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Tumour phenotypes in Prss56Cre, R26tdTom, Nf1flox/flox and Prss56Cre, R26tdTom, Nf1flox/- mice

Supplementary Materials and Methods

Supplementary Figures S1-S6

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

Patients carrying an inactive NF1 allele develop tumors of Schwann cell origin called neurofibromas (NF). Genetically engineered mouse models have significantly enriched our understanding of plexiform forms of NFs (pNF). However, this has not been the case for cutaneous neurofibromas (cNF), observed in all NF1 patients, as no previous model recapitulates their development. Here, we show that conditional Nf1 inactivation in Prss56-positive boundary cap cells leads to bona fide pNFs and cNFs. This work identifies subepidermal glia as a likely candidate for the cellular origin of cNFs and provides insights on disease mechanisms, revealing a long, multistep pathologic process in which inflammation-related signals play a pivotal role. This new mouse model is an important asset for future clinical and therapeutic investigations of NF1-associated neurofibromas. Significance: Patients affected by NF1 develop numerous cNFs. We present a mouse model that faithfully recapitulates cNFs, identify a candidate cell type at their origin, analyze the steps involved in their formation, and show that their development is dramatically accelerated by skin injury. These findings have important clinical/therapeutic implications. This article is highlighted in the In This Issue feature, p. 1

Summary Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration., Graphical Abstract, Highlights • Reliable method for generation of astrocytes from human iPSCs and ESCs • Generated astrocytes are functional and inflammation-responsive • Generated astrocytes share properties with primary astrocytes in vitro • This method is a valuable tool for disease modeling of neuroinflammation, In this article, Gage and colleagues developed a reliable method for generating functional astrocytes from human pluripotent stem cells. This protocol uses an intermediate glial progenitor stage and generates inflammation-responsive astrocytes, providing an important tool to model neurological diseases in-a-dish, enabling the study of neurological disorders with an inflammatory component.

Bipolar disorder (BD) is a progressive psychiatric disorder with more than 3% prevalence worldwide. Affected individuals experience recurrent episodes of depression and mania, disrupting normal life and increasing the risk of suicide greatly. The complexity and genetic heterogeneity of psychiatric disorders have challenged the development of animal and cellular models. We recently reported that hippocampal dentate gyrus (DG) neurons differentiated from induced pluripotent stem cell (iPSC)-derived fibroblasts of BD patients are electrophysiologically hyperexcitable. Here we used iPSCs derived from Epstein-Barr virus-immortalized B-lymphocytes to verify that the hyperexcitability of DG-like neurons is reproduced in this different cohort of patients and cells. Lymphocytes are readily available for research with a large number of banked lines with associated patient clinical description. We used whole-cell patch-clamp recordings of over 460 neurons to characterize neurons derived from control individuals and BD patients. Extensive functional analysis showed that intrinsic cell parameters are very different between the two groups of BD neurons, those derived from lithium (Li)-responsive (LR) patients and those derived from Li-non-responsive (NR) patients, which led us to partition our BD neurons into two sub-populations of cells and suggested two different subdisorders. Training a Naïve Bayes classifier with the electrophysiological features of patients whose responses to Li are known allows for accurate classification with more than 92% success rate for a new patient whose response to Li is unknown. Despite their very different functional profiles, both populations of neurons share a large, fast after-hyperpolarization (AHP). We therefore suggest that the large, fast AHP is a key feature of BD and a main contributor to the fast, sustained spiking abilities of BD neurons. Confirming our previous report with fibroblast-derived DG neurons, chronic Li treatment reduced the hyperexcitability in the lymphoblast-derived LR group but not in the NR group, strengthening the validity and utility of this new human cellular model of BD.

