Hippocampal neurons at 21 DIV were incubated for 3 h with Wnt-3a (150 ng/ml) or vehicle at 37°C. Neurons on coverslips were then washed with Tyrode modified solu- tion, mounted in a microscope perfusion chamber, and incubated for 30 s with 10 μM FM1-43 (Molecular Probes) followed by 1 minute of loading by mild depo- larization with 30 mM KCl. Nonspecific and non-synaptic FM1-43 staining was diminished by washing with 10 min- utes of continued perfusion of Tyrode solution at 1 to 2 ml/minute controlled with a peristaltic pump (Cole Palmer, Vernon Hills, IL, USA). The chamber was adapted at the stage of a Zeiss Axiovert 200 M microscope coupled to Pascal LSM5 confocal laser scanning system. Neurons were imaged with a 63 × 1.4 NA oil objective at 512 × 512 full-frame resolution using a 488-nm argon laser to excite the FM1-43 probe, andthe fluorescence signals were col- lected over 505 nm. Then, after a period of 50 s of basal fluorescence acquisition, neurons were depolarized with 90 mM KCl and imaged for 300 s at 1-s intervals. Images from presynaptic loaded puncta were selected for measur- ing fluorescence intensities using areas ofthe region of interest of 1.5 × 1.5 μm. Images ofWnt-3a-treated neurons and control neurons were obtained using identical set- tings for laser power, confocal thickness, and detector sen- sitivity. All measurements were taken at room temperature (25°C).
Wnt signaling has been implicated in hippocampal neurogenesis (Lie et al., 2005) andin neurotransmitter release, the modulation of synaptic activity, and syn- aptogenesis in mossy fibers (Ahmad- Annuar et al., 2006). Moreover, adult rat hippocampus and mature hippocampal neurons in culture express Wnt ligand, and we have observed that Wnt-7a induces hippocampal neuronal synaptic vesicle exocytosis (Cerpa, Alfaro, Farias, Fuent- ealba, Metcalfe, Godoy, Bonansco, and Inestrosa, unpublished observations). Here, we demonstrate that Wnt-7a signal- ing induces the clustering of APC and ␣7- nAChR on mature neurons inpresynaptic membranes as indicated by their cointer- action with SV-2, p-synapsin, VAMP-1/2, and synaptotagmin 1a/b. This suggests that Wnt-7a may dynamically modulate neurotransmitter release by altering ␣7- nAChRs levels at synaptic terminals, thereby contributing to synaptic plasticity. Wnt-7a exposure also affects the number and cluster size of several other essential, presynaptic proteins involved in neuro- transmitter release, providing ways to af- fect synaptic functionand plasticity in ad- dition to or independent of effects on ␣7- nAChRs. APC plays an essential rolein localization of ␣7-nAChR at the synapse, but there are some APC clusters that do not contain ␣7-nAChRs, so APC may also facilitate localization of other proteins to synapses at sites distinct from those at which ␣7-nAChRs cluster. Nevertheless, whether in maturing or mature neurons, ␣7-nAChRs could be an important synap- tic target ofWnt signaling and/or an effec- tor oftheWnt pathway.
ten membrane receptors and a plethora of cofactors and regulators are known. Different mechanisms ofWnt sign- aling have also been identified. The best understood of these is the "canonical" pathway, in which β-catenin transduces theWnt signal to the nucleus . In this case, the signaling cascade by Wnts involves an interaction with a receptor complex comprising members oftheFrizzled (Fz) class of 7-transmembrane receptors and a member ofthe low density lipoprotein receptor 5/6 (LRP 5/6) family of single-pass membrane proteins. Wnt interaction with its receptor results in an increase inthe stability of β-cat- enin, whose accumulation results in translocation to the nucleus where it can interact with members ofthe TCF/ LEF class of transcription factors and therefore modulate gene expression. The stability of β-catenin is controlled by Wnt through the modulation of a large cytoplasmic pro- tein complex comprised ofthe protein Axin (axis inhibi- tion protein), APC (adenomatosis polyposis coli), CK1α (casein kinase 1 alpha), GSK-3β(glycogen synthase kinase 3 beta) and GβP/frat . GSK-3β directly controls the level of β-catenin phosphorylation, which leads to its con- sequent degradation by the proteasome pathway . Wnt signaling is regulated by a wide range of proteins, which act either intracellularly by affecting signal transduction, or extracellularly by interfering with the interaction between Wnt ligands and their membrane co-receptors . Different families of extracellular antagonists ofthe canonical Wnt pathway have been described, such as Wise, the secreted frizzled-related protein (sFRP), theWnt inhibitory factor 1 (Wif1), Cerberus, andthe Dickkopf (Dkk) family of secreted proteins. Ofthe four known Dkk family members, Dkk-1 is uniquely described as a nega- tive modulator ofthe canonical Wnt signaling, whereas, Dkk-2 for example may activate or inhibit the pathway depending on the cellular context. Dkk-1 is expressed at very low levels inthe adult brain , and binds to LRP 5/6 andthe transmembrane protein Kremen-2, promot- ing the endocytosis and subsequent degradation of LRP 5/ 6, which is no longer available as a co-receptor for Wnt .
