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The SOX2 transcription factor is a key regulator of nervous system development, and its mutation in humans leads to a rare disease characterized by severe eye defects, cognitive defects, hearing defects, abnormalities of the CNS and motor control problems. SOX2 has an essential role in neural stem cell maintenance in specific regions of the brain, and it is one of the master genes required for the generation of induced pluripotent stem cells. Sox2 is expressed in sensory organs, and this review will illustrate how it regulates the differentiation of sensory cell types required for hearing, touching, tasting and smelling in vertebrates and, in particular, in mice.

Background Multiple sclerosis (MS) is an autoimmune disease which results from the invasion of the brain by activated immune cells across the endothelial cells (ECs) of the blood-brain barrier (BBB), due to loss of immune self-tolerance. Many reports define the metabolic profile of immune cells in MS, however little is known about the metabolism of the BBB ECs during the disease. We aim to determine whether circulating factors in MS induce metabolic alterations of the BBB ECs compared to a healthy state, which can be linked with disruption of BBB integrity and subsequent immune cell extravasation. Methods and results In this report, we used an in vitro model to study the effect of sera from naïve-to-treatment, relapsing-remitting MS (RRMS) patients on the human brain microvascular endothelium, comparing effects to age/sex-matched healthy donor (HD) sera. Our data show that RRMS serum components affect brain endothelial cells by impairing intercellular tightness through the down-modulation of occludin and VE-cadherin, and facilitating immune cell extravasation through upregulation of intercellular adhesion molecules (ICAM-1) and P-glycoprotein (P-gp). At a metabolic level, the treatment of the endothelial cells with RRMS sera reduced their glycolytic activity (measured through the extracellular acidification rate-ECAR) and oxygen consumption rate (oxidative phosphorylation rate-OCR). Such changes were associated with the down-modulation of endothelial glucose transporter 1 (GLUT-1) expression and by altered mitochondrial membrane potential. Higher level of reactive oxygen species released from the endothelial cells treated with RRMS sera indicate a pro-inflammatory status of the cells together with the higher expression of ICAM-1, endothelial cell cytoskeleton perturbation (stress fibres) as well as disruption of the cytoskeleton signal transduction MSK1/2 and β-catenin phosphorylation. Conclusions Our data suggest that circulating factors present in RRMS patient serum induce physiological and biochemical alterations to the BBB, namely reducing expression of essential tightness regulators, as well as reduced engagement of glycolysis and alteration of mitochondrial potential. As these last changes have been linked with alterations in nutrient usage and metabolic function in immune cells; we propose that the BBB endothelium of MS patients may similarly undergo metabolic dysregulation, leading to enhanced permeability and increased disease susceptibility.

SOX2 is a transcription factor conserved throughout vertebrate evolution, whose expression marks the central nervous system from the earliest developmental stages. In humans, SOX2 mutation leads to a spectrum of CNS defects, including vision and hippocampus impairments, intellectual disability, and motor control problems. Here, we review how conditional Sox2 knockout (cKO) in mouse with different Cre recombinases leads to very diverse phenotypes in different regions of the developing and postnatal brain. Surprisingly, despite the widespread expression of Sox2 in neural stem/progenitor cells of the developing neural tube, some regions (hippocampus, ventral forebrain) appear much more vulnerable than others to Sox2 deletion. Furthermore, the stage of Sox2 deletion is also a critical determinant of the resulting defects, pointing to a stage-specificity of SOX2 function. Finally, cKOs illuminate the importance of SOX2 function in different cell types according to the different affected brain regions (neural precursors, GABAergic interneurons, glutamatergic projection neurons, Bergmann glia). We also review human genetics data regarding the brain defects identified in patients carrying mutations within human SOX2 and examine the parallels with mouse mutants. Functional genomics approaches have started to identify SOX2 molecular targets, and their relevance for SOX2 function in brain development and disease will be discussed.