International audience; Figure 1. Le trouble bipolaire est une mala-die psychiatrique relevant des troubles de l'humeur. Les troubles de l'humeur se dis-tribuent selon un spectre, en fonction de la durée et l'intensité des épisodes dépressifs et maniaques. Le trouble unipolaire, ou dépression majeure, présente seulement des épisodes dépressifs ; le trouble bipo-laire de type II présente en plus des épi-sodes hypomaniaques ; le trouble bipolaire de type I comporte des phases dépressives et maniaques sévères. La cyclothymie est caractéri-sée par une succession de phases dépressives et euphoriques de faible intensité. La dysthymie correspond à un état dépressif chronique, moins intense que la dépression majeure.

Schwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behavior ex vivo and in vivo after exogenous transplantation in the demyelinated spinal cord. The data highlight for the first time that SC migrate preferentially along blood vessels in perivascular extracellular matrix (ECM), avoiding CNS myelin. We demonstrate in vitro and in vivo that this migration route occurs by virtue of a dual mode of action of Eph/ephrin signaling. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinβ1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair. Electronic supplementary material The online version of this article (10.1007/s00401-019-02011-1) contains supplementary material, which is available to authorized users.

Patients carrying an inactive

International audience; Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20(-/-) mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.

Interleukin-27 (IL-27) suppresses immune responses through inhibition of the development of IL-17 producing Th17 cells and induction of IL-10 production. We previously showed that forced expression of early growth response gene 2 (Egr-2), a transcription factor required for T-cell anergy induction, induces IL-10 and lymphocyte activation gene 3 expression and confers regulatory activity on CD4(+) T cells in vivo. Here, we evaluated the role of Egr-2 in IL-27-induced IL-10 production. Among various IL-10-inducing factors, only IL-27 induced high levels of Egr-2 and lymphocyte activation gene 3 expression. Intriguingly, IL-27 failed to induce IL-10 in Egr-2-deficient T cells. IL-27-mediated induction of Prdm1 that codes B lymphocyte induced maturation protein-1, a transcriptional regulator important for IL-10 production in CD4(+) T cells, was also impaired in the absence of Egr-2. Although IL-27-mediated IL-10 induction was dependent on both STAT1 and STAT3, only STAT3 was required for IL-27-mediated Egr-2 induction. These results suggest that IL-27 signal transduction through Egr-2 and B lymphocyte induced maturation protein-1 plays an important role in IL-10 production. Furthermore, Egr-2-deficient CD4(+) T cells showed dysregulated production of IFN-γ and IL-17 in response to IL-27 stimulation. Therefore, Egr-2 may play key roles in controlling the balance between regulatory and effector cytokines.

In this study, we have investigated the expression and function of the transcription factor early growth response factor 2 (Egr2)/Krox20 in the developing anterior pituitary. Egr2 is initially expressed in all differentiating hormonal cells types, but its expression is mostly restricted to the somatotroph lineage after birth. Egr2 knockout results in anterior pituitary hypoplasia. However, the analysis of a conditional mutant demonstrates that this phenotype does not originate from a lack of Egr2 expression in the pituitary. Using an Egr2 allele driving a Cre-activable toxin gene, we performed a genetic ablation of Egr2-positive cells in the pituitary. During the postnatal period, this ablation leads to specific and progressive depletion of the somatotroph population, creating a novel model of early-onset isolated GH deficiency (GHD). Mutant animals were subjected to a complete metabolic analysis, revealing atypical and expected features. Consistent with an adult-onset isolated GHD model, mutant animals are hypoglycemic and display increased insulin sensitivity and glucose clearance. This latter phenotype is in contrast to the glucose intolerance observed in another early-onset GHD model. Surprisingly, increased insulin sensitivity is not accompanied by a modified balance between fat and lean tissues, but by reduced metabolic adaptability between glucose and lipid oxidation conditions. This suggests that the relationship between these metabolic features and insulin sensitivity should be reconsidered. In conclusion, our mutant may be a valuable genetic model with which to study the effects of long-term GH deficiency, in conditions of normal pancreatic function and unaffected balance between fat and glucose metabolism.

