compared with D0 control. ...
Background: Rosmarinus officinalis L.is traditionally used as an infusion in the treatment of several diseases and in particular against neuropsychiatric disorders, such as anxiety and depression. It was established that rosemary extracts show an antidepressant effect on animal models. However, to the best of our knowledge, there is no scientific data that highlights the therapeutic effects of rosemary intake on human mental health.
Aim:This study investigated whether rosemary tea consumption affects the plasma levels of anxiety and depression biomarkers in healthy volunteers.
Methods:Twenty-two healthy volunteers aged between 20 and 50 years old consumed rosemary tea prepared from 5 g of dried rosemary in 100 mL boiled water once a day for 10 days. Plasma concentrations of Brain-Derived Neurotrophic Factor (BDNF), Interleukine-6 (IL-6), Interleukine-4 (IL-4), Tumor Necrosis Factor- alpha (TNF-α), Interferon-gamma (IFNϒ), and cortisol were measured by enzyme-linked immunosorbent assay using commercial ELISA kits (R&D systems) before rosemary consumption and at the end of the experiment.
Results:Rosemary tea consumption significantly increased the concentration of BDNF([BDNF]D0 = 22363.86 ± 12987.66 pg/mL, [BDNF]D10 = 41803.64 ± 28109.19, p = 0.006) and TNF-α([TNF-α] D0 = 39.49 ± 14.44 pg/mL, [TNF-α] D10 = 56.24 ± 39.01, p = 0.016). However, a slight variation that was statistically non-significant in INFϒ, cortisol, IL-4, IL-6 levels and in the ratio IL-4/INFϒ was observed (p > 0.05).
Conclusion:Our findings highlight the promising anxiolytic and/or antidepressant effects of rosemary tea consumption in healthy volunteers since it increases the level of the most reliable depression biomarker BDNF. However, more powerful studies with larger sample size, carefully-chosen target population and, an extended intervention period are required.
Keywords: Rosemary teaBDNFanxietydepressioncytokinecortisol
... In the present study, we found that the cultured DRG-SGCs did not express sensory neuron markers Ngn1 (Neurogenin1), Ngn2 (Neurogenin2), Brn3a, Isl1, TrkA, TrkB, and TrkC (Fig. S3). We then treated these cells with CHIR99021, SU5402 and RO4929097 to activate Wnt pathway, and inhibit FGF and Notch pathway, a protocol that was reported to promote the production of sensory neurons [28,[31][32][33][34][35][36]. After 7 d, qRT-PCR results showed an increased expression of Brn3a, Runx3, TrkA, TrkB, and TrkC, the sensory neuron-related genes. ...
Dorsal root ganglia (DRG) sensory neurons can transmit information about noxious stimulus to cerebral cortex via spinal cord, and play an important role in the pain pathway. Alterations of the pain pathway lead to CIPA (congenital insensitivity to pain with anhidrosis) or chronic pain. Accumulating evidence demonstrates that nerve damage leads to the regeneration of neurons in DRG, which may contribute to pain modulation in feedback. Therefore, exploring the regeneration process of DRG neurons would provide a new understanding to the persistent pathological stimulation and contribute to reshape the somatosensory function. It has been reported that a subpopulation of satellite glial cells (SGCs) express Nestin and p75, and could differentiate into glial cells and neurons, suggesting that SGCs may have differentiation plasticity. Our results in the present study show that DRG-derived SGCs (DRG-SGCs) highly express neural crest cell markers Nestin, Sox2, Sox10, and p75, and differentiate into nociceptive sensory neurons in the presence of histone deacetylase inhibitor VPA, Wnt pathway activator CHIR99021, Notch pathway inhibitor RO4929097, and FGF pathway inhibitor SU5402. The nociceptive sensory neurons express multiple functionally-related genes (SCN9A, SCN10A, SP, Trpv1, and TrpA1) and are able to generate action potentials and voltage-gated Na⁺ currents. Moreover, we found that these cells exhibited rapid calcium transients in response to capsaicin through binding to the Trpv1 vanilloid receptor, confirming that the DRG-SGC-derived cells are nociceptive sensory neurons. Further, we show that Wnt signaling promotes the differentiation of DRG-SGCs into nociceptive sensory neurons by regulating the expression of specific transcription factor Runx1, while Notch and FGF signaling pathways are involved in the expression of SCN9A. These results demonstrate that DRG-SGCs have stem cell characteristics and can efficiently differentiate into functional nociceptive sensory neurons, shedding light on the clinical treatment of sensory neuron-related diseases.
Graphical Abstract
... On the contrary, upon differentiation β-catenin binds a TCF/LEF binding site in the Ngn2 gene promoter in association with active chromatin markers AcH3 and H3K4me3 (Qu et al., 2013). Ngn2 is a proneural basic helixloop-helix (bHLH) transcription factor that promotes neuronal differentiation (Israsena et al., 2004). Prior to differentiation, no β-catenin was detected in the TCF/LEF binding site of the Ngn2 promoter in NPCs. ...
Neurogenesis persists during adulthood in the dentate gyrus of the hippocampus. Signals provided by the local hippocampal microenvironment support neural stem cell proliferation, differentiation, and maturation of newborn neurons into functional dentate granule cells, that integrate into the neural circuit and contribute to hippocampal function. Increasing evidence indicates that Wnt signaling regulates multiple aspects of adult hippocampal neurogenesis. Wnt ligands bind to Frizzled receptors and co-receptors to activate the canonical Wnt/β-catenin signaling pathway, or the non-canonical β-catenin-independent signaling cascades Wnt/Ca²⁺ and Wnt/planar cell polarity. Here, we summarize current knowledge on the roles of Wnt signaling components including ligands, receptors/co-receptors and soluble modulators in adult hippocampal neurogenesis. Also, we review the data suggesting distinctive roles for canonical and non-canonical Wnt signaling cascades in regulating different stages of neurogenesis. Finally, we discuss the evidence linking the dysfunction of Wnt signaling to the decline of neurogenesis observed in aging and Alzheimer’s disease.
... For growth factor stimulation experiments in PC12/TrkB cells and primary mixed cortical neurons, growth factors BDNF (50 ng/mL) [23,24], FGF2 (50 ng/mL) [25], EGF (100 ng/mL) [26], IGF (100 ng/mL) [27,28], NGF (100 ng/ mL) [29], VEGF (100 ng/mL) [30], BMP4 (100 ng/mL) [31], TGFβ (100 ng/mL) [32], Bicuculline (50 μM) [33], and KCl (55 mM) [34], all from Sigma, were acutely applied onto cells. Samples were collected at 2 h to assess c-Fos or c-Jun expression, and samples were collected at 12 and 24 h for p11 expression in PC12-TrkB cells and primary mixed cortical neurons, respectively. ...
Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed drugs for mood disorders. While the mechanism of SSRI action is still unknown, SSRIs are thought to exert therapeutic effects by elevating extracellular serotonin levels in the brain, and remodel the structural and functional alterations dysregulated during depression. To determine their precise mode of action, we tested whether such neuroadaptive processes are modulated by regulation of specific gene expression programs. Here we identify a transcriptional program regulated by activator protein-1 (AP-1) complex, formed by c-Fos and c-Jun that is selectively activated prior to the onset of the chronic SSRI response. The AP-1 transcriptional program modulates the expression of key neuronal remodeling genes, including S100a10 (p11), linking neuronal plasticity to the antidepressant response. We find that AP-1 function is required for the antidepressant effect in vivo. Furthermore, we demonstrate how neurochemical pathways of BDNF and FGF2, through the MAPK, PI3K, and JNK cascades, regulate AP-1 function to mediate the beneficial effects of the antidepressant response. Here we put forth a sequential molecular network to track the antidepressant response and provide a new avenue that could be used to accelerate or potentiate antidepressant responses by triggering neuroplasticity.
... FGF (acidic) phosphorylates and deactivates glycogen synthase kinase-3β leading to the accumulation of β-catenin (54). β-catenin also promote proliferation of NSCs along with higher levels of bFGF, although, conditions like an early cell cycle exit may induce neuronal differentiation (55). Thus, β-catenins turns out to be neurogenic by activating basic helix-loop-helix (bHLH) family of transcription factors in stem cells (56). ...
The promise of engineering specific cell types from stem cells and rebuilding damaged or diseased tissues has fascinated stem cell researchers and clinicians over last few decades. Mesenchymal Stem Cells (MSCs) have the potential to differentiate into non-mesodermal cells, particularly neural-lineage, consisting of neurons and glia. These multipotent adult stem cells can be used for implementing clinical trials in neural repair. Ongoing research identifies several molecular mechanisms involved in the speciation of neuroglia, which are tightly regulated and interconnected by various components of cell signalling machinery. Growing MSCs with multiple inducers in culture media will initiate changes on intricately interlinked cell signalling pathways and processes. Net result of these signal flow on cellular architecture is also dependent on the type of ligands and stem cells investigated in vitro. However, our understanding about this dynamic signalling machinery is limited and confounding, especially with spheroid structures, neurospheres and organoids. Therefore, the results for differentiating neurons and glia in vitro have been inconclusive, so far. Added to this complication, we have no convincing evidence about the electrical conductivity and functionality status generated in differentiating neurons and glia. This review has taken a step forward to tailor the information on differentiating neuroglia with the common methodologies, in practice.