Wnt ligands have been linked to the assembly of structural com- ponents inpresynaptic compartments. Inthe cerebellum, Wnt7a is expressed in granular cells (GC) at the same time as the mossy ﬁber (MF) axon, which is thepresynaptic contact (Lucas and Salinas, 1997). Several changes remodel the connectivity between both areas to increase the contact surface. Wnt7a induces axonal spread- ing and incremental growth of cone size and branching, leading to the accumulation of synaptic proteins (Hall et al., 2000; Lucas and Salinas, 1997). Wnt7a probably contributes to the formation of active zones because it increases the clustering of synapsin I, a protein located inthepresynaptic membrane involved in synapse formation andfunction (Hall et al., 2000). Because this effect of Wnt7a is mimicked by lithium application, it seems to involve GSK-3 ␤ (Lucas and Salinas, 1997). This effect has been blocked by theWnt antagonist sFRP and a mutant mice deﬁcient in Wnt7a shows a delayed synaptic maturation (Hall et al., 2000). Then inthe cerebellum, Wnt7a can act as a retrograde signal from GC to induce presynapticdifferentiationin MF, working as a synapto- genic factor (Ahmad-Annuar et al., 2006; Hall et al., 2000). Like Wnt7a, Wnt7b and Wnt3a increase the number of pre-synaptic puncta suggesting a role for these ligands inpresynaptic assem- bly (Ahmad-Annuar et al., 2006; Cerpa et al., 2008; Davis et al., 2008). Wnt7a also increases the clustering ofpresynaptic proteins such as synaptophysin, synaptotagmin and SV-2, but does not affect postsynaptic clustering of proteins like PSD-95 (Cerpa et al., 2008). Despite Wnt7a clustering induction correlates with ␤ -catenin sta- bilization, this does not involve Wnt gene target expression – an effect that is also mimicked by Wnt3a. Unexpectedly, GSK-3 ␤ is also not required for presynaptic clustering induced by Wnt7a, suggesting that an upstream mechanism is involved (Cerpa et al., 2008). It has been suggested that Wnt7a requires
FZD1 receptor mediates the activation ofthe canonical Wnt pathway by different Wnt ligands [25–29]; in agree- ment, we determined in N2a cells and AHPs that FZD1- knockdown reduced Wnt/β-catenin signaling activity. Therefore, it is possible that inthe SGZ FZD1 mediates the activation of canonical Wnt signaling in progenitor cells to induce neural differentiation. In agreement, it is known from Wnt/β-catenin reporter mice that the ca- nonical Wnt pathway is active in neural progenitor cells inthe SGZ , and it was recently determined that knocking down the expression of LRP6, which is one ofthe co-receptors required for the activation oftheWnt/ β-catenin signaling pathway, decreased neuronal fate de- termination of newborn cells . The mechanism in- volved inthe regulation of neuronal differentiation by Wnts involves the expression of proneural Wnt target genes [32, 36, 40], including the transcription factors NeuroD1, which stimulates neuronal differentiationand survival of neural progenitor cells inthe adult dentate gyrus [32, 45] and Prox1, which is required for initial granule cell differentiationinthe adult hippocampus . Interestingly, we determined that both genes were downregulated by FZD1 knockdown. Therefore, it is possible that FZD1 regulates neuronal differentiationinthe adult hippocampus by mediating the activation ofthe canonical Wnt/β-catenin signaling pathway andthe expression of proneural genes.