Adult neurogenesis in the mammalian brain is restricted to specific regions, such as the dentate gyrus (DG) in the hippocampus and the subventricular zone (SVZ) in the walls of the lateral ventricles. Here, we used a mouse line carrying a knock-in of Cre recombinase in the Prss56 gene, in combination with two Cre-inducible fluorescent reporters (Rosa26 mTmG and Rosa26 tdTom ), to perform genetic tracing of Prss56-expressing cells in the adult brain. We found reporter-positive cells in three neurogenic niches: the DG, the SVZ and the hypothalamus ventricular zone. In the prospective DG, Prss56 is expressed during embryogenesis in a subpopulation of radial glia. The pattern of migration and differentiation of reporter-positive cells during development recapitulates the successive steps of DG neurogenesis, including the formation of a subpopulation of adult neural stem cells (NSC). In the SVZ, Prss56 is expressed postnatally in a subpopulation of adult NSC mainly localized in the medial-ventral region of the lateral wall. This subpopulation preferentially gives rise to deep granule and Calbindin-positive periglomerular interneurons in the olfactory bulb. Finally, Prss56 is also expressed in a subpopulation of α2-tanycytes, which are potential adult NSCs of the hypothalamus ventricular zone. Our observations suggest that some α2-tanycytes translocate their soma into the parenchyma and may give rise to a novel cell type in this territory. Overall, this study establishes the Prss56 Cre line as an efficient and promising new tool to study multiple aspects of adult neurogenesis in the mouse.

Frutos, Cristina A. de et al., The neocortex undergoes extensive developmental growth, but how its architecture adapts to expansion remains largely unknown. Here, we investigated how early born Cajal-Retzius (CR) neurons, which regulate the assembly of cortical circuits, maintain a dense superficial distribution in the growing neocortex. We found that CR cell density is sustained by an activity-dependent importation of olfactory CR cells, which migrate into the neocortex after they have acted as axonal guidepost cells in the olfactory system. Furthermore, using mouse genetics, we showed that CR cell density severely affects the architecture of layer 1, a key site of input integration for neocortical networks, leading to an excitation/inhibition ratio imbalance. Our study reveals that neurons reenter migration several days after their initial positioning, thereby performing sequential developmental roles in olfactory cortex and neocortex. This atypical process is essential to regulate CR cell density during growth, which in turn ensures the correct wiring of neocortical circuitry. Video Abstract, We are grateful to B. Mathieu and the IBENS Imaging Facility (France BioImaging, supported by ANR-10-INBS-04, ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-0001-02 PSL∗ Research University, “Investments for the future”; NERF N°2011-45; FRM DGE 20111123023; and FRC Rotary International France). We also thank N. Boggetto and the Flow Cytometry platform of the ImagoSeine facility (France BioImaging, supported by ANR-10-INSB-04, “Investments for the future”). C.A.F. was supported by the IEF Marie Curie program and the Labex Memolife, “Investements for the future” (ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001-02 PSL∗ Research University). G.B. was funded by Labex Memolife, FRM, and Région Ile de France. Y.A. was the recipient of a fellowship from ARC and FRM. The work in P.C. group was supported by FRM (DEQ20121126545) and work in A.P. group by grants from the ANR (ANR-2011-BSV4-023-01) and FRM (DEQ20130326521). This work was supported by grants from MRT, CNRS, INSERM, and the EURYI program to S.G. S.G. is an EMBO YIP awardee and part of the ENP program.

Endoneurial fibroblast-like cells (EFLCs) have been described for more than 60 years, but the embryology, functions, and pathology of these cells are not well defined. Several hypotheses of their origin have been proposed. A previous study suggesting that they were of neural crest origin is supported by our data in humans. This lineage might account for EFLCs having multiple biologic functions and involvement in pathological processes. Here, we review what is known about the origin; functions in collagen synthesis, phagocytosis, inflammatory responses, and immune surveillance; and the pathological alterations of EFLCs based on the literature and on our personal observations.