DNA sequence variants (single nucleotide polymorphisms or variants, SNPs/SNVs; copy number variants, CNVs) associated to neurodevelopmental disorders (NDD) and traits often map on putative transcriptional regulatory elements, including, in particular, enhancers. However, the genes controlled by these enhancers remain poorly defined. Traditionally, the activity of a given enhancer, and the effect of its possible alteration associated to the sequence variants, has been thought to influence the nearest gene promoter. However, the obtainment of genome-wide long-range interaction maps in neural cells chromatin challenged this view, showing that a given enhancer is very frequently not connected to the nearest promoter, but to a more distant one, skipping genes in between. In this Perspective, we review some recent papers, who generated long-range interaction maps (by HiC, RNApolII ChIA-PET, Capture-HiC, or PLACseq), and overlapped the identified long-range interacting DNA segments with DNA sequence variants associated to NDD (such as schizophrenia, bipolar disorder and autism) and traits (intelligence). This strategy allowed to attribute the function of enhancers, hosting the NDD-related sequence variants, to a connected gene promoter lying far away on the linear chromosome map. Some of these enhancer-connected genes had indeed been already identified as contributive to the diseases, by the identification of mutations within the gene’s protein-coding regions (exons), validating the approach. Significantly, however, the connected genes also include many genes that were not previously found mutated in their exons, pointing to novel candidate contributors to NDD and traits. Thus, long-range interaction maps, in combination with DNA variants detected in association with NDD, can be used as “pointers” to identify novel candidate disease-relevant genes. Functional manipulation of the long-range interaction network involving enhancers and promoters by CRISPR-Cas9-based approaches is beginning to probe for the functional significance of the identified interactions, and the enhancers and the genes involved, improving our understanding of neural development and its pathology.

The hippocampus is a brain area central for cognition. Mutations in the human SOX2 transcription factor cause neurodevelopmental defects, leading to intellectual disability and seizures, together with hippocampal dysplasia. We generated an allelic series of Sox2 conditional mutations in mouse, deleting Sox2 at different developmental stages. Late Sox2 deletion (from E11.5, via Nestin-Cre) affects only postnatal hippocampal development; earlier deletion (from E10.5, Emx1-Cre) significantly reduces the dentate gyrus (DG), and the earliest deletion (from E9.5, FoxG1-Cre) causes drastic abnormalities, with almost complete absence of the DG. We identify a set of functionally interconnected genes (Gli3, Wnt3a, Cxcr4, p73 and Tbr2), known to play essential roles in hippocampal embryogenesis, which are downregulated in early Sox2 mutants, and (Gli3 and Cxcr4) directly controlled by SOX2; their downregulation provides plausible molecular mechanisms contributing to the defect. Electrophysiological studies of the Emx1-Cre mouse model reveal altered excitatory transmission in CA1 and CA3 regions.

The neocortex, the most recently evolved brain region in mammals, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the presence of graded expression of transcription factors and molecular determinants defining neuronal identity. However, little is known about the expression of key master genes orchestrating human cortical development. In this study, we explored the expression dynamics of NR2F1 and SOX2, key cortical genes whose mutations in human patients cause severe neurodevelopmental syndromes. We focused on physiological conditions, spanning from mid-late gestational ages to adulthood in unaffected specimens, but also investigated gene expression in a pathological context, a developmental cortical malformation termed focal cortical dysplasia (FCD). We found that NR2F1 follows an antero-dorsallow to postero-ventralhigh gradient as in the murine cortex, suggesting high evolutionary conservation. While SOX2 is mainly expressed in neural progenitors next to the ventricular surface, NR2F1 is found in both mitotic progenitors and post-mitotic neurons at GW18. Interestingly, both proteins are highly co-expressed in basal radial glia progenitors of the outer sub-ventricular zone (OSVZ), a proliferative region known to contribute to cortical expansion and complexity in humans. Later on, SOX2 becomes largely restricted to astrocytes and oligodendrocytes although it is also detected in scattered mature interneurons. Differently, NR2F1 maintains its distinct neuronal expression during the whole process of cortical development. Notably, we report here high levels of NR2F1 in dysmorphic neurons and NR2F1 and SOX2 in balloon cells of surgical samples from patients with FCD, suggesting their potential use in the histopathological characterization of this dysplasia.