Genes oftheWntandFrizzled class, expressed in HNSCC tissue and cell lines, have an established rolein cell morphogenesis anddifferentiation, and also they have oncogenic properties. We studied Wntand Fz genes as potential tumor-associated markers in HNSCC by qPCR. Expression levels ofWntand Fz genes in 22 unique frozen samples from HNSCC were measured. We also assessed possible correlation between the expression levels obtained in cancer samples in relation to clinicopathologic outcome. Wnt-1 was not expressed inthe majority ofthe HNSCC studied, whereas Wnt-5A was the most strongly expressed by the malignant tumors. Wnt-10B expression levels were related with higher grade of undifferentiation. Related to Fz genes, Fz-5 showed more expression levels in no-affectation of regional lymph nodes. Kaplan–Meier survival analyses suggest a reduced time of survival for low and high expression ofWnt-7A and Fz-5 mRNA, respectively. qPCR demonstrated that HNSCC express Wntand Fz members, and suggested that Wntand Fz signaling is activated in HNSCC cells.
In addition to the participation in neuronal polarization and morphogenesis, it is known that Wnt signalling regulates syn- apse formation and neurotransmission. Electrophysiological recordings in rat hippocampal slices showed that a blockade ofWnt signalling impairs long-term potentiation (LTP) (Chen et al. 2006), indicating the importance of this signalling path- way in synaptic plasticity (Vargas et al. 2014). Importantly, the expression and release of Wnts are regulated by neuronal activity (Chen et al. 2006; Wayman et al. 2006), supporting the notion that these ligands may play a role during neuronal transmission. Regarding the synaptic effects of Wnts, several years ago it was shown in cerebellar neurons that Wnt-7a regulates the clustering ofthe synaptic vesicle protein synapsin I (Hall et al. 2000; Lucas and Salinas 1997). The effect ofWnt signalling on presynaptic assembly has also been observed in hippocampal neurons, in which Wnt-7a, Wnt-3a andWnt-7b increase thepresynaptic puncta andthe synaptic vesicle cycle (Ahmad-Annuar et al. 2006; Cerpa et al. 2008; Davis et al. 2008). Moreover, electrophysiological recordings on adult rat hippocampal slices demonstrated that Wnt-7a increases neuro- transmitter release in CA3-CA1 synapses (Cerpa et al. 2008). Thepresynaptic effects of Wnts may be mediated by Wnt receptors located at the synaptic region. In hippocampal neu- rons, we determined that theWntreceptor FZD1 is present at thepresynaptic site where it regulates thepresynaptic assembly (Varela-Nallar et al. 2009). Also, FZD5 was shown to mediate synaptogenesis induced by Wnt-7a (Sahores et al. 2010). These findings have demonstrated a relevant role for theWnt signalling pathway inpresynapticdifferentiationandfunction. In addition, non-canonical Wnt signalling cascades have been associated with the postsynaptic apparatus. Electrophysiological recordings showed that Wnt-5a in- creases the amplitude of field excitatory postsynaptic poten- tials (fEPSP) and upregulates synaptic NMDAR currents, facilitating the induction of LTP (Cerpa et al. 2010, 2011), a two-step effect that is independently mediated by protein kinase C (PKC) and JNK (Cerpa et al. 2011). At the structural level, Wnt-5a modulates postsynaptic assembly, increasing clustering ofthe postsynaptic density protein-95 (PSD-95) (Farias et al. 2009), which is a key scaffold protein ofthe postsynaptic density.