The transcription factor EGR2 is expressed in Schwann cells, where it controls peripheral nerve myelination. Mutations of EGR2 have been found in patients with congenital hypomyelinating neuropathy or Charcot-Marie-Tooth disease type 1D. In a patient with congenital amyelinating neuropathy, we observed pathological abnormalities recapitulating the peripheral nervous system phenotype of homozygous Egr2-null mice. This patient, born from consanguineous parents, showed no EGR2 immunoreactivity in Schwann cells and harbored a homozygous 10.7-kilobase-long deletion encompassing a myelin-specific enhancer of EGR2. This regulatory mutation is the first genetic abnormality associated with congenital amyelinating neuropathy in humans.

The transcription factor Sox10 is expressed throughout Schwann cell development and has already been shown to be essential for specification and for the identity and further development of immature Schwann cells. Here, we show that Sox10 is also required in Schwann cells for establishing the myelinating state. This is concluded from the fact that a peripheral neuropathy develops in mice in which Sox10 is deleted by a Cre recombinase whose expression is under control of Krox20 regulatory elements. This neuropathy is characterized by altered marker gene expression along the peripheral nerve, decreased conductivity, and severe persistent hypomyelination. As the Cre recombinase is additionally active in boundary cap cells, we also analyzed the role of Sox10 during embryogenesis in establishment and maintenance of the boundary between central and peripheral nervous systems. Sox10 deletion did not affect establishment or survival of boundary cap cells but appeared to compromise barrier function as cells expressing oligodendrocyte and astrocyte markers were no longer restricted to the central nervous system, and instead found in peripheral nerves. We infer that in addition to its many roles in Schwann cells, Sox10 is also important for the integrity of the boundary between central and peripheral nervous systems. © 2012 Wiley Periodicals, Inc.

Boundary cap cells (BC), which express the transcription factor Krox20, participate in the formation of the boundary between the central nervous system and the peripheral nervous system. To study BC stemness, we developed a method to purify and amplify BC in vitro from Krox20 Cre/+ , R26R YFP/+ mouse embryos. We show that BC progeny are EGF/FGF2-responsive, form spheres, and express neural crest markers. Upon growth factor withdrawal, BC progeny gave rise to multiple neural crest and CNS lineages. Transplanted into the developing murine forebrain, they successfully survived, migrated, and integrated within the host environment. Surprisingly, BC progeny generated exclusively CNS cells, including neurons, astrocytes, and myelin-forming oligodendrocytes. In vitro experiments indicated that a sequential combination of ventralizing morphogens and glial growth factors was necessary to reprogram BC into oligodendrocytes. Thus, BC progeny are endowed with differentiation plasticity beyond the peripheral nervous system. The demonstration that CNS developmental cues can reprogram neural crest-derived stem cells into CNS derivatives suggests that BC could serve as a source of cell type-specific lineages, including oligodendrocytes, for cell-based therapies to treat CNS disorders.

During development, boundary cap cells (BC) and neural crest cell (NCC) derivatives generate Schwann cells (SC) of the spinal roots and a subpopulation of neurons and satellite cells in the dorsal root ganglia. Despite their stem-like properties, their therapeutic potential in the diseased central nervous system (CNS) was never explored. The aim of this work was to explore BC therapeutic potential for CNS remyelination. We derived BC from Krox20Cre × R26RYfp embryos at E12.5, when Krox20 is exclusively expressed by BC. Combining microdissection and cell fate mapping, we show that acutely isolated BC are a unique population closely related but distinct from NCC and SC precursors. Moreover, when grafted in the demyelinated spinal cord, BC progeny expands in the lesion through a combination of time-regulated processes including proliferation and differentiation. Furthermore, when grafted away from the lesion, BC progeny, in contrast to committed SC, show a high migratory potential mediated through enhanced interactions with astrocytes and white matter, and possibly with polysialylated neural cell adhesion molecule expression. In response to demyelinated axons of the CNS, BC progeny generates essentially myelin-forming SC. However, in contact with axons and astrocytes, some of them generate also myelin-forming oligodendrocytes. There are two primary outcomes of this study. First, the high motility of BC and their progeny, in addition to their capacity to remyelinate CNS axons, supports the view that BC are a reservoir of interest to promote CNS remyelination. Second, from a developmental point of view, BC behavior in the demyelinated CNS raises the question of the boundary between central and peripheral myelinating cells.

Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

International audience; The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

The zinc finger transcription factor Krox20 plays an essential role in the vertebrate hindbrain segmentation process. It positively or negatively controls a large variety of other regulatory genes, coordinating delimitation of segmental territories, specification of their identity, and maintenance of their integrity. We have investigated the molecular mechanisms of Krox20 transcriptional control by performing a detailed structure-function analysis of the protein in the developing chick hindbrain. This revealed an unsuspected diversity in the modes of action of a transcription factor in a single tissue, since regulation of each of the five tested target genes requires different parts of the protein and/or presumably different co-factors. The multiplicity of Krox20 functions might rely on this diversity. Investigation of known Krox20 co-factors was initiated in relation to this analysis. Nab was shown to act as a negative feedback modulator of the different Krox20 activating functions in the hindbrain. HCF-1 was found to bind to a Krox20 N-terminal region, which was shown to rely on multiple elements, including acidic domains, to convey Nab activation and Krox20 autoregulation.

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis. © 2008 Elsevier Inc. All rights reserved., This work was supported by the grant BFU2006-05604 from the Spanish Ministry of Education and Science to C.P. and BFU2007-61030, Tercel, Ciberned and Junta de Castilla y León to T.S. C.V.E. was supported by a FI fellowship from AGAUR (Generalitat de Catalunya), and by CONACYT (Mexico); E.D.F. was supported by a FPI fellowship from the MEC, Spain.

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Resume Les cellules de la crete neurale donnent naissance a la quasi-totalite des derives neuronaux et gliaux du systeme nerveux peripherique (SNP). Notre groupe a recemment montre qu’au sein du SNP les cretes neurales generent egalement un autre type de derivees, appelees les cellules de capsules frontieres (CF). Ces cellules sont localisees a l’interface entre le systeme nerveux central (SNC) et le SNP, au niveau des points d’entree et de sortie des nerfs et jouent un role cle dans la formation et le maintien du SNP. L’ablation genetique des CF conduit a un phenotype surprenant : alors que la croissance axonale des motoneurones n’est pas perturbee, leurs corps cellulaires migrent a l’exterieur de la moelle epiniere en se translocant le long des axones. Ainsi, les CF sont necessaires au confinement des motoneurones dans le SNC. Les experiences de tracage in vivo des cellules CF ont revele qu’elles migrent le long des racines dorsales et ventrales des nerfs peripheriques et atteignent les ganglions sensoriels. Le long des racines, les cellules derivees des CF donnent naissance a la totalite des precurseurs de cellules de Schwann. Dans les ganglions, ces cellules se differencient d’une part en cellules gliales satellites et d’autre part en neurones nociceptifs. Ainsi la capsule frontiere represente une population de cellules potentiellement pluripotentes qui pourrait constituer un reservoir de cellules souches neurales susceptibles d’intervenir dans le developpement ou la reparation du SNP.

The homeodomain transcription factor vHNF1 plays an essential role in the patterning of the caudal segmented hindbrain, where it participates in the definition of the boundary between rhombomeres (r) 4 and 5 and in the specification of the identity of r5 and r6. Understanding the molecular basis of vHnf1 own expression therefore constitutes an important issue to decipher the regulatory network governing hindbrain patterning. We have identified a highly conserved 800-bp enhancer element located in the fourth intron of vHnf1 and whose activity recapitulates vHnf1 neural expression in transgenic mice. Functional analysis of this enhancer revealed that it contains two types of essential motifs, a retinoic acid response element and two half T-MARE sites, indicating that it integrates direct inputs from the retinoic acid signaling cascade and MAF-related factors. Our data suggest that MAFB, which is itself regulated by vHNF1, acts as a positive modulator of vHnf1 in r5 and r6, whereas another MAF-related factor is absolutely required for the expression of vHnf1 in both the hindbrain and the spinal cord. We propose a model accounting for the initiation and maintenance phases of vHnf1 expression and for the establishment of the r4/r5 boundary, based on cooperative contributions of Maf factors and retinoic acid signaling.