The Sox2 transcription factor, encoded by a gene conserved in animal evolution, has become widely known because of its functional relevance for stem cells. In the developing nervous system, Sox2 is active in neural stem cells, and important for their self-renewal; differentiation to neurons and glia normally involves Sox2 downregulation. Recent evidence, however, identified specific types of fully differentiated neurons and glia that retain high Sox2 expression, and critically require Sox2 function, as revealed by functional studies in mouse and in other animals. Sox2 was found to control fundamental aspects of the biology of these cells, such as the development of correct neuronal connectivity. Sox2 downstream target genes identified within these cell types provide molecular mechanisms for cell-type-specific Sox2 neuronal and glial functions. SOX2 mutations in humans lead to a spectrum of nervous system defects, involving vision, movement control, and cognition; the identification of neurons and glia requiring Sox2 function, and the investigation of Sox2 roles and molecular targets within them, represents a novel perspective for the understanding of the pathogenesis of these defects.

Summary Visual system development involves the formation of neuronal projections connecting the retina to the thalamic dorso-lateral geniculate nucleus (dLGN) and the thalamus to the visual cerebral cortex. Patients carrying mutations in the SOX2 transcription factor gene present severe visual defects, thought to be linked to SOX2 functions in the retina. We show that Sox2 is strongly expressed in mouse postmitotic thalamic projection neurons. Cre-mediated deletion of Sox2 in these neurons causes reduction of the dLGN, abnormal distribution of retino-thalamic and thalamo-cortical projections, and secondary defects in cortical patterning. Reduced expression, in mutants, of Sox2 target genes encoding ephrin-A5 and the serotonin transport molecules SERT and vMAT2 (important for establishment of thalamic connectivity) likely provides a molecular contribution to these defects. These findings unveil thalamic SOX2 function as a novel regulator of visual system development and a plausible additional cause of brain-linked genetic blindness in humans., Graphical Abstract, Highlights • Sox2 is expressed in postmitotic neurons of the visual thalamic nucleus (dLGN) • Sox2 ablation in the dLGN perturbs retino-thalamic and thalamo-cortical projections • The visual cortex is not correctly patterned in Sox2 thalamic mutants • Downregulation of EphrinA5 and SERT expression may mediate these defects, Biological Sciences; Neuroscience; Developmental Neuroscience

Hemichordates (Enteropneusta and Pterobranchia) belong to a small deuterostome invertebrate group that may offer insights on the origin and evolution of the chordate nervous system. Among them, the colonial pterobranch Rhabdopleuridae are recognized as living representatives of Graptolithina, a taxon with a rich fossil record. New information is provided here on the substrate selection and the life cycle of Rhabdopleura recondita Beli, Cameron and Piraino, 2018, and for the first time, we describe the nervous system organization of the larva and the adult zooid, as well as the morphological, neuroanatomical and behavioural changes occurring throughout metamorphosis. Immunohistochemical analyses disclosed a centralized nervous system in the sessile adult zooid, characterized by different neuronal subsets with three distinct neurotransmitters, i.e. serotonin, dopamine and RFamide. The peripheral nervous system comprises GABA-, serotonin-, and dopamine-immunoreactive cells. These observations support and integrate previous neuroanatomical findings on the pterobranch zooid of Cephalodiscus gracilis. Indeed, this is the first evidence of dopamine, RFamide and GABA neurotransmitters in hemichordates pterobranchs. In contrast, the lecithotrophic larva is characterized by a diffuse basiepidermal plexus of GABAergic cells, coupled with a small group of serotonin-immunoreactive cells localized in the characteristic ventral depression. It is envisaged the use of R. recondita as a novel and easily accessible hemichordate model organism to shed light on the evolution of hemichordates and more generally on the origin of deuterostome developmental mechanisms.