... Many extrinsic and intrinsic factors regulate the neurogenic potential of neocortical NPCs. For instance, Wnt signaling instructs neuronal differentiation of NPCs during the neurogenic phase (Hirabayashi et al., 2004;Israsena, Hu, Fu, Kan, & Kessler, 2004;Kuwahara et al., 2010;Lyu, Yamamoto, & Lu, 2008;Munji, Choe, Li, Siegenthaler, & Pleasure, 2011;Zhou, Borello, Rubenstein, & Pleasure, 2006), whereas signaling by bone morphogenetic protein (BMP) and members of the leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) family of proteins induces ...
Neural progenitor cells (NPCs, also known as radial glial progenitors) produce neurons and then glial cells such as astrocytes during development of the mouse neocortex. Given that this sequential generation of neural cells is critical for proper brain formation, the neurogenic potential of NPCs must be precisely controlled. Here we show that the transcription factor Plag1 plays an important role in the regulation of neurogenic potential in mouse neocortical NPCs. We found that Hmga2, a key neurogenic factor in neocortical NPCs, induces expression of the Plag1 gene. Analysis of the effects of overexpression or knockdown of Plag1 indicated that Plag1 promotes the production of neurons at the expense of astrocyte production in embryonic neocortical cultures. Furthermore, overexpression of Plag1 promoted and knockdown of Plag1 suppressed neuronal differentiation of neocortical NPCs in vivo. Transcriptomic analysis showed that Plag1 increases the expression of a set of neuronal genes in NPCs. Our results thus identify Plag1 as a regulator of neuronal gene expression and neuronal differentiation in NPCs of the developing mouse neocortex. This article is protected by copyright. All rights reserved.
... The decision to symmetrically maintain potency or differentiate is likely context-dependent and not completely cell intrinsic [74]. Indeed, opposite neurogenic outcomes have been previously described from in vivo and in vitro studies and could reflect a change in environmental cues: loss of cell polarity [75], mitogen exposure [76] and feedback mechanisms [77]. Conditionally deleting the Pten tumor suppressor gene in adult hematopoietic cells led to myeloproliferative disease within days and leukemias within weeks and also promoted hematopoietic stem cell (HSC) proliferation. ...
Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential β-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/β-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases β-catenin Kat3 coactivator usage and enhances β-catenin/CBP transcription at the expense of β-catenin/p300 transcription. Biased β-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced β-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/β-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of β-catenin Kat3 coactivator and the mode of stem cell division.
... To fully understand the heterogeneity of the neuromast domain, it is necessary to identify all of the transcriptional targets that are activated or repressed by FGF and Wnt regulation during neuromast proliferation in zebrafish. Recently, many studies have shown that bFGF activates several intracellular pathways, including the RAS/ERK and PI3K/AKT cascades [47][48][49] . Therefore, defining which signals act downstream of the Wnt-FGF interactions during proliferation in neuromasts is of great interest. ...
Studying sensory organs on the skin of zebrafish is revealing details of molecular signaling pathways that may be relevant to our own sensory systems, especially the hair cells of the ear. These cells have fine hair-like structures that move in response to sound waves and help generate electrical signals to the brain that result in perception of sound. Huawei Li and colleagues at Fudan University, Shanghai, China, studied the roles of two well-known cellular signaling pathways in regulating the proliferation of similar sensory hair cells in zebrafish, a commonly used model organism. These pathways involve cell surface proteins that interact with small extracellular molecules to stimulate molecular changes within cells. Learning how the pathways control hair cell generation and multiplication may assist modification of similar systems in humans to study and treat hearing loss.
... Cohen et al. showed that FGF signalling in hESC induced early neural specification (Cohen et al., 2010). Israsena et al., reported that cells cultured in the presence of FGF2 aided in maintaining the cells at the neural progenitor stage (Israsena et al., 2004) and Hu et al. described increased numbers of PAX6 cells when NBM was supplemented with FGF however their data was cell line dependant (Hu et al., 2010). Increased heterogeneity and rosette malformation has been reported during supplementation of neural stem cell cultures with FGF2 and EGF (Denham and Dottori, 2009;Elkabetz et al., 2008). ...
Production of 3-dimensional neural progenitor cultures from human pluripotent stem cells offers the potential to generate large numbers of cells. We utilised our nanobridge system to generate 3D hPSC aggregates for differentiation towards the neural lineage, and investigate the ability to passage aggregates while maintaining cells at a stem/progenitor stage. Over 38 days, aggregate cultures exhibited upregulation and maintenance of neural-associated markers and demonstrated up to 10 fold increase in cell number. Aggregates undergoing neural induction in the presence or absence of nanobridges demonstrated no differences in marker expression, proliferation or viability. However, aggregates formed without nanobridges were statistically significantly fewer and smaller by passage 3. Organoids, cultured from aggregates, and treated with retinoic acid or rock inhibitor demonstrated terminal differentiation as assessed by immunohistochemistry. These data demonstrate that nanobridge 3D hPSC can differentiate to neural stem/progenitor cells, and be maintained at this stage through serial passaging and expansion.
... The developmental switch from neurogenesis to gliogenesis during CNS development is promoted by many factors, including JAK/STAT signaling (Bonni et al., 1997;He et al., 2005;, Notch signaling (Namihira et al., 2009), coordination of Sox9 and Nfia (Kang et al., 2012;Stolt et al., 2003), and microRNAs (Li et al., 2016;Meares et al., 2018;Zhao et al., 2015;Zheng, Li, Huang, & Qiu, 2012;Zheng, Li, Zhu, Zhu, & Qiu, 2010). WNT/βcatenin signaling was previously shown to regulate the balance between the proliferation and differentiation of NPCs during neurogenesis (Ille et al., 2007;Israsena, Hu, Fu, Kan, & Kessler, 2004;Zechner et al., 2003). However, whether WNT/β-catenin signaling is involved in the precision control of the initiation of gliogenesis remains elusive. ...
Neural progenitor cells (NPCs) are sequentially specified into neurons and glia during the development of central nervous system. WNT/β‐catenin signaling is known to regulate the balance between the proliferation and differentiation of NPCs during neurogenesis. However, the function of WNT/β‐catenin signaling during gliogenesis remains poorly defined. Here, we report that activation of WNT/β‐catenin signaling disrupts astrogliogenesis in the developing spinal cord. Conversely, inhibition of WNT/β‐catenin signaling leads to precocious astrogliogenesis. Further analysis reveals that activation of WNT/β‐catenin pathway results in a dramatic increase of neurogenin 2 (Ngn2) expression in transgenic mice, and knockdown of Ngn2 expression in neural precursor cells can reverse the inhibitory effect of WNT/β‐catenin on astrocytic differentiation. Moreover, Ngn2 can directly bind to the promoters of several astrocyte specific genes and suppress their expression independent of STATs activity. Together, our studies provide the first in vivo evidence that WNT/β‐catenin signaling inhibits early astrogliogenesis via an Ngn2‐dependent transcriptional repression mechanism.
... This suggests that the activation of the Wnt/β-catenin pathway may also contribute to the neuroprotective mechanism of CPE-NFα1-N in embryonic neurons (Qin et al., our unpublished data). Additionally, β-catenin has been reported to mediate neuronal proliferation and differentiation of stem cells (Israsena et al., 2004). Thus, CPE-NFα1-N may also regulate these processes during development via the Wnt signaling pathway. ...
Carboxypeptidase E, also known as neurotrophic factor-α1 (CPE-NFα1), was first discovered as an exopeptidase and is known to work by cleaving C-terminal basic amino acids from prohormone intermediates to produce mature peptide hormones and neuropeptides in the endocrine and central nervous systems, respectively. CPE-NFα1 also plays a critical role in prohormone sorting and secretory vesicle transportation. Recently, emerging studies have indicated that CPE-NFα1 exerts multiple non-enzymatic physiological roles in maintaining normal central nervous system function and in neurodevelopment. This includes potent neuroprotective and anti-depressant activities, as well as stem cell differentiation functions. In addition, N-terminal truncated variants of CPE-NFα1 have been identified to regulate expression of important neurodevelopmental genes. This mini-review summarizes recent advances in understanding the mechanisms underlying CPE-NFα1’s function in neuroprotection during stress and aspects of neurodevelopment.
... In the developing brain, Wnt/βcatenin signaling promotes NSC self-renewal and proliferation (Chenn and Walsh 2002;Lie et al. 2005;Kalani et al. 2008) and regulates the anterior-posterior patterning of the nervous system (Ciani and Salinas 2005). During later stages of neurogenesis, Wnt/βcatenin signaling is required for specification of neuronal subtypes (Israsena et al. 2004;Kuwabara et al. 2009;Munji et al. 2011). Several studies have implicated dysregulated Wnt signaling in GBM despite the absence of genetic alterations within known pathway components such as APC or βcatenin (Reya and Clevers 2005). ...
Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/βcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/βcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnthi GBM cells (which exhibit high levels of βcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wntlo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/βcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.
... In the developing mouse neocortex, neural stem/progenitor cells (NPCs) undergo a progressive cell fate restriction that underlies the ordered production of various neuronal and glial cell types (Hirabayashi and Gotoh, 2010). During the neurogenic phase, neurogenic genes such as Neurog1 and Fezf2 are temporarily repressed but are poised for expression in response to differentiation-inducing cues that include Wnt signaling as well as inhibition of Notch or fibroblast growth factor (FGF) signaling (Hirabayashi et al., 2004;Paridaen and Huttner, 2014;Martynoga et al., 2012;Pierfelice et al., 2011;Israsena et al., 2004). PRC2 and PRC1 play pivotal roles in this suppression of premature differentiation of NPCs in the early neurogenic phase (Fasano et al., 2007;Fasano et al., 2009;Pereira et al., 2010). ...