Next we set out to determine whether these two ligands also influence VM development at a proliferative progenitor level. We therefore investigated whether CXCL6 or CXCL8 could be used to promote cell division and/or differentiationof progeni- tors in VM neurospheres. Mouse VM E11.5 neurospheres were grown inthe presence of bFGF and EGF. Similar to our results in primary precursor cultures, incorporation of BrdU inthe presence of mitogens was increased threefold by CXCL8, but not by CXCL6 (Fig. 3A), indicating that CXCL8 indeed pro- motes cell division in VM neurosphere cultures. Interestingly, the number of Ki67 ⫹ cells did not change (Fig. 3B), but when the labeling index (percentage BrdU⫹ and ki67⫹/total Ki67⫹) was examined, we found that CXCL8 clearly increased the proportion of Ki67 ⫹ cells that went through DNA synthesis (Fig. 3C). Since the pool of Ki67⫹ progenitors did not increase, our results suggest that the increase in BrdU by CXCL8 does not reflect an increase in proliferation. We then examined the num- ber of nestin-positive cells inthe neurospheres (Fig. 3D) andthe number of spheres (data not shown). However, these parameters did not change, indicating that the effect of CXCL8 was not on
To my thesis supervisors, Dr. Susana Balcells and Dr. Natalia Garcia Giralt. To Susana, for guiding me throughout this journey, sharing your experience with me. Your advices and comments have improved my work in every possible way as from the first Skype video call which started all this - . To Natalia, Nats… where shall I begin? I don’t think I ever mentioned to you how lucky I think I am to have had the opportunity to work with you. I learned so much, in so many different levels, that the professional and lab skills lab are only a fraction of it. You taught me to analyse and diagnose; you didn’t let me give up even when I felt I had enough. I know I failed you since I never cried during my thesis, but it will happened one day, and I promise I`ll send you a video - . You were always there to answer any question I had, professional or personal, in every hour ofthe day (even if it was during your vacations...). I have no doubt your advices and ideas, your views and thoughts will continue to guide me inthe future. I’m sure I’ll find myself many times thinking- What would have Nats done? (Or to make it less dramatic- I’ll just send you an email or call you to ask for your advice). Thank you. To Dani Grinberg, for letting me be a part of this group and for the patience to answer each and every question I had before I arrived andin my first months here (and I had many of them…) and for passing on so much of a your knowledge to me. It is
Figura 7. Fases de la patogènesi de la lipotoxicitat cardíaca. A) Fase compensada. L’elevada captació d’àcids grassos provoca l’increment de la biogènesi mitocondrial i de l’oxidació lipídiques a través del PPARα i del PGC-1α, de manera que es redueix l’ús de glucosa com a substrat. B) Fase de transició. El desequilibri entre la β-oxidació i la respiració mitocondrial condueix a un increment del contingut de triglicèrids cardíacs, així com a la formació d’espècies lipídiques tòxiques i de ROS que provoquen resistència cardíaca a la insulina. D’entre els mecanismes de resposta dels cardiomiòcits a aquest ambient tòxic destaquen les UCPs les quals, a través del desacoblament, redueixen la producció mitocondrial de ROS. C) Fase descompensada. Els mecanismes de resposta són insuficients i s’incrementa la formació de ROS, provocant disfunció mitocondrial i l’activació de vies inflamatòries que agreugen la situació lipotòxica. CD36, clúster de diferenciació 36; DAG, diacilglicerol; GLUT4, transportador de glucosa de tipus 4; IRS1, substrat 1 del receptor de la insulina; PDK4, cinasa 4 del complex de la piruvat deshidrogenasa; PGC-1α, coactivador 1α del receptor activitat per proliferadors peroxisomals γ; PPARα, receptor activat per proliferadors peroxisomals α; ROS, espècies reactives de l’oxigen; TG, triglicèrids; UCP2, UCP3, proteïnes desacoblants 2 i 3. Adaptat de Schilling, 2015.
However, the site from which the vascular progenitors for placental and embryo vasculogenesis emerge is still debated. It is now broadly accepted that inthe embryo vascular progenitors emerge from intra- and extra-embryonic mesodermal tissues , whilst inthe placenta they arise from the extra-embryonic meso- derm . Additionally, there is growing evidence for a crucial roleofthe yolk sac in embryo and placental vascular development . Indeed, using Ncx-1 knockout mice which fail to initiate cardiac contraction Lux et al.  showed that all the hematopoietic progenitor cells emerge from the yolk sac. Furthermore, a study in mice yolk sac demonstrated that vasculogenesis at this level is initiated by NO . These authors described the spatio-temporal expression pattern of iNOS and eNOS, which was related to vas- culogenesis inthe yolk sac. Inthe ﬁ rst stage, at 7 days of embryonic development (E7.0), iNOS-derived NO synthesized by endodermal cells induces thedifferentiationof adjacent extra-embryonic mesodermal cells to form a primary capillary plexus. After that, eNOS expression increases inthe yolk sac mesodermal cells [36,42] accompanied by a decrease in iNOS expression inthe endoderm . Experimental inhibition of NOS activity at E6.5 completely arrests the development ofthe primary capillary plexus . Altogether, these data suggest that NO could be crucial for placental vasculogenesis.