The Sox2 transcription factor is expressed in different neural tumors. In particular, it is active within the “cancer stem cell” (CSC) subpopulation of tumor cells, able to reinitiate tumorigenesis after conventional chemotherapy (to which it is usually resistant). This led to hypothesize that Sox2 (and its downstream regulated genes) may qualify as promising targets for therapeutic strategies directed against CSC. However, the potential relevance of Sox2 in this regard depends on whether it is functionally important to maintain CSC. Here, we comparatively examine the effects of Sox2 genetic ablation within mouse models of different neural tumor types. Sox2 ablation in mouse glioma (and in human glioblastomaderived CSC) demonstrated a critical function for Sox2 in the maintenance of CSC. Surprisingly, however, Sox2 ablation in two different mouse models of melanoma (a neural crest-related tumor), and in a mouse model of medulloblastoma of the Sonic Hedgehog subgroup, showed that, in these contexts, Sox2 is dispensable for tumorigenesis. This heterogeneous situation has a parallel in the normal development of the nervous system, where generalized Sox2 ablation in neural stem/progenitor cells selectively affects the development of some neural regions, but not other ones. Molecular mechanisms underlying these specificities may involve the regulation, by Sox2, of different sets of target genes in different tumors, but also a redundant regulation of the same targets by different Sox transcription factors, differentially coexpressed with Sox2 in different tumors. Collectively, these findings point to the need to experimentally address the requirement for Sox2, and its downstream targets, within different tumor types, as a prerequisite to fully exploit its potential as a target for novel therapeutic approaches.

The transcription factor Sox2 is necessary to maintain pluripotency of embryonic stem cells, and to regulate neural development. Neurogenesis in the vertebrate olfactory epithelium persists from embryonic stages through adulthood. The role Sox2 plays for the development of the olfactory epithelium and neurogenesis within has, however, not been determined. Here, by analysing Sox2 conditional knockout mouse embryos and chick embryos deprived of Sox2 in the olfactory epithelium using CRISPR-Cas9, we show that Sox2 activity is crucial for the induction of the neural progenitor gene Hes5 and for subsequent differentiation of the neuronal lineage. Our results also suggest that Sox2 activity promotes the neurogenic domain in the nasal epithelium by restricting Bmp4 expression. The Sox2-deficient olfactory epithelium displays diminished cell cycle progression and proliferation, a dramatic increase in apoptosis and finally olfactory pit atrophy. Moreover, chromatin immunoprecipitation data show that Sox2 directly binds to the Hes5 promoter in both the PNS and CNS. Taken together, our results indicate that Sox2 is essential to establish, maintain and expand the neuronal progenitor pool by suppressing Bmp4 and upregulating Hes5 expression., Summary: Analysis of Sox2 mutant mouse and Sox2 CRISPR-targeted chick embryos reveals that Sox2 controls the establishment of sensory progenitors in the olfactory epithelium by suppressing Bmp4 and upregulating Hes5 expression.

We recently investigated the prognostic impact of a Chronic Care Model (CCM)-based healthcare program applied in primary care in Tuscany Region mainly run by multidisciplinary teams composed of general practitioners (GPs) and nurses. The project included proactively planned follow-up visits for each patient, individualized counselling to optimize lifestyle modifications and adherence to appropriate diagnostic and therapeutic pathways. 1761 patients with Chronic heart failure (CHF) directly enrolled by the GPs were matched with 3522 CHF controls not involved in the project. Over a 4-year follow-up in the CCM group a higher CHF hospitalization rate was found (12.1 vs 10.3 events/100 patient-years; incidence rate ratio [IRR] 1.15, p=0.0030), whereas mortality was lower (10.8 vs 12.6 events/100 patient-years; IRR 0.82, p0.0001). The CCM status was independently associated with a 34% increase in the risk of CHF hospitalization and a 18% reduction in the risk of death (p0.0001 for both). The CCM status was associated with a 50% increase in the rate of planned Heart failure (HF) hospitalizations whereas the rate of 1-month CHF readmissions showed no differences. Such a divergent trend could be explained by the direct involvement of GPs in the CCM program, leading them to a better awareness of patients' clinical status, and then to a more frequent use of clinical pathways and facilities, including hospitalization. It is reasonable to argue that not all hospitalizations must necessarily be considered as a poor outcome, as they often provide additional opportunities to improve therapies, optimize patient education, or define follow-up strategies. The evidence of a divergent trend between mortality and hospitalization in our population might support the clinical importance of a multidisciplinary approach for the management of patients with HF.