Polycomb repressive complex (PRC) 1 maintains developmental genes in a poised state through monoubiquitination (Ub) of histone H2A. Although Ub-independent functions of PRC1 have also been suggested, it has remained unclear whether Ub-dependent and -independent functions of PRC1 operate differentially in a developmental context. Here, we show that the E3 ubiquitin ligase activity of Ring1B, a core component of PRC1, is necessary for the temporary repression of key neuronal genes in neurogenic (early-stage) neural stem or progenitor cells (NPCs) but is dispensable for the persistent repression of these genes associated with the loss of neurogenic potential in astrogliogenic (late-stage) NPCs. Our results also suggest that histone deacetylase (HDAC) activity of the NuRD/MBD3 complex and Phc2-dependent PRC1 clustering are necessary for the transition from the Ub-dependent to -independent function of PRC1. Together, these results indicate that Ub-independent mode of repression by PRC1 plays a key role in mammalian development during cell fate restriction.
... The role of β-catenin in regulating NPC self-renewal versus differentiation is context dependent, for instance, varying with growth factor signaling. [36] Future studies should explore the effects of material properties such as 2D versus 3D dimensionality to elucidate possible mechanisms underlying these two different roles for β-catenin and YAP signaling in the context of biomaterial culture platforms for NPCs. ...
Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use. Matrix remodeling of engineered 3D hydrogels is required for efficient differentiation and maturation of neural progenitor cells (NPCs) into neurons and astrocytes. Provided sufficient remodeling time, NPCs degrade the surrounding hydrogel material, form cell–cell contacts, and activate β‐catenin signaling, which maintains expression of Yes‐associated protein and primes NPCs for differentiation.
... We mapped the identified neuronal-promoting factors, Ezh2, Foxo1, Ngn1, onto the STRING database (Szklarczyk et al., 2015) and found that they directly interacted with b-catenin, a core factor that regulates neuronal differentiation and function (Hoogeboom et al., 2008;Otero et al., 2004;Wisniewska, 2013). Furthermore, b-catenin activates Ngn1 and Brn2 as downstream genes (Goodall et al., 2004;Israsena et al., 2004). We incorporated the observations in this study into a hypothetical gene interaction network, in which the Wnt/b-catenin pathway acts as a hub ( Figure S7). ...
Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.
... In vitro and in vivo studies demonstrate that the Wnt/ -catenin pathway regulates the proliferation and differentiation of neural progenitor cells [88]. Neuronal differentiation is induced by overexpression of -catenin or the pharmacological inhibition of GSK3 (the phosphorylating enzyme of -catenin) [89,90]. This pathway also promotes blood vessel formation during vascular development, as well as the vascular repair process after TBI [91]. ...
Objectives:
Xuefu Zhuyu decoction (XFZYD), a traditional Chinese medicine (TCM) formula, has been demonstrated to be effective for the treatment of traumatic brain injury (TBI). However, the underlying pharmacological mechanisms remain unclear. This study aims to explore the potential action mechanisms of XFZYD in the treatment of TBI and to elucidate the combination principle of this herbal formula.
Methods:
A network pharmacology approach including ADME (absorption, distribution, metabolism, and excretion) evaluation, target prediction, known therapeutic targets collection, network construction, and molecule docking was used in this study.
Results:
A total of 119 bioactive ingredients from XFZYD were predicted to act on 47 TBI associated specific proteins which intervened in several crucial pathological processes including apoptosis, inflammation, antioxidant, and axon genesis. Almost each of the bioactive ingredients targeted more than one protein. The molecular docking simulation showed that 91 pairs of chemical components and candidate targets had strong binding efficiencies. The "Jun", "Chen", and "Zuo-Shi" herbs from XFZYD triggered their specific targets regulation, respectively.
Conclusion:
Our work successfully illuminates the "multicompounds, multitargets" therapeutic action of XFZYD in the treatment of TBI by network pharmacology with molecule docking method. The present work may provide valuable evidence for further clinical application of XFZYD as therapeutic strategy for TBI treatment.
... before differentiation, which is the self-renewal marker of NSCs, whereas there was no expression of EGFP for other differentiation markers, indicating the stemness characteristics of ReNcell VM in the miniaturized 3D culture on the chip, which is in accordance with previous studies [38,39,42]. NSCs maintain their pluripotency in the presence of EGF and bFGF signaling (supplemented in the growth medium) [59], and their removal encourages NSCs to differentiate into various lineages. In our study, removal of growth factors induced EGFP expression for MBP, GFAP and synapsin1, indicating the early stage differentiation of ReNcell VM into glial and neuronal lineages. ...
Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1mg/mL growth factor reduced (GFR) Matrigel with 25mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system.
(https://authors.elsevier.com/c/1Xb0B_L4pkbuR)
... The b-catenin molecule then enters the nucleus and forms a complex with T-cell factors (TCFs) and lymphocyte enhance factors (LEFs) [29]. b-Catenin-TCF-LEF complex increases expression of basic helix-loop-helix (bHLH) proteins family, neurogenin 1 (Ngn 1) and Ngn 2, by activating the promoter of Ngn 1 and Ngn 2, and ultimately increase neurogenesis [30][31][32][33]. When the Wnt signalling pathway is not active, b-catenin is phosphorylated by the axin-APC-GSK3b-conductin complex and degrades by the ubiquitin-proteasome system [31,33]. ...
CD133 (prominin-1), a pentaspan membrane glycoprotein, is one of the most well-characterized biomarkers used for the isolation of cancer stem cells (CSCs). The presence of CSCs is one of the main causes of tumor reversal and resilience. Accumulating evidence has shown that CD133 might be responsible for CSCs tumorigenesis, metastasis, and chemoresistance. It is now understood that CD133 interacts with the Wnt/β-catenin and PI3K-Akt signaling pathways. Moreover, CD133 can upregulate the expression of FLICE-like inhibitory protein (FLIP) in CD133-positive cells, inhibiting apoptosis. Additionally, CD133 can increase angiogenesis by activating the Wnt signaling pathway and increasing the expression of vascular endothelial growth factor-A (VEGF-A) and interleukin-8. Therefore, CD133 could be considered to be an “Achilles’ heel” for CSCs, because by inhibiting this protein, the signaling pathways that are involved in cell proliferation will also be inhibited. By understanding the molecular biology of CD133, we can not only isolate stem cells, but can also utilize it as a therapeutic strategy. In this review, we summarize new insights into the fundamental cell biology of CD133 and discuss the involvement of CD133 in metastasis, metabolism, tumorigenesis, drug-resistance, apoptosis, and autophagy.
... Wnt signaling is also likely a direct regulator of Neurog1, as Lef binding sites reside within the Neurog1 regulatory region (Hirabayashi et al., 2009). Indeed, Wnt signaling components are sufficient to induce ectopic expression of both Neurog1 and Neurog2 in the ventral telencephalon (Gunhaga et al., 2003;Israsena et al., 2004;Machon et al., 2005;Watanabe et al., 2005). In the case of Neurog2, regulation by Wnt signaling is temporally restricted, as disruption of Wnt signaling reduces Neurog2 expression during early but not late cortical neurogenesis (Bluske et al., 2012;Machon et al., 2005). ...
The formation of functional neural circuits in the vertebrate central nervous system (CNS) requires that appropriate numbers of the correct types of neuronal and glial cells are generated in their proper places and times during development. In the embryonic CNS, multipotent progenitor cells first acquire regional identities, and then undergo precisely choreographed temporal identity transitions (i.e. time-dependent changes in their identity) that determine how many neuronal and glial cells of each type they will generate. Transcription factors of the basic-helix-loop-helix (bHLH) family have emerged as key determinants of neural cell fate specification and differentiation, ensuring that appropriate numbers of specific neuronal and glial cell types are produced. Recent studies have further revealed that the functions of these bHLH factors are strictly regulated. Given their essential developmental roles, it is not surprising that bHLH mutations and de-regulated expression are associated with various neurological diseases and cancers. Moreover, the powerful ability of bHLH factors to direct neuronal and glial cell fate specification and differentiation has been exploited in the relatively new field of cellular reprogramming, in which pluripotent stem cells or somatic stem cells are converted to neural lineages, often with a transcription factor-based lineage conversion strategy that includes one or more of the bHLH genes. These concepts are reviewed herein.
... This latter role appears to be mediated by tonic expression of stabilized β-catenin which inhibits cellcycle exit among cortical progenitors, whereas inhibition of canonical Wnt signaling through β-catenin and increasing activity of the Wnt/Planar Cell Polarity pathway induces maturation (Chenn and Walsh 2002;Mutch et al. 2010;Schafer et al. 2015). However, these effects of Wnt signaling are closely tied to developmental stage, and therefore presumably the temporal expression patterns of coregulators of neuronal development, such as FGF2, which modulate cellular sensitivity to Wnt ligands (Israsena et al. 2004). For instance, during later developmental stages, Wnt7a has been shown to negatively regulate cellular expansion in the neocortex and instead promotes neural progenitor cell differentiation (Hirabayashi et al. 2004). ...
The manipulation of neural plasticity as a means of intervening in the onset and progression of stress-related disorders retains its appeal for many researchers, despite our limited success in translating such interventions from the laboratory to the clinic. Given the challenges of identifying individual genetic variants that confer increased risk for illnesses like depression and post-traumatic stress disorder, some have turned their attention instead to focusing on so-called "master regulators" of plasticity that may provide a means of controlling these potentially impaired processes in psychiatric illnesses. The mammalian homolog of Tailless (TLX), Wnt, and the homeoprotein Otx2 have all been proposed to constitute master regulators of different forms of plasticity which have, in turn, each been implicated in learning and stress-related disorders. In the present review, we provide an overview of the changing distribution of these genes and their roles both during development and in the adult brain. We further discuss how their distinct expression profiles provide clues as to their function, and may inform their suitability as candidate drug targets in the treatment of psychiatric disorders.