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs1–3. A recent genome-wide association study4 identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls revealed 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1–dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

The zinc-finger transcription factor Krox20 constitutes a key regulator of hindbrain development, essential for the formation and specification of rhombomeres (r) 3 and 5. It is in particular responsible for the respective activation and repression of odd- and even-numbered rhombomere-specific genes, which include Hox genes. In this study, we have identified PIASxbeta as a novel direct interactor of Krox20. In addition, we found that PIASxbeta is able to activate the r4-specific gene Hoxb1. Binding of Krox20 prevents this activation, providing a molecular basis for the repression of Hoxb1 by Krox20. The same domain in the Krox20 protein, the zinc-fingers, is involved in DNA binding for transcriptional activation and in interaction with PIASxbeta for transcriptional repression, although the actual precise contacts are different. Our findings add an additional level in the complexity of Hox gene regulation and provide an example of how a single regulator can coordinate the activation and repression of a set of genes by very different mechanisms, acting as a molecular switch to specify cell identity and fate.

PMID:16615895, Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain., We have been supported by grants from Spanish Government BMC2002-03337, GVA GRUPOS03/053, NARSAD, the European Commission through STREP contract number 005139 (INTERDEVO), and the EURYI program to O.M.; by grants from INSERM, MENRT, ARC, and AFM to P.C.; by NIH grant NS29071 to L.R. and D.T.; and by the Picasso PAI/Programa de Acciones Integradas to O.M. and S.G. G.L.-B. is a “Ramón y Cajal” Investigator from the CSIC. F.B. was supported by a fellowship from the Académie Nationale de Médecine. S.G. is a recipient of the Human Frontier Science Program Organization CDA. O.M. is an EMBO Young Investigator, a NARSAD Young Investigator, and an EURYI Awardee.

Boundary cap (BC) cells are neural crest derivatives that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various knock-in alleles of the mouse gene Egr2 (also known as Krox20), the expression of which, in trunk regions, is initially restricted to BC cells, we were able to trace BC cell progeny during development and analyze their fate. Trunk BC-derived cells migrated along peripheral axons and colonized spinal nerve roots and dorsal root ganglia (DRG). All Schwann cell precursors occupying the dorsal roots were derived from BC cells. In the DRG, BC-derived cells were the progenitors of both neurons, mainly nociceptive afferents, and satellite cells. These data indicate that BC cells constitute a source of peripheral nervous system (PNS) components that, after the major neural crest ventrolateral migratory stream, feeds a secondary wave of migration to the PNS.

Helix-loop-helix transcription factors of the Ebf/Olf1 family have previously been implicated in the control of neurogenesis in the central nervous system in both Xenopus laevis and the mouse, but their precise roles have remained unclear. We have characterised two family members in the chick, and have performed a functional analysis by gain- and loss-of-function experiments. This study revealed several specific roles for Ebf genes in the spinal cord and hindbrain regions of higher vertebrates, and enabled their precise positioning along the neurogenic cascade.During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. We show that antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis.Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilisation of the committed state. Finally,gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS.

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.

Neuroepithelial b2T cells were derived from the hindbrain and the spinal cord of mouse transgenic embryos, which expressed SV40 T antigen under the control of a Hoxb2 enhancer. Strikingly, b2T cell lines of either origin exhibit a very similar gene expression pattern, including markers of the hindbrain and the spinal cord, such as Hox genes, but not of more anterior cephalic regions. In addition, the broad expression pattern of b2T cells, probably linked to culture conditions, appeared to be appropriately modulated when the cells were reimplanted at different longitudinal levels into chick host embryos, suggesting that these cells are responsive to exogenous signalling mechanisms. Further support for these allegations was obtained by culturing b2T cells in defined medium and by assessing the expression of Krox20, an odd-numbered rhombomere marker, which appeared to be modulated by a complex interplay between FGF, retinoic acid (RA), and noggin. With respect to these as yet unique properties, b2T cells constitute an original alternative tool to in vivo models for the analysis of molecular pathways involved in the patterning of the neural tube.