SummaryMutations in the human NOTCH3 gene cause CADASIL syndrome (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). CADASIL is an inherited small vessel disease characterized by diverse clinical manifestations including vasculopathy, neurodegeneration and dementia. Here we report two mutations in the zebrafish notch3 gene, one identified in a previous screen for mutations with reduced expression of myelin basic protein (mbp) and another caused by a retroviral insertion. Reduced mbp expression in notch3 mutant embryos is associated with fewer oligodendrocyte precursor cells (OPCs). Despite an early neurogenic phenotype, mbp expression recovered at later developmental stages and some notch3 homozygous mutants survived to adulthood. These mutants, as well as adult zebrafish carrying both mutant alleles together, displayed a striking stress-associated accumulation of blood in the head and fins. Histological analysis of mutant vessels revealed vasculopathy, including: an enlargement (dilation) of vessels in the telencephalon and fin, disorganization of the normal stereotyped arrangement of vessels in the fin, and an apparent loss of arterial morphological structure. Expression of hey1, a well-known transcriptional target of Notch signaling, was greatly reduced in notch3 mutant fins, suggesting that Notch3 acts via a canonical Notch signaling pathway to promote normal vessel structure. Ultrastructural analysis confirmed the presence of dilated vessels in notch3 mutant fins and revealed that the vessel walls of presumed arteries showed signs of deterioration. Gaps evident in the arterial wall and the presence of blood cells outside of vessels in mutants indicated that compromised vessel structure led to hemorrhage. In notch3 heterozygotes, we found elevated expression of both notch3 itself and target genes, indicating that specific alterations in gene expression caused by a partial loss of Notch3 function might contribute to the abnormalities observed in heterozygous larvae and adults. Our analysis of zebrafish notch3 mutants indicates that Notch3 regulates OPC development and mbp gene expression in larvae, and maintains vascular integrity in adults.

The essential role of SOX2 in embryogenesis was first demonstrated by the Sox2 gene knockout in mouse, leading to early embryonic lethality by loss of the epiblast, the founder tissue of the embryo proper. However, later in development, SOX2 expression is also a conserved marker of the developing central nervous system (CNS), and therein of neural stem cells (NSC), the self-renewing precursors of neurons, astrocytes, and oligodendrocytes. Genetic manipulation of Sox2 in mouse and other animal systems revealed an essential role for SOX2 in the development of multiple CNS regions and in the maintenance of NSC. Some differentiated neural cell types also require SOX2. Brain defects in mouse mutants mirror defects found in patients heterozygous for SOX2 mutations. Multiple transcriptional regulators, including cell cycle-control genes, act on SOX2 neural expression; in turn, SOX2 target genes have begun to be identified that have important roles in its biological function.

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of β-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor,lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

The distribution of EMX2, the protein product of the homeobox gene Emx2, was analyzed in the developing mouse CNS by means of a polyclonal antibody we raised against it. The protein is present in the rostral brain, the olfactory area and a set of scattered cells lying between the nasal pits and the telencephalon. In the cortical neuroepithelium EMX2 is expressed all along the rostro-caudal axis in a graded distribution with a caudal-medial maximum and a rostral-lateral minimum. Anti-EMX2 immunoreactivity is also detectable in Cajal-Retzius cells as well as in apical dendrites of marginal neurons of the cortical plate. We also observe that the EMX2 and EMX1 homeoproteins display complementary expression patterns in olfactory bulbs and amygdaloid complex. Here, they demarcate different neuronal populations, involved in processing olfactory information coming from the vomero-nasal organ and from the main olfactory epithelium, respectively. EMX2 is also detectable in mesencephalic structures, such as the optic tectum and tegmentum. The graded distribution of EMX2 along antero-posterior and medial-lateral axes of the primitive cortex prefigures a role of this protein in the subdivision of the cortex in cytoarchitectonic regions and possibly functional areas, whereas its presence in Cajal-Retzius cells suggests a role in the process of cortical lamination.

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE- derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction. © 2013. Published by The Company of Biologists Ltd., Cariplo Foundation (grant 20100673); ASTIL Regione Lombardia (SAL-19, N Prot FL 16874); Telethon (GGP12152); AssociazioneItaliana Ricerca sul Cancro (AIRC IG-5801); Spanish Ministerio de Economia y Competividad (BFU2010-16031); Comunidad Autonoma de Madrid (BDM-2315)

Making Myelin The myelin sheath insulates neurons and facilitates rapid axonal conduction, and its disruption is characteristic of some neurological disorders, such as multiple sclerosis. Axonal signals stimulate Schwann cells to form myelin in peripheral nerves, but the mechanism is not completely known. By characterizing a mutation identified in zebrafish, Monk et al. (p. 1402 ; see the Perspective by Meijer ) show that Gpr126, a member of the G protein–coupled receptor superfamily, is essential for myelin formation. It appears that Gpr126 acts as a receptor for axonal signals to elevate cAMP levels in Schwann cells and trigger myelination.