... Expression of Wnt7a using retroviral transduction in a culture of mouse forebrain explant, promoted proliferation and suppressed differentiation of neural precursors [24]. Similarly, overexpression of b-catenin in the presence of fibroblast growth factor 2 (FGF2) in NSCs isolated from the mouse cortex remarkably increased the formation of secondary neurospheres [25]. ...
The effects of Wnt signaling modifiers on cell proliferation, seem to be cell specific. Enhancing the proliferation of subventricular zone (SVZ) progenitors has been in the focus of research in recent years. Here we investigate the effect of CHIR99021, a Glycogen Synthase Kinase 3 (GSk-3) inhibitor, on SVZ progenitor’s proliferation both in vivo and in vitro. Neural stem cells were extracted from the adult C57bl/6 by mincing and trypsin treatment followed by culturing in specific medium. Sphere cells formed within about 7–10 days and were characterized by immunostaining. Number of spheres and their size was assessed following exposure to different concentration of CHIR99021 or vehicle. For in vivo studies, animals received intracerebroventricular (i.c.v.) injection of CHIR99021 or vehicle for four days. A subgroup of animals, after 4 days treatment with CHIR99021 received intranasal kainic acid to induce local neurodegeneration in CA3 area of hippocampus. Inhibition of GSk-3 by CHIR99021 increased neural progenitor proliferation and the effect of CHIR99021 was long lasting so that the treated cells showed higher proliferation even after CHIR99021 removal. In vivo administration of CHIR99021 increased the number of neural progenitors at the rims of lateral ventricles especially when the treatment was followed by kainic acid administration which induces neural insult. Results showed that direct administration of CHIR99021 into the culture medium or animal brain increased the number of SVZ progenitors, especially when a neural insult was induced in the hippocampus.
... Cross talk of Wnt and FGF pathways in HCV-related HCC is supported by observations that FGF signaling leads to the release of b-catenin from the b-catenin-E-cadherin complexes because of the phosphorylation of Tyr654 as described above. Furthermore, FGF2 increases expression of b-catenin messenger RNA, upregulates b-catenin nuclear translocation, and inactivates GSK3b [131], probably mediated through activation of the PI3K/Akt and Ras/Raf/MEK/ERK pathways. Conversely, Wnt/b-catenin signaling is able to activate FGF signaling by increasing FGF18 and FGF20 expression [132] (Fig. 3). ...
Hepatitis C virus (HCV) infection is one of the leading causes of hepatocellular carcinoma (HCC) worldwide but the mechanistic basis as to how chronic HCV infection furthers the HCC process remains only poorly understood. Accumulating evidence indicates that HCV core and nonstructural proteins provoke activation of the Wnt/β-catenin signaling pathway, and the evidence supporting a role of Wnt/β-catenin signaling in the onset and progression of HCC is compelling. Convincing molecular explanations as to how expression of viral effectors translates into increased activity of the Wnt/β-catenin signaling machinery are still largely lacking, hampering the design of rational strategies aimed at preventing HCC. Furthermore, how such increased signaling is especially associated with HCC oncogenesis in the context of HCV infection remains obscure as well. Here we review the body of contemporary biomedical knowledge on the role of the Wnt/β-catenin pathway in the progression from chronic hepatitis C to cirrhosis and HCC and explore potential hypotheses as to the mechanisms involved.
... Cross talk of Wnt and FGF pathways in HCV-related HCC is supported by observations that FGF signaling leads to the release of b-catenin from the b-catenin–E-cadherin complexes because of the phosphorylation of Tyr654 as described above. Furthermore , FGF2 increases expression of b-catenin messenger RNA, upregulates b-catenin nuclear translocation, and inactivates GSK3b [131], probably mediated through activation of the PI3K/Akt and Ras/Raf/MEK/ERK pathways . Conversely, Wnt/b-catenin signaling is able to activate FGF signaling by increasing FGF18 and FGF20 expression [132] (Fig. 3). ...
... Young and mature rats are intermingled in the same cluster regulation of adult neurogenesis remain unclear. It has been published that β-catenin is able to affect both proliferation and differentiation processes depending on the activation state of other signaling cascades [76]. Generally, Wnt/β-catenin signaling regulates the proliferative state of NSCs and progenitors in different adult niches, such as hippocampus or the SVZ [49,72,[77][78][79]. ...
The Wnt family of proteins plays key roles during central nervous system development and in several physiological processes during adulthood. Recently, experimental evidence has linked Wnt-related genes to regulation and maintenance of stem cells in the adult neurogenic niches. In the spinal cord, the ependymal cells surrounding the central canal form one of those niches, but little is known about their Wnt expression patterns. Using microdissection followed by TaqMan® low-density arrays, we show here that the ependymal regions of young, mature rats and adult humans express several Wnt-related genes, including ligands, conventional and non-conventional receptors, co-receptors, and soluble inhibitors. We found 13 genes shared between rats and humans, 4 exclusively expressed in rats and 9 expressed only in humans. Also, we observed a reduction with age on spontaneous proliferation of ependymal cells in rats paralleled by a decrease in the expression of Fzd1, Fzd8, and Fzd9. Our results suggest a role for Wnts in the regulation of the adult spinal cord neurogenic niche and provide new data on the specific differences in this region between humans and rodents.
... The importance of β-catenin in neurogenesis is illustrated by the fact that β-catenin deletion is associated with impaired neurogenesis from immature neuron progenitor cells 23 . Among genes downstream of Wnt signaling, Ngn1 and Ngn2 are key regulators that are highly expressed transcriptional factors during the neurogenesis phase 24,25 . Ngn regulates neurogenesis and gliogenesis in a complex way. ...
Brain vasculature acts in synergism with neurons to maintain brain function. This neurovascular coupling, or trophic coupling between cerebral endothelium and neuron, is now well accepted as a marker for mapping brain activity. Neurovascular coupling is most active in the perivascular region, in which there are ample opportunities for cell–cell interactions within the neurovascular unit. This trophic coupling between cells maintains neurovascular function and cellular plasticity. Recent studies have revealed that even adult brains contain multiple stem cells of various lineages, which may provide cellular plasticity through the process of differentiation among these stem cell populations. In this chapter, we provide an overview of the process by which neurovascular components contribute to cellular plasticity in the cerebral perivascular regions, focusing on mechanisms of cell–cell interaction in adult brain.
The neocortex is the site of higher cognitive functions and its development is tightly regulated by cell signalling pathways. Wnt signalling is inexorably linked with neocortex development but its precise role remains unclear. Most studies demonstrate that Wnt/β-catenin regulates neural progenitor self-renewal but others suggest it can also promote differentiation. Wnt/STOP signalling is a novel branch of the Wnt pathway that stabilizes proteins during G2/M by inhibiting glycogen synthase kinase 3 (GSK3)-mediated protein degradation. Recent data from Da Silva et al. (2021) demonstrate that Wnt/STOP is involved in neocortex development where, by stabilizing the neurogenic transcription factors Sox4 and Sox11, it promotes neural progenitor differentiation. The authors also show that Wnt/STOP regulates asymmetric cell division and cell cycle dynamics in apical and basal progenitors, respectively. This study reveals a division of labour in the Wnt signalling pathway by suggesting that Wnt/STOP is the primary driver of cortical neurogenesis while Wnt/β-catenin is mainly responsible for self-renewal. These results resolve a decades-old question on the role of Wnt signalling in cortical neural progenitors.
Background:
Deregulation of transcription in the pathogenesis of sporadic Amyotrophic Lateral Sclerosis (sALS) is taking central stage with RNA-sequencing analyses from sALS patients tissues highlighting numerous deregulated long non-coding RNAs (lncRNAs). The oncogenic lncRNA ZEB1-AS1 is strongly downregulated in peripheral blood mononuclear cells of sALS patients. In addition, in cancer-derived cell lines, ZEB1-AS1 belongs to a negative feedback loop regulation with hsa-miR-200c, acting as a molecular sponge for this miRNA. The role of the lncRNA ZEB1-AS1 in sALS pathogenesis has not been characterized yet, and its study could help identifying a possible disease-modifying target.
Methods:
the implication of the ZEB1-AS1/ZEB1/hsa-miR-200c/BMI1 pathway was investigated in multiple patients-derived cellular models (patients-derived peripheral blood mononuclear cells and induced pluripotent stem cells-derived neural stem cells) and in the neuroblastoma cell line SH-SY5Y, where its function was inhibited via RNA interference. Molecular techniques such as Real Time PCR, Western Blot and Immunofluorescence were used to assess the pathway dysregulation.
Results:
Our results show a dysregulation of a signaling pathway involving ZEB1-AS1/hsa-miR-200c/β-Catenin in peripheral blood mononuclear cells and in induced pluripotent stem cells-derived neural stem cells from sALS patients. These results were validated in vitro on the cell line SH-SY5Y with silenced expression of ZEB1-AS1. Moreover, we found an increase for ZEB1-AS1 during neural differentiation with an aberrant expression of β-Catenin, highlighting also its aggregation and possible impact on neurite length.
Conclusions:
Our results support and describe the role of ZEB1-AS1 pathway in sALS and specifically in neuronal differentiation, suggesting that an impairment of β-Catenin signaling and an alteration of the neuronal phenotype are taking place.