Animals were supplied by Harlan Laboratories. All animals were naïve before any treatment. For radioligand binding experiments, fresh tissue from a total of 21 young adult male Macaca fascicularis primates (between 4 and 5.5 years old) were used. Proximity ligation assays were performed in samples from 6 additional monkeys (see details in 2.2). Animal handling was conducted in accordance with the European Council Directive 86/609/EEC as well as in agreement with the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research. The experi- mental design was approved by the Ethical Committee for Animal Testing ofthe University of Navarra (ref: 018/2008) as well as by the Department of Health from the Government of Navarra (ref: NA-UNAV-04-08). Ofthe 21 animals devoted to perform binding assays in freshly isolated tissue, 11 were naïve and 10 monkeys were treated with systemic delivery ofthe dopaminergic neurotoxin MPTP (1- Methyl-4-phenyl-1,2,3,6-tetrahydropyridine from Sigma, Madrid, Spain) to induce a bilateral parkinsonian syndrome (Rico et al., 2010). Animals received a weekly injection of MPTP (0.2 mg/kg i/v; accumulated doses ranging from 5 to 7 mg/kg) until reaching a non-reversible parkinsonian syndrome. The severity ofthe MPTP- induced parkinsonism was evaluated using a clinical rating scale (Kurlan et al., 1991). This scale rates parkinsonian motor symptoms such as facial expression (0e3), resting tremor (0e3), action or intention tremor (0e3), posture (0e2), bra- dykinesia (0e4), balance coordination (0e3), gait (0e3), gross motor skills ofthe upper limb (0e3) and lower limb (0e3), and defense reaction (0e2) in an accu- mulating scale where the maximum score (i.e., highest severity) is 29. Once primates reached minimum score of 21 points and above, the MPTP treatment was dis- continued for a wash-out period of 2 months to ensure that the parkinsonian syn- drome was fully stabilized. At the end ofthe stabilization period, the PD
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive abilities, mainly caused by synaptic impairments and neuronal death in specific regions ofthe brain (Mattson, 2004; Selkoe, 2001). Accumulation of amyloid-β peptide (Aβ) in senile plaques mostly located in hippocampus, cortex and other brain areas linked to cognitive processes, is considered one ofthe major pathological hallmarks of AD brains (Selkoe, 2001). Most recent evidence indicates that soluble Aβ oligomers rather than plaques determine cognitive decline (Lacor et al., 2007; Shankar et al., 2007). Actually, the severity of dementia in AD patients is strongly correlated with the levels of soluble Aβ oligomers (Lue et al., 1999; McLean et al., 1999). Thus, the current view of AD considers Aβ oligomers as a key factor in synaptic dysfunction linked to early stages of AD (Sakono and Zako, 2010). Indeed, Aβ oligomers isolated of AD brains can affect memory and disrupt hippocampal synaptic plasticity through inhibition of long-term potentiation (LTP) and increasing long-term depression (LTD) (Cleary et al., 2005; Shankar et al., 2008). However, Aβ-induced blockade of LTP can be overcome by inhibitors of Aβ oligomerization (Walsh et al., 2005), suggesting that AD cognitive impairment might be due to a direct effect of Aβ oligomers on the synaptic region. In fact, Aβ oligomers can affect excitatory synaptic transmission by reducing the amplitude ofthe field excitatory postsynaptic potentials (fEPSP) at hippocampal synapses (Cerpa et al., 2010; Hermann et al., 2009).