The development of vertebrate basal forebrain dopaminergic (DA) neurons requires the conserved zinc finger protein Too Few (Tof/Fezl) in zebrafish. However, how Tof/Fezl regulates the commitment and differentiation of these DA neurons is not known. Proneural genes encoding basic helix–loop–helix transcription factors regulate the development of multiple neuronal lineages, but their involvement in vertebrate DA neuron determination is unclear. Here we show that neurogenin 1 ( ngn1 ), a vertebrate proneural gene related to the Drosophila atonal , is expressed in and required for specification of DA progenitor cells, and when overexpressed leads to supernumerary DA neurons in the forebrain of zebrafish. Overexpression of ngn1 is also sufficient to induce tyrosine hydroxylase expression in addition to the pan-neuronal marker Hu in nonneural ectoderm. We further show that Tof/Fezl is required to establish basal forebrain ngn1 -expressing DA progenitor domains. These findings identify Ngn1 as a determinant of brain DA neurons and provide insights into how Tof/Fezl regulates the development of these clinically important neuronal types.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos,we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves.Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphénucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix,they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be because of its recently identified ability to interact with BMP4 and TGFβ. We demonstrate, using Xenopus embryos, that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF,which is distinct from the BMP- and TGFβ-interacting domain. Our results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis.

Cyr61 is a secreted, heparin-binding, extracellular matrix-associated protein whose activities include the promotion of adhesion and chemotaxis, and the stimulation of fibroblast and endothelial cell growth. Many, if not all,of these activities of Cyr61 are mediated through interactions with integrins. We explore the role of Cyr61 in the early development of Xenopus laevis. Gain- and loss-of-function experiments show that Xcyr61 is required for normal gastrulation movements. This role is mediated in part through the adhesive properties of Xcyr61 and its related ability to modulate assembly of the extracellular matrix. In addition, Xcyr61 can, in a context-dependent manner, stimulate or inhibit signalling through the Wnt pathway. These properties of Xcyr61 provide a mechanism for integrating cell signalling, cell adhesion and cell migration during gastrulation.

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.

Neocorticogenesis in mice homozygous for anEmx2null allele is the topic of this article. The development of both main components of neocortex, primordial plexiform layer derivatives and cortical plate, was analyzed, paying special attention to radial migration of neurons forming the cortical plate. The products of theReelingene, normally playing a key role in orchestrating radial migration of these neurons, display normal distribution at the beginning of the cortical neuronogenesis but are absent in the neocortical marginal zone of the mutant mice at the time when the cortical plate is laid down. As a consequence, the development of radial glia is impaired, and neurons making up the cortical plate display abnormal migration patterns. In addition, restricted defects along the rostrocaudal and the mediolateral axes are present in the subplate, suggesting anEmx2-specific role in priming the proper development of this layer.

The appropriate control of proliferation of neural precursors has fundamental implications for the development of the central nervous system and for cell homeostasis/replacement within specific brain regions throughout adulthood. The role of genetic determinants in this process is largely unknown.We report the expression of the homeobox transcription factor Emx2 within the periventricular region of the adult telencephalon. This neurogenetic area displays a large number of multipotent stem cells. Adult neural stem cells isolated from this region do express Emx2 and down-regulate it significantly upon differentiation into neurons and glia. Abolishing or, increasing Emx2 expression in adult neural stem cells greatly enhances or reduces their rate of proliferation, respectively. We determined that altering the expression of Emx2 affects neither the cell cycle length of adult neural stem cells nor their ability to generate neurons and glia. Rather, when Emx2 expression is abolished, the frequency of symmetric divisions that generate two stem cells increases, whereas it decreases when Emx2 expression is enhanced.