Hippocampal neurogenesis is known to be related to depressive symptoms. Increasing evidence indicates that Wnt/β-catenin signaling regulates multiple aspects of adult hippocampal neurogenesis. Baicalin is a major flavonoid compound with multiple pharmacological effects such as anti-inflammatory, anti-apoptotic, and neuroprotective effects. The current study aimed to explore the antidepressant effects of baicalin and its possible molecular mechanisms affecting hippocampal neurogenesis via the regulation of the Wnt/β-catenin signaling pathway. A chronic mild unpredictable stress (CUMS) model of depression was used in the study. The CUMS-induced mice were treated with baicalin (50 and 100 mg/kg) for 21 days, orally, and the fluoxetine was used as positive control drug. The results indicated that baicalin alleviated CUMS-induced depression-like behaviour, and improved the nerve cells’ survival of the hippocampal dentate gyrus (DG) in CUMS-induced depression of model mice and increased Ki-67- and doublecortin (DCX)-positive cells to restore CUMS-induced suppression of hippocampal neurogenesis. The related proteins in the Wnt/β-catenin signaling pathway, which declined in the CUMS-induced depression model of mice, were upregulated after baicalin treatment, including Wingless3a (Wnt3a), dishevelled2 (DVL2), and β-catenin. Further study found that the phosphorylation rate of glycogen synthase kinase-3β (GSK3β) and β-catenin nuclear translocation increased, as the levels of the β-catenin target genes cyclinD1, c-myc, NeuroD1, and Ngn2 upregulated after baicalin treatment. In conclusion, these findings suggest that baicalin may promote hippocampal neurogenesis, thereby exerting the antidepressant effect via regulation of the Wnt/β-catenin signaling pathway.
The nervous system consists of several hundred neuronal subtypes and glial cells that show specific gene expression and are generated from common ancestors, neural stem cells (NSCs). As the experimental techniques and molecular tools to analyze epigenetics and chromatin structures are rapidly advancing, the comprehensive events and genome‐wide states of DNA methylation, histone modifications, and chromatin accessibility in developing NSCs are gradually being unveiled. Here, we review recent advances in elucidating the role of epigenetic and chromatin regulation in NSCs, especially focusing on the acquisition of glial identity and how epigenetic regulation enables the temporal regulation of NSCs during murine cortical development. Main Points: Recent studies have unveiled the roles of epigenetics and chromatin during neural development. Global and local regulation of histone and DNA modifications and chromatin accessibility orchestrate transcription of neuronal and glial genes for glial identity.
Brain's neurodegenerative diseases are one of the most actual problems of neurology and neurobiology. The lack of the modern methods of treating this diseases stimulates to develop new effective approaches based on neuronal and glial cells, which requires studying the signaling mechanisms of neural differentiation. This review considers the key mechanisms and substances involved in the formation of the neuroepithelium in vivo, as well as for obtaining the neural stem cells from iPSCs and its further differentiation in various types of neuronal and glial cells in vitro.
During fetal and postnatal development, the human brain generates 160 billion neuronal and glial cells, each with precise cellular phenotypes. To effectively manage such a complicated task, intrinsic (e.g., transcription factors) and extrinsic (environmental signals) cues cooperate to regulate the decision by neural progenitors to continue to proliferate or to differentiate. Loss- and gain-of-function studies in the mouse brain have been instrumental in identifying these cues, leading to a fairly well-developed and well-integrated model of neocortical development. This research has revealed that the neurons, astrocytes, and oligodendrocytes that populate the mature neocortex are generated sequentially from neural progenitor pools in both the dorsal (pallial) and ventral (subpallial) telencephalon. Understanding how cellular diversity is established during neocortical development is critical, as appropriate numbers of inhibitory and excitatory neurons, oligodendrocytes, and astrocytes are required for normal neural function. Indeed, an imbalance in excitatory vs inhibitory neurotransmission or alterations in glial cell number are hallmark features of neuropsychological and intellectual disorders such as schizophrenia, bipolar disorder, and autism. Moreover, these fundamental studies are beginning to pave the way for the rational design of neural cell reprogramming strategies, which are of value for the assessment of disease etiology, and for the possible development of novel cell-based therapies. We review herein our current understanding of the intrinsic cues and environmental signals that govern cell fate specification and differentiation decisions during development of neuronal and glial lineages in the murine neocortex.
Neural stem cells are multipotent stem cells that have an unlimited capacity to proliferate and self-renew but whose progeny are restricted to neural lineages. Neural stem cells can generate large numbers of mature neuronal and glial progeny, often through transient amplification of intermediate progenitor pools, similar to the pattern observed in other organ systems. Cells that do not selfrenew indefinitely but that nevertheless proliferate and have the capacity to generate multiple phenotypes are often referred to as multipotential progenitor cells, but they will be included in a broad definition of stem cells for the purposes of this review. While invertebrate model systems including C. elegans and Drosophila have contributed substantially to the understanding of neural stem cell biology, this chapter will focus on the properties of mammalian neural stem cells. Other stem cell-derived precursor populations that are able to proliferate but that have more restricted lineage potential (e.g., glial restricted or neuronal restricted cells) will be a part of the discussion on lineage commitment in this chapter, but will also be discussed in greater depth later in this volume.
Wnt/β-catenin signaling is activated repeatedly during an animal's lifespan, and it controls gene expression through its essential nuclear effector, β-catenin, to regulate embryogenesis, organogenesis, and adult homeostasis. Although the β-catenin transcriptional complex has the ability to induce the expression of many genes to exert its diverse roles, it chooses and transactivates a specific gene set from among its numerous target genes depending on the context. For example, the β-catenin transcriptional complex stimulates the expression of cell cycle-related genes and consequent cell proliferation in neural progenitor cells, while it promotes the expression of neural differentiation-related genes in differentiating neurons. Recent studies using animal and cell culture models have gradually improved our understanding of the molecular basis underlying such context-dependent actions of the β-catenin transcriptional complex. Here, we describe eight mechanisms that support β-catenin-mediated context-dependent gene regulation, and their spatio-temporal regulation during vertebrate development. In addition, we discuss their contribution to the diverse functions of Wnt/β-catenin signaling.
The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant’s action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin—a gene involved in neuronal stem cell regeneration—were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.
The intracellular Raf-Erk signaling pathway is activated during neural stem cell (NSC) proliferation, and neuronal and astrocytic differentiation. A key question is how this signal can evoke multiple and even opposing NSC behaviors. We show here, using a constitutively active Raf (ca-Raf), that Raf-Erk activation in NSCs induces neuronal differentiation in a cell-autonomous manner. By contrast, it causes NSC proliferation and the formation of astrocytes in an extrinsic autocrine/paracrine manner. Thus, treatment of NSCs with medium (CM) conditioned in ca-Raf-transduced NSCs (Raf-CM; RCM) became activated to form proliferating astrocytes resembling radial glial cells (RGCs) or adult-type NSCs. Infusion of Raf-CM into injured mouse brains caused expansion of the NSC population in the subventricular zone, followed by the formation of new neurons that migrated to the damaged site. Our study shows an example how molecular mechanisms dissecting NSC behaviors can be utilized to develop regenerative therapies in brain disorders.
Recent advances in stem cell biology raise our hope that loss of neurons can be replaced to restore disrupted neural circuits. To translate it into effective clinical applications, we need to gain a deep knowledge about how to generate, navigate, and integrate new neurons at the right time and in the right place. Neural stem cells exist in the adult mammalian brain throughout life and contribute to the production of new neurons every day. Understanding the underlying biology of adult neurogenesis will provide a framework for the development of stem cell therapy against various neurological disorders. Recent studies highlight the glial nature of adult neural stem cells, which is well conserved among vertebrates. The behavior of these glia-like stem cells is regulated by complex molecular mechanisms, but the Wnt/beta-catenin signaling is one of the most important pathways involved in multiple aspects of adult neurogenesis.
Cell cycle progression and exit must be precisely patterned during development to generate tissues of the correct size, shape and symmetry. Here we present evidence that dorsal-ventral growth of the developing spinal cord is regulated by a Wnt mitogen gradient. Wnt signaling through the β-catenin/TCF pathway positively regulates cell cycle progression and negatively regulates cell cycle exit of spinal neural precursors in part through transcriptional regulation of cyclin D1 and cyclin D2. Wnts expressed at the dorsal midline of the spinal cord, Wnt1 and Wnt3a, have mitogenic activity while more broadly expressed Wnts do not. We present several lines of evidence suggesting that dorsal midline Wnts form a dorsal to ventral concentration gradient. A growth gradient that correlates with the predicted gradient of mitogenic Wnts emerges as the neural tube grows with the proliferation rate highest dorsally and the differentiation rate highest ventrally. These data are rationalized in a ‘mitogen gradient model’ that explains how proliferation and differentiation can be patterned across a growing field of cells. Computer modeling demonstrates this model is a robust and self-regulating mechanism for patterning cell cycle regulation in a growing tissue.
Supplemental data available on-line
β-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator.
Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of β-catenin, decreases its binding to E-cadherin.
We show here that phosphorylation of Tyr-654 also stimulates the association of β-catenin to the basal transcription factor
TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the
β-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to
the TATA-binding protein or to E-cadherin. Phosphorylation of β-catenin Tyr-654 decreases armadillo-C-terminal tail association,
uncovering the last armadillo repeats. In a C-terminal-depleted β-catenin, the presence of a negative charge at Tyr-654 does
not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment
of ion pairs dominates its association with β-catenin, and its binding is greatly dependent on the absence of a negative charge
at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-β-catenin interaction, even though the steric hindrance
of the C-tail is no longer present. These results explain how phosphorylation of β-catenin in Tyr-654 modifies the tertiary
structure of this protein and the interaction with its different partners.
Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N-cadherin with the actin containing cytoskeleton is lost and N-cadherin-mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin-containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N-cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co-precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development.
The Drosophila gene product Wingless (Wg) is a secreted glycoprotein and a member of the Wnt gene family. Genetic analysis of Drosophila epidermal development has defined a putative paracrine Wg signalling pathway involving the zeste-white 3/shaggy (zw3/sgg) gene product. Although putative components of Wg- (and by inference Wnt-) mediated signalling pathways have been identified by genetic analysis, the biochemical significance of most factors remains unproven. Here we show that in mouse 10T1/2 fibroblasts the activity of glycogen synthase kinase-3 (GSK-3), the murine homologue of Zw3/Sgg, is inactivated by Wg. This occurs through a signalling pathway that is distinct from insulin-mediated regulation of GSK-3 in that Wg signalling to GSK-3 is insensitive to wortmannin. Additionally, Wg-induced inactivation of GSK-3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31-8220 and prolonged pre-treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK-3/Zw3 during Wnt/Wg signal transduction.
We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.
During development of the mammalian brain, both neurons and glia are generated from multipotent neural stem cells. Although neurogenesis ceases in most areas at birth, stem cells continue to generate neurons within the subventricular zone and hippocampal dentate gyrus throughout adult life. In this work, we provide the first demonstration that precursors native to regions of the adult brain that generate only glia can also generate neurons after exposure to FGF-2 in vitro. When progenitors isolated from hippocampal tissue were directly compared with cells isolated from the neocortex, both populations were able to initiate a program of proliferative neurogenesis. Genetic marking and lineage analysis showed that a majority of the cells able to generate neurons were multipotent precursors; however, progeny from these precursors acquired the competence to differentiate into neurons only after exposure to FGF-2. The recruitment of similar FGF-2-responsive cells from the adult optic nerve, a structure well isolated from the neurogenic zones within the brain, confirmed that neuron-competent precursors naturally exist in widely divergent tissues of the adult brain.
Little is known about regionally specific signals that control the number of neuronal progenitor cells in vivo. We have previously shown that the germline mutation of the basic fibroblast growth factor (Fgf2) gene results in a reduction in the number of cortical neurons in the adult. We show here that Fgf2 is expressed in the pseudostratified ventricular epithelium (PVE) in a dorsoventral gradient and that Fgf2 and its receptor, Fgfr-1, are downregulated by mid to late stages of neurogenesis. In Fgf2 knockout mice, the volume and cell number of the dorsal PVE (the cerebral cortical anlage) are substantially smaller, whereas the volume of the basal PVE is unchanged. The dorsal PVE of Fgf2 knockout mice has a 50% decrease in founder cells and a reduced expansion of the progenitor pool over the first portion of neurogenesis. Despite this reduction, the degree of apoptosis within the PVE is not changed in the Fgf2 knockouts. Cortical neuron number was decreased by 45% in Fgf2 knockout mice by the end of neurogenesis, whereas the number of neurons in the basal ganglia was unaffected. Microscopically, the frontal cerebral cortex of neonatal Fgf2 null mutant mice lacked large neurons in deep cortical layers. We suggest that Fgf2 is required for the generation of a specific class of cortical neurons arising from the dorsal PVE.
In the early Xenopus embryo, the Xiro homeodomain proteins of the Iroquois (Iro) family control the expression of proneural genes and the size of the neural plate. We report that Xiro1 functions as a repressor that is strictly required for neural differentiation, even when the BMP4 pathway is impaired. We also show that Xiro1 and Bmp4 repress each other. Consistently, Xiro1 and Bmp4 have complementary patterns of expression during gastrulation. The expression of Xiro1 requires Wnt signaling. Thus, Xiro1 is probably a mediator of the known downregulation of Bmp4 by Wnt signaling.
A growing body of work indicates that neural induction may be initiated prior to the establishment of the gastrula mesodermal organizer. Here, we examine neural induction in Xenopus embryos in which mesoderm induction has been blocked by Cerberus-short, a reagent that specifically inhibits Nodal-related (Xnr) signals. We find that extensive neural structures with cyclopic eyes and brain tissue are formed despite the absence of mesoderm. This neural induction correlates with the expression of chordin and other BMP inhibitors-such as noggin, follistatin, and Xnr3-at the blastula stage, and requires beta-Catenin signaling. Activation of the beta-Catenin pathway by mRNA microinjections or by treatment with LiCl leads to differentiation of neurons, as well as neural crest, in ectodermal explants. Xnr signals are required for the maintenance, but not for the initiation, of BMP antagonist expression. Recent work has demonstrated a role for beta-Catenin signaling in neural induction mediated by the transcriptional down-regulation of BMP-4 expression. The present results suggest an additional function for beta-Catenin, the early activation of expression of secreted BMP antagonists, such as Chordin, in a preorganizer region in the dorsal side of the Xenopus blastula.
Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of β-catenin. While
Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic
region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding
sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by β-catenin, and mutation of the
Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that
Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct
the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding
conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.
Wnt1 and Wnt3a are signaling factors known to play a role in the induction of myogenesis in the myotome of the differentiating
somite. Both factors may transduce their signal by a conserved pathway that leads to transcriptional regulation by β-catenin/Lef1.
β-Catenin and Lef1 are found in the myotome prior to MyoD expression. We have utilized the P19 cell system to study the mechanisms
by which Wnt3a may activate MyoD expression and subsequent skeletal muscle development. We have isolated P19 cell lines that
stably express either Wnt3a or activated β-catenin and found that aggregation of these cells results in the induction of myogenesis
compared with control cells. Pax3, Gli2, Mox1, and Six1 were expressed during Wnt3a and β-catenin-induced differentiation
prior to MyoD expression. Furthermore, we have shown that the nuclear function of β-catenin was essential for skeletal myogenesis
in P19 cells by overexpression of a dominant negative β-catenin/engrailed chimera. Primitive streak factors were present,
but expression of Pax3, Mox1, Gli2, and Six1 was lost in these cells, indicating that nuclear β-catenin is essential for specification
of mesodermal precursors to the myogenic lineage. Therefore, Wnt signaling, acting via β-catenin, is necessary and sufficient
for skeletal myogenesis in P19 cells.
In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.
The Drosophila gene product Wingless (Wg) is a secreted glycoprotein and a member of the Wnt gene family. Genetic analysis of Drosophila epidermal development has defined a putative paracrine Wg signalling pathway involving the zeste‐white 3/shaggy (zw3/sgg) gene product. Although putative components of Wg‐ (and by inference Wnt‐) mediated signalling pathways have been identified by genetic analysis, the biochemical significance of most factors remains unproven. Here we show that in mouse 10T1/2 fibroblasts the activity of glycogen synthase kinase‐3 (GSK‐3), the murine homologue of Zw3/Sgg, is inactivated by Wg. This occurs through a signalling pathway that is distinct from insulin‐mediated regulation of GSK‐3 in that Wg signalling to GSK‐3 is insensitive to wortmannin. Additionally, Wg‐induced inactivation of GSK‐3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31–8220 and prolonged pre‐treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK‐3/Zw3 during Wnt/Wg signal transduction.
In the embryonic mouse cerebral cortex, progenitors in the ventricular zone (VZ) undergo a developmental change between embryonic day 13 (E13) and E15. This results in the generation of a secondary proliferative population and the appearance of a second germinal layer, the subventricular zone (SVZ). We have shown previously that bone morphogenetic proteins (BMPs) and fibroblast growth factor 2 (FGF2) act antagonistically to regulate the development of a subset of SVZ progenitors that normally express a high level of epidermal growth factor (EGF) receptors and divide in response to EGF. In the present study, we show that Wnt 7a, Wnt 7b, and Sonic hedgehog (Shh) promote progenitor maturation in explant cultures, as reported for FGF2. Wnts 7a and 7b also stimulate the proliferation of neurogenic progenitors and increase the number of cells that can generate primary neurospheres. To determine whether Wnts, FGF2, and Shh act independently or in a common pathway, each factor was inhibited in cortical explants. This revealed that endogenous Wnts, FGF2, and Shh normally contribute to progenitor maturation. Moreover, Wnt 7a depends on FGF2 or Shh to promote maturation but not proliferation. Maturation induced by blocking BMPs also depends on Shh. In contrast, FGF2 promotes maturation by a Shh-independent mechanism. In vivo, progenitors infected with a Wnt 7a retrovirus at E10.5 were found preferentially in the SVZ at E16.5. These findings suggest that Wnts depend on Shh or FGF2 to promote progenitor maturation to an SVZ state in the embryonic cortex.
To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.
In order to identify factors involved in posteriorization of the central nervous system, we undertook a functional screen in Xenopus animal cap explants which involved coinjecting noggin RNA together with pools of RNA from a chick somite cDNA library. In the course of this screen, we isolated a clone encoding a truncated form of β-catenin, which induced posterior neural and dorsal mesodermal markers when coinjected with noggin in animal caps. Similar results were obtained with Xwnt-8 and Xwnt-3a, suggesting that these effects are a consequence of activating the canonical Wnt signalling pathway. To investigate whether the activation of posterior neural markers requires mesoderm induction, we performed experiments using a chimeric inducible form of β-catenin. Activation of this protein during blastula stages resulted in the induction of both posterior neural and mesodermal markers, while activation during gastrula stages induced only posterior neural markers. We show that this posteriorizing activity occurs by an indirect and noncell-autonomous mechanism requiring FGF signalling.