According to SJT, when people are chronically or situationally high on need for control, they would be more motivated to justify the social arrangements, which in turn would predict higher psychological well-being. If we extend this argument, when individuals legitimate the social systems to which they belong, they would perceive a higher personal control, because they have already fulfilled their need for control through system justification. Andthe perception of control is the distinctive mechanism that explains an increase in psychological well-being. This argument is coherent with several studies which propose that loss of control is associated with anxiety (e.g., Tullet, Kay, & Inzlicht, 2015) and perceiving personal control is related to well-being (e.g., DeNeve & Cooper, 1998; Lang & Heckhausen, 2001; Spector et al., 2017; Taylor & Brown, 1988), and it is relatively understudied within SJT literature because most studies have been focused on the direct association (e.g., Godfrey et al., 2017; Harding & Sibley, 2013; O ’ Brien & Major, 2005; Osborne & Sibley, 2013; Sengupta, Greaves, Osborne, & Sibley, 2017; Vargas-Salfate, 2017). One ofthe studies that has contrasted personal control as the mechanism involved inthe palliative functionof ideology did not find a significant mediation of control inthe effect of Protestant ethic (i.e., a system justifying ideology) on psychological well- being (Quinn & Crocker, 1999, Study 1), although other research has found that control mediated the effect of meritocratic beliefs on self-esteem (McCoy, Wellman, Cosley, Saslow, & Epel, 2013, Study 1).
Two issues underlie the democratic character ofthe ECB and its obligation to accountability. It is first necessary to consider whether to impose the supremacy of economics over politics, or vice versa. This matter is a core principle inthe creation of supranational institutions andthe definition ofthe functions to be developed. Must be the conduct of monetary policy inthe hands of a team of technocrats who have not been elected by the people or, on the contrary, must lie inthe political arena? No doubt there are arguments for the former in order to keep people away monetary policy short-term political interests, especially at election time. The argument that monetary policy should be designed to maintain price stability over the medium and long term, protecting the EU economy infla- tion risk, brings a high degree of consensus inthe Eurozone. However, it is also necessary to note that the jobs and growth should not be excluded in any case the objectives of economic policy. If we believe that monetary policy is part of eco- nomic policy and having an impact on employment and growth, it seems reason- able that the latter among the objectives ofthe first. If no goals are for the ECB so compromising containment of inflation, at least expect from this institution that does business without harming them inthe extreme.
As we have already mentioned, THs are critical for many processes like cell growth, differentiation, metabolism, and homeostasis maintenance (1). The classical effects of THs are initiated when T3 binds to their nuclear receptors (TRs) that interact with specific responding elements (TREs) inthe promoters of target genes. The conformational change promoted by the binding of T3 to TRs induces the exchange of corepressors for coactivators, thus leading to gene transcription on responsive genes (2, 3). TRs are encoded by two different genes: the THRA located in chromosome 17, andthe THRB located in chromosome 3, codifying for the TRα and TRβ proteins, respectively (2, 3). The expression of these isoforms differs during the embryonic development andin adult tissues (1). Mutations of TRs have been detected in several cancers, such as erythroleukemia and liver, kidney and thyroid cancers (13). These mutations have been suggested to be a selective advantage for malignant transformation (85). Thus, the mutation (86, 87) or aberrant expression (88) of TRs has been demonstrated in several cancer cell lines. Also, biopsies of patients with gastrointestinal tumors showed increased levels of TR α 1 that correlate with Wnt pathway activation and tumor proliferation (89).