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.

The bZip transcription factor Mafb is expressed in two segments of the developing vertebrate hindbrain: the rhombomeres 5 and 6. Loss of Mafb expression in the mouse mutant kreisler leads to elimination of r5 and to alterations of r6 regional identity. Here, we further investigated the role of Mafb in hindbrain patterning using gain-of-function experiments in the chick embryo. Our work has revealed novel functions for Mafb, including a positive autoregulatory activity, the capacity to repress Hoxb1 expression, and the capacity to synergise with or antagonise Krox20 activity. These different activities appear to be spatially restricted in the hindbrain, presumably due to interactions with other factors. Reinvestigation of the kreisler mutation indicated that it also results in an ectopic activation of Mafb in rhombomere 3, accounting for the previously described molecular alterations of this rhombomere in the mutant. Together, these data allow us to refine our view of the dual function of Mafb in both segmentation and specification of anteroposterior identity in the hindbrain.

The calcium-dependent adhesion protein E-cadherin is present in noncompacted regions of myelin sheaths in the peripheral nervous system. There, it is localized to electron-dense structures between membranes of the same Schwann cell referred to as autotypic adherens junctions. It has been suggested that the failure of E-cadherin-mediated adhesion might cause demyelination that proceeds in certain pathological states. To test the requirement of E-cadherin in peripheral nerves, we used tissue-specific gene ablation techniques based on the Cre/LoxP system. We show that E-cadherin deficiency does not cause significant demyelination up to the age of 15 months. Immunostainings for nodal sodium channels, the paranodal protein Caspr1, and the juxtaparanodal potassium channels Kv1.1 and Kv1.2 revealed that E-cadherin is not necessary to maintain the general functional architecture of the nodal region. On the ultrastructural level, we detected a widening of the outer mesaxon accompanied by a loss of electron-dense cytoplasmic areas. We conclude that E-cadherin is required for the proper establishment and/or the maintenance of the outer mesaxon in myelinated PNS fibers but is dispensable for proper nerve function.

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed at high relative levels in the rhombomeres (r) 5 and 6, and 5, respectively. The single enhancer elements responsible for these activities have been identified previously and shown to constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. Here, we have analysed the contribution of the transcription factor Krox20, present in r3 and r5. Genetic analyses demonstrated that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, revealed that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to their domain of overlap, r5. These analyses also suggested contributions from an Ets-related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development.

During embryonic development, restricted expression of the regulatory genes Krox20 and kreisler are involved in segmentation and antero-posterior patterning of the hindbrain neural tube. The analysis of transgenic mice in which specific rhombomeres (r) are eliminated points to an important role of segmentation in the generation of neuronal networks controlling vital rhythmic behaviours such as respiration. Thus, elimination of r3 and r5 in Krox20-/- mice suppresses a pontine antiapneic system (Jacquin et al., 1996). We now compare Krox20-/- to kreisler heterozygous (+/kr) and homozygous (kr/kr) mutant neonates. In +/kr mutant mice, we describe hyperactivity of the antiapneic system: analysis of rhythm generation in vitro revealed a pontine modification in keeping with abnormal cell specifications previously reported in r3 (Manzanares et al., 1999b). In kr/kr mice, elimination of r5 abolished all +/kr respiratory traits, suggesting that +/kr hyperactivity of the antiapneic system is mediated through r5-derived territories. Furthermore, collateral chemosensory pathways that normally mediate delayed responses to hypoxia and hyperoxia were not functional in kr/kr mice. We conclude that the pontine antiapneic system originates from r3r4, but not r5. A different rhythm-promoting system originates in r5 and kreisler controls the development of antiapneic and chemosensory signal transmission at this level.