β-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. β-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of β-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in β-catenin and in components regulating its stability can contribute to the formation of certain tumors.
Beta-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. beta-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of beta-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in beta-catenin and in components regulating its stability can contribute to the formation of certain tumors.
To identify molecules that regulate the transition of dividing neuroblasts to terminally differentiated neurons in the CNS, conditions have been developed that allow the neuronal differentiation of cortical precursor cells to be examined in vitro. In these cultures, the proliferation of undifferentiated precursor cells is controlled by basic fibroblast growth factor (bFGF). The proliferative effects of bFGF do not preclude the action of signals that promote differentiation, since addition of neurotrophin-3 (NT-3) antagonizes the proliferative effects of bFGF and enhances neuronal differentiation. In addition, blocking NT-3 function with neutralizing antibodies leads to a marked decrease in the number of differentiated neurons, without affecting the proliferation of cortical precursors or the survival of postmitotic cortical neurons. These observations suggest that bFGF and NT-3, by their distinct effects on cell proliferation and differentiation, are key regulators of neurogenesis in the CNS.
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The P19 embryonal carcinoma (EC) cell line represents a useful model system for analysis of neural development and differentiation processes that are difficult to study in mammalian embryos. Since many members of the Wnt family of signaling molecules are expressed in the developing as well as adult nervous system, we have examined expression of these genes in P19 cells. Analysis of the mRNA accumulation profiles for Wnt genes during retinoic acid (RA)-induced neural differentiation of P19 cells showed that nine Wnt family members were expressed in a regulated manner during this process. Most were induced by RA treatment, and some were also expressed in undifferentiated P19 cells. Since Wnt-1 is not expressed in undifferentiated P19 cells but is induced during neuroectodermal differentiation we have generated P19 cell lines that overexpress Wnt-1 in the absence of RA treatment, in order to address the role of Wnt-1 in P19 differentiation. In the presence of ectopic Wnt-1, expression of other endogenous Wnt genes, which serve as early differentiation markers in this system, were induced without RA, which is normally required for appearance of these gene products. Furthermore, ectopic expression of Wnt-1 resulted in a loss of SSEA-1 antigen expression, a marker of undifferentiated P19 cells. Similarly to the parental cell line, addition of RA to P19 cells overexpressing Wnt-1 induced the neuroectodermal pathway, but expression of cell type-specific markers such as MASH-1, HNK-1, and GAP-43 was diminished and the morphology of neuronal processes, stained with an antibody to neurofilament, was abnormal. These data suggest that Wnt-1 itself can induce some aspects of early neuroectodermal differentiation and, furthermore, that the correct timing of Wnt-1 expression is necessary for proper RA-induced expression of the neural phenotype.
Previous studies have described Wnt-1 protein synthesized by cell lines transfected with Wnt-1 cDNA. This report presents the first identification and biochemical characterization of native Wnt-1 protein, from P19 embryonal carcinoma cells undergoing neuroectodermal differentiation. The data demonstrate that P19 cells efficiently process and secrete glycosylated Wnt-1 protein species of 40 kD and 42 kD. Similar to recombinant Wnt-1 proteins, native Wnt-1 proteins are associated with the cell surface upon secretion and can be released by suramin treatment of the cells. Non-reducing gel analysis showed that Wnt-1 proteins are secreted as monomers.
The embryonic cerebral cortex contains a population of stem-like founder cells capable of generating large, mixed clones of neurons and glia in vitro. We report that the default state of early cortical stem cells is neuronal, and that stem cells are heterogeneous in the number of neurons that they generate. In low fibroblast growth factor (FGF2) concentrations, most maintain this specification, generating solely neuronal progeny. Oligodendroglial production within these clones is stimulated by a higher, threshold level of FGF2, and astrocyte production requires additional environmental factors. Because most cortical neurons are born before glia in vivo, these data support a model in which the scheduled production of cortical cells involves an intrinsic neuronal program in the early stem cells and exposure to environmental, glia-inducing signals.
Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.
The recent discovery that the HMG box transcription factor XTCF-3 is involved in early axis specification in Xenopus laevis (Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J. Godsave, S., Korinek, V., Roose, J., Destree, O., Clevers, H., 1996. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399) led us to search for other members of the TCF/LEF family in this species. A newly identified HMG box factor was cloned with highest homology to human LEF-1, called XLEF-1. Unlike XTcf-3, XLef-1 is not expressed maternally, but its transcripts become detectable directly after the mid blastula transition (MBT). At later stages, both genes are expressed in the central nervous system (CNS), eyes, otic vesicles, head mesenchyme, neural crest and derivatives, branchial arches, developing heart, tailbud and limb buds. The expression pattern of Lef-1 during later stages of development is evolutionarily conserved.
We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3beta, which inhibits NeuroD function in vivo, blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.
The serine threonine protein kinase encoded by the shaggy locus has been implicated in neurogenesis in Drosophila. In vertebrates, the shaggy homolog, GSK3beta, is involved in early pattern formation, specifically in setting up the dorsal ventral axis. In the present study we have cloned the Xenopus homolog of the shaggy kinase and show (1) that GSK3beta is expressed in the right time and place to play a role in primary neurogenesis in Xenopus; (2) that overexpression of wild-type GSK3beta leads to a decrease in the number of primary neurons; (3) that inhibition of endogenous GSK3beta activity with overexpression of a dominant negative GSK3beta construct leads to an increase in the number of primary neurons; and (4) that GSK3beta inhibits the ability of neurogenin and NeuroD to produce ectopic tubulin expression, but does not inhibit the ability of neurogenin to produce ectopic NeuroD. On the basis of these data we propose that GSK3beta inhibits the function of NeuroD and therefore prevents neuronal differentiation at a relatively late stage in the developmental pathway.
Environmental signals are important in the development of neural crest, during which process multipotent progenitor must choose from several fates. However, the nature of these environmental signals is unknown. A previous fate map of zebrafish cranial neural crest showed that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. Here we determine the role of Wnt signals in modulating the fate of neural crest by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signalling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. We conclude that endogenous Wnt signalling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates.
Multipotent, self-renewing neural stem cells reside in the embryonic mouse telencephalic germinal zone. Using an in vitro neurosphere assay for neural stem cell proliferation, we demonstrate that FGF-responsive neural stem cells are present as early as E8.5 in the anterior neural plate, but EGF-responsive neural stem cells emerge later in development in a temporally and spatially specific manner. By separately blocking EGF and FGF2 signaling, we also show that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation and at relatively high cell densities separate cell nonautonomous effects can substantially enhance the mitogen-induced proliferation. At lower cell densities, neural stem cell proliferation is additive in the presence of EGF and FGF2 combined, revealing two different stem cell populations. However, both FGF-responsive and EGF-responsive neural stem cells retain their self-renewal and multilineage potential, regardless of growth factor conditions. These results support a model in which separate, lineage-related EGF- and FGF-responsive neural stem cells are present in the embryonic telencephalic germinal zone.
The fibroblast growth factor (FGF) family comprises several members with distinct patterns of expression in the developing central nervous system. FGFs regulate the early specification and the subsequent growth of central nervous system regions. These different actions require the coordinated activation of distinct sets of target genes by FGFs at the appropriate stage of development. The role of FGF2 in the growth and morphogenesis of the cerebral cortex is reviewed in detail. The cellular and molecular mechanisms that underlie the action of FGF2 on cortical development are discussed.
The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.
The co-activation of Wnt signaling and concomitant inhibition of BMP signaling has previously been implicated in vertebrate neural patterning, as evidenced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined that XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, connexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 gene is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus.
Although Sonic Hedgehog (Shh) plays a critical role in brain development, its actions on neural progenitor cell proliferation and differentiation have not been clearly defined. Transcripts for the putative Shh-receptor genes patched (Ptc) and smoothened (Smo) are expressed by embryonic, postnatal, and adult progenitor cells, suggesting that Shh can act directly on these cells. The recombinant human amino-terminal fragment of Shh protein (Shh-N) alone did not support the survival of cultured progenitor cells, but treatment with Shh-N in the presence of bFGF increased progenitor cell proliferation. Furthermore, treatment of embryonic rat progenitor cells propagated either in primary culture or after mitogen expansion significantly increased the proportions of both beta-tubulin- (neuronal marker) and O4- (oligodendroglial marker) immunoreactive cells and reduced the proportion of nestin- (uncommitted neural progenitor cell marker) immunoreactive cells. By contrast Shh-N had no effect on the elaboration of GFAP- (astroglial marker) immunoreactive cells. Cotreatment with Shh-N and bone morphogenetic protein-2 (BMP2) inhibited the anti-proliferative, astroglial-inductive, and oligodendroglial-suppressive effects of BMP2. Our observations suggest that Shh-N selectively promotes the elaboration of both neuronal and oligodendroglial lineage species and inhibits the effects of BMP2 on progenitor cell proliferation and astroglial differentiation.