neurotransmitter (Tsuru et al., 2002) and several studies indicate that this neuronal signal is involved inthe control of gap junction-mediated intercellular communication in different cell types. Twenty years ago, Giaume et al. found that b - and a - adrenergic signaling possesses a dual regulatory effect on gap junction channels in primary cultures of mouse striatal astrocytes (Giaume et al., 1991). While the increase in cAMP associated to b -adrenoceptor activation enhanced dye- coupling in these cells, stimulation of a 1-adrenergic receptor with noradrenaline inhibited the intercellular gap junction communication through a phospholipase C-dependent signal- ing pathway (Giaume et al., 1991). Likewise, the electrical coupling of ventricular cardiac cells was observed to be strongly reduced by the stimulation with phenylephrine, an a 1- adrenoceptor agonist (De Mello, 1997). Consistent with these ﬁ ndings, electrical stimulation of perivascular nerves or exogenous application of noradrenaline inhibited the longitu- dinal conduction of acetylcholine-induced vasodilation in retractor muscle arterioles of hamster (Haug and Segal, 2005). As conduction of vasomotor responses depends on spread of an electrical signal along the vessel length through gap junctions (Figueroa et al., 2003, 2004; Figueroa and Duling, 2009), the inhibition ofthe conducted vasodilation induced by acetyl- choline suggest that sympathetic activation reduces the gap
The authors wish to thank the members of their laboratories for comments on this Review. Work in N.C.I.’s laboratory is supported by grants from Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) through a Base Centre for Excellence in Science and Technology, FONDAP (Fondo De Areas Prioritarias)-Biomedicine number 13980001 andthe Millennium Institute for Fundamental and Applied Biology (MIFAB). Work in E.A.’s laboratory is supported by grants from the Swedish Foundation for Strategic Research (INGVAR (individual grant for the advancement of research leaders) and CEDB (Center of Excellence in Developmental Biology)), t h e S we d i s h R e s e a rc h C o u n c i l ( V R 2 0 0 8 : 2 811 , VR2008:3287 and DBRM (Developmental Biology for Regenerative Medicine)), the Norwegian Research Council, the Karolinska Institutet andthe European Commission (Neurostemcell).
In steady-state or inthe absence ofWnt stimulation, the cyto- plasmic cellular levels of β -catenin are low since casein kinase-1 α (CK-1 α ) and glycogen synthase kinase-3 β (GSK-3 β ) sequentially phosphorylates the protein, targeting β -catenin for ubiquitination and proteasome degradation (Aberle et al., 1997; Liu et al., 2002; Nusse and Varmus, 2012). Instead, inthe presence ofWnt ligand, Wnt binds both Fz and LRP5/6 forming a Wnt-receptor com- plex that recruits the protein disheveled (Dvl), which oligomerizes inthe plasma membrane making a platform for the recruitment and allocation ofthe “β-catenin destruction complex” (Bilic et al., 2007). This complex is formed by the scaffold protein Axin, GSK- 3 β , CK-1 α , andthe tumor suppressor adenomatous polyposis coli (APC; Cliffe et al., 2003; Schwarz-Romond et al., 2007; Gao and Chen, 2010). Once the complex is recruited, CK-1 α phos- phorylates LRP5/6 which causes the inhibition ofthe “β-catenin destruction complex” (Figure 1). As consequence of this inhibi- tion, β-catenin is stabilized and accumulated inthe cytoplasm and can enter to the nucleus to activate the transcription ofWnt target genes (Logan and Nusse, 2004) under the control ofthe TCF/LEF transcription factors (T-cell factor, TCF /lymphoid enhancer fac- tor, LEF; Clevers and Nusse, 2012). There are several Wnt target genes that are activated in this process, including c-Myc, cyclin D1, Axin2, and Ca 2 + -calmodulin-dependent protein kinase type IV (CamKIV; Toledo et al., 2008; Arrazola et al., 2009; Hodar et al., 2010; Nusse and Varmus, 2012).
Interleukin-1b (IL-1b) is an important trophic factor inthe nervous system (NS). IL-1b is ubiquitously expressed from very early stages during the development ofthe amphibian NS and its action has been demonstrated in vitro on survival, proliferation and diﬀer- entiation in mammalian embryos. In this report, we show that IL-1b is immunocytochemically expressed in embryonic spinal cord from early stages, both in rat (embryonic day 12) andin chicken (stage 17-HH), in neuroepithelial cells and nerve ﬁbres, dorsal root ganglia, anterior and posterior roots ofthe spinal nerves, andinthe ﬁbres of these nerves. Our in vivo experiments on chick embryos, with micro- beads impregnated with IL-1b implanted laterally to the spinal cord at the level ofthe wing anlage, demonstrate that this cytokine pro- duces a statistically signiﬁcant increase in nuclear incorporation of BrdU at the dorsal level and a reduction of this at the ventral level, whereas local immunoblocking with anti-IL-1b antibodies causes a dorsal reduction of BrdU incorporation and alters ventral diﬀeren- tiation. These data demonstrate that IL-1b plays a part in controlling proliferation and early diﬀerentiation during the development ofthe spinal cord in chick embryos.