We report a new role for Wnt signaling in the vertebrate embryo: the induction of neural tissue from ectoderm. Early expression of mouse wnt8, Xwnt8, beta-catenin, or dominant-negative GSK3 induces the expression of neural-specific markers and inhibits the expression of Bmp4 in Xenopus ectoderm. We show that Wnt8, but not the BMP antagonist Noggin, can inhibit Bmp4 expression at early gastrula stages. Furthermore, inhibition of beta-catenin activity in the neural ectoderm of whole embryos by a truncated TCF results in a decrease in neural development. Therefore, we suggest that a cleavage-stage Wnt signal normally contributes to an early repression of Bmp4 on the dorsal side of the embryo and sensitizes the ectoderm to respond to neural inducing signals from the organizer. The Wnt targets Xnr3 and siamois have been shown previously to have neuralizing activity when overexpressed. However, antagonists of Wnt signaling, dnXwnt8 and Nxfrz8, inhibit Wnt-mediated Xnr3 and siamois induction, but not neural induction, suggesting an alternative mechanism for Bmp repression and neuralization. Conversely, dnTCF blocks both Wnt-mediated Xnr3 and neural induction, suggesting that both pathways require this transcription factor.
The basic helix-loop-helix transcription factor neurogenin1 is required for proper nervous system development in vertebrates. It is expressed in neuronal precursors during embryonic development and is thought to play a role in specifying neuronal fate. To investigate the regulation of neurogenin1 expression, the transcriptional start site of the gene was identified and a 2.7-kb fragment ending in the first exon was shown to possess basal promoter activity. This 2.7-kb fragment was able to promote expression of reporter genes in P19 cells under conditions in which expression of endogenous neurogenin1 was induced. Importantly, the 2.7-kb fragment was able to drive expression of a lacZ reporter gene in transgenic mice in most tissues in which neurogenin1 is normally expressed, including those peripheral ganglia that fail to develop in neurogenin1 "knockout" mice. These findings identify a regulatory region containing elements responsible for appropriate expression of a gene with a crucial role in generating the vertebrate nervous system.
beta-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of beta-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of beta-catenin to the basal transcription factor TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the beta-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to E-cadherin. Phosphorylation of beta-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted beta-catenin, the presence of a negative charge at Tyr-654 does not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment of ion pairs dominates its association with beta-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-beta-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of beta-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.
A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.
As a component of adherens junctions and the Wnt signaling pathway, beta-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. We have determined the crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of beta-catenin. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices which interact with both ends of the arm repeat domain and an extended stretch of 14 residues which closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of beta-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly.
To clarify the molecular mechanisms of human carcinogenesis associated with abnormal Wnt/wingless signaling, we searched for genes the expression of which was significantly altered by introduction of wild-type AXIN1 into LoVo colon cancer cells. By means of a cDNA microarray, we compared expression profiles of LoVo cells infected with either adenoviruses expressing wild-type AXIN1 (Ad-Axin) or those expressing a control gene (Ad-LacZ). Among the genes showing altered expression, the ectodermal-neural cortex 1 (ENC1) gene was down-regulated in response to Ad-Axin. The promoter activity of ENC1 was elevated approximately 3-fold by transfection of an activated form of beta-catenin together with wild-type T-cell factor (Tcf)4 in HeLa cells. Semiquantitative reverse transcription-PCR experiments revealed that expression of ENC1 was increased in more than two-thirds of 24 primary colon cancer tissues that we examined compared with corresponding noncancerous mucosae. Introduction of exogenous ENC1 increased the growth rate of HCT116 colon cancer cells in serum-depleted medium. In other experiments, overexpression of ENC1 in HT-29 colon cancer cells suppressed the usual increase of two differentiation markers, in response to treatment with sodium butyrate, a differentiation-inducible agent. These data suggest that ENC1 is regulated by the beta-catenin/Tcf pathway and that its altered expression may contribute to colorectal carcinogenesis by suppressing differentiation of colonic cells.
Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 -/- embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic beta-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.
Patterning of the chick telencephalon has been debated, especially in regard to a ventral (subpallial) or dorsal (pallial) nature of the dorsal ventricular ridge (DVR). Here we report the expression patterns of chick homologues of molecules known to be involved in telencephalic patterning in other vertebrate species. We show here that the transcription factors Ngn1, Ngn2, Cash1, Gsh2 and the secreted frizzled related protein 1 (sfrp1), a wnt receptor, are expressed in characteristic telencephalic domains during chick development. At embryonic day 7 (E7) Ngn1 and Ngn2 are localized in the dorsal pallium and the DVR, similar to the region of Pax6 expression. In contrast, Gsh2 and Cash1 are restricted to the subpallium and some DVR cells. Interestingly Sfrp, a dorsoventral boundary marker in mouse telencephalon, is expressed between the DVR and subpallium. Gsh2 and Pax6 double-positive cells further characterize this boundary region in the developing chick telencephalon.
E-cadherin is a transmembrane protein that mediates Ca2+-dependent cell-cell adhesion and is implicated in a number of biologic processes, including cell growth and differentiation, cell recognition and cell sorting during development. We have previously demonstrated that both cell-cell adhesion and invasion are modulated by fibroblast growth factor (FGF)-1 and FGF-2 in a panel of pancreatic adenocarcinoma cell lines (BxPc3, T3M4 and HPAF). Here, we examine further the role of FGFs in the expression and activation of the E-cadherin/catenin system. We demonstrate that both FGF-1 and FGF-2 upregulate E-cadherin and beta-catenin at the protein level in the BxPc3 and HPAF cell lines and modestly in T3M4 cells. FGF-1 and FGF-2 facilitate the association of E-cadherin and alpha-catenin with the cytoskeleton, as demonstrated by the increase in the detergent-insoluble fraction of E-cadherin in BxPc3 and HPAF cells. Since the correct function of the E-cadherin/catenin complex requires its association with the cytoskeleton, our data suggest that FGF-1 and FGF-2 contribute to the integrity and thus the function of the complex. Furthermore, FGFs facilitate the assembly of the E-cadherin/catenin axis. The effect is associated with elevation of tyrosine phosphorylation of E-cadherin, alpha-catenin, beta-4051 mu-catenin and gamma-catenin, but not p120ctn. These findings indicate that the E-cadherin/catenin system is a target of the FGF/FGFR system and that coordinated signals from both systems may determine the ultimate biologic responses.
A study on glycogen synthase kinase (GSK)-3 was presented. The isolation, characterization and regulation of GSK-3 activity was discussed. The role of GSK-3 in the Wnt signaling pathway was studied. The inhibition of GSK-3 by GBP/FRAT, GSK-3 phosphorylation of β-catenin and APC and regulation of GSK-3 by other signaling pathways was also described.
In early Ciona savignyi embryos, nuclear localization of beta-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of beta-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of beta-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in beta-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.
Cell cycle progression and exit must be precisely patterned during development to generate tissues of the correct size, shape and symmetry. Here we present evidence that dorsal-ventral growth of the developing spinal cord is regulated by a Wnt mitogen gradient. Wnt signaling through the beta-catenin/TCF pathway positively regulates cell cycle progression and negatively regulates cell cycle exit of spinal neural precursors in part through transcriptional regulation of cyclin D1 and cyclin D2. Wnts expressed at the dorsal midline of the spinal cord, Wnt1 and Wnt3a, have mitogenic activity while more broadly expressed Wnts do not. We present several lines of evidence suggesting that dorsal midline Wnts form a dorsal to ventral concentration gradient. A growth gradient that correlates with the predicted gradient of mitogenic Wnts emerges as the neural tube grows with the proliferation rate highest dorsally and the differentiation rate highest ventrally. These data are rationalized in a 'mitogen gradient model' that explains how proliferation and differentiation can be patterned across a growing field of cells. Computer modeling demonstrates this model is a robust and self-regulating mechanism for patterning cell cycle regulation in a growing tissue. Supplemental data available on-line
Wnt signals regulate differentiation of neural crest cells through the beta-catenin associated with a nuclear mediator of the lymphoid-enhancing factor 1 (LEF-1)/T-cell factors (TCFs) family. Here we show the interaction between the basic helix-loop-helix and leucine-zipper region of microphthalmia-associated transcription factor (MITF) and LEF-1. MITF is essential for melanocyte differentiation and its heterozygous mutations cause auditory-pigmentary syndromes. Functional cooperation of MITF with LEF-1 results in synergistic transactivation of the dopachrome tautomerase (DCT) gene promoter, an early melanoblast marker. This activation depends on the separate cis-acting elements, which are also responsible for the induction of the DCT promoter by lithium chloride that mimics Wnt signaling. beta-catenin is required for efficient transactivation, but dispensable for the interaction between MITF and LEF-1. The interaction with MITF is unique to LEF-1 and not detectable with TCF-1. LEF-1 also cooperates with the MITF-related proteins, such as TFE3, to transactivate the DCT promoter. This study therefore suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF-1, which may ensure efficient propagation of Wnt signals in many types of cells.
The Wnt signal transduction pathway regulates various aspects of embryonal development and has been implicated in promoting cancer. Signalling by Wnts leads to the stabilization of cytosolic beta-catenin, which then associates with TCF transcription factors to regulate expression of Wnt-target genes. The Wnt pathway is further subject to cross-regulation at various levels by other components.
Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. In particular, it has been shown that TAK1 activates NLK, which phosphorylates TCFs bound to beta-catenin. This phosphorylation down-regulates the DNA-binding activity of a TCF-4/beta-catenin complex, and blocks activation of their target genes. To investigate the role of NLK in Xenopus development, we isolated xNLK, a Xenopus homologue of NLK. Our findings indicate that xNLK is expressed in neural tissues and induces the anterior-neural marker gene, Otx-2. Moreover, xSox11, which is induced by the expression of Chordin, co-operates with xNLK to induce neural development. These molecules also interact in mammalian cells, and expression of a mutant of xNLK lacking kinase activity was found to suppress the induction of neural marker gene expression by xSox11.
Our findings indicate that xNLK may play a role in neural development together with xSox11 during early Xenopus embryogenesis.
Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.