Dihydrofolate reductase activity controls neurogenic transitions in the developing neocortex.

One-carbon/folate (1C) metabolism supplies methyl groups required for DNA and histone methylation, and is involved in the maintenance of self-renewal in stem cells. Dihydrofolate reductase (DHFR), a key enzyme in 1C metabolism, is highly expressed in human and mouse neural progenitors at the early stages of neocortical development. Here, we have investigated the role of DHFR in the developing neocortex and report that reducing its activity in human neural organoids and mouse embryonic neocortex accelerates indirect neurogenesis, thereby affecting neuronal composition of the neocortex. Furthermore, we show that decreasing DHFR activity in neural progenitors leads to a reduction in one-carbon/folate metabolites and correlates with modifications of H3K4me3 levels. Our findings reveal an unanticipated role for DHFR in controlling specific steps of neocortex development and indicate that variations in 1C metabolic cues impact cell fate transitions.

Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex.

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly or indirectly, through the production of intermediate basal progenitors. The balance between these major progenitor types is critical for the production of the proper number and types of neurons, and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that, in the developing mouse neocortex of both sex, deleting CDC25B in apical progenitors leads to a transient increase in the production of TBR1+ neurons at the expense of TBR2+ basal progenitors. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is because of the G2 phase lengthening in CDC25B mutants. Together, this study identifies a new role for CDC25B and G2 phase length in direct versus indirect neurogenesis at early stages of cortical development.SIGNIFICANCE STATEMENT This study is the first analysis of the function of CDC25B, a G2/M regulator, in the developing neocortex. We show that removing CDC25B function leads to a transient increase in neuronal differentiation at early stages, occurring simultaneously with a decrease in basal intermediate progenitors (bIPs). Conversely, a CDC25B gain of function promotes production of bIPs, and this is directly related to CDC25B's ability to regulate CDK1 activity. This imbalance of neuron/progenitor production is linked to a G2 phase lengthening in apical progenitors; and using pharmacological treatments on cortical slice cultures, we show that shortening the G2 phase is sufficient to enhance bIP production. Our results reveal the importance of G2 phase length regulation for neural progenitor fate determination.

Heterogeneity in the size of the apical surface of cortical progenitors.

BACKGROUND: The apical surface (AS) of epithelial cells is highly specialized; it is important for morphogenetic processes that are essential to shape organs and tissues and it plays a role in morphogen and growth factor signaling. Apical progenitors in the mammalian neocortex are pseudoepithelial cells whose apical surface lines the ventricle. Whether changes in their apical surface sizes are important for cortical morphogenesis and/or other aspects of neocortex development has not been thoroughly addressed. RESULTS: Here we show that apical progenitors are heterogeneous with respect to their apical surface area. In Efnb1 mutants, the size of the apical surface is modified and this correlates with discrete alterations of tissue organization without impacting apical progenitors proliferation. CONCLUSIONS: Altogether, our data reveal heterogeneity in apical progenitors AS area in the developing neocortex and shows a role for Ephrin B1 in controlling AS size. Our study also indicates that changes in AS size do not have strong repercussion on apical progenitor behavior.

Efnb2 haploinsufficiency induces early gap junction plaque disassembly and endocytosis in the cochlea.

Chromosome 13q deletions encompassing EFNB2, which encodes the transmembrane protein ephrin-B2, are likely to cause syndromic forms of sensorineural hearing loss of unclear origin. Thus, unravelling the pathogenic mechanisms could help to improve therapeutic strategies. In the cochlea, adjacent non-sensory epithelial cells are connected via gap junction channels, the activity of which is critical to maintain cochlear homeostasis. Here we show that ephrin-B2 promotes the assembly of connexin 30 (Cx30) gap junction plaques (GJPs) between adjacent non-sensory Deiters' cells. An in situ proximity ligation assay revealed that ephrin-B2 preferentially interacts with Cx30 in the periphery of the GJPs, i.e. where newly synthesized connexin hemichannels accrue to the GJP. Moreover, we observed that heterozygous mice encoding an Efnb2 null allele display excessive clathrin-mediated internalization of Cx30 GJPs in early postnatal stages. Finally, an in vitro organotypic assay revealed that ectopic activation of ephrin-B2 reverse signalling promotes the internalization of Cx30 GJPs. These data argue in favor of a cell-autonomous, Eph receptor-independent role of ephrin-B2 in the assembly of Cx30 GJPs. According to recent observations, early GJP degradation could certainly play a role in the pathogenic process leading to progressive sensorineural hearing loss due to Efnb2/EFNB2 haploinsufficiency.

Inhibition of DHFR targets the self-renewing potential of brain tumor initiating cells.

Brain tumors are a heterogeneous group of benign and malignant tumors arising from the brain parenchyma and its surrounding structures, with in general a poor clinical outcome due to high recurrence. One of the underlying causes for this somber prognostic is the presence of brain tumor initiating cells (BTIC) endowed with self-renewal potential, multi-lineage differentiation and resistance to treatment. One promising therapeutic avenue for brain tumors is targeting BTIC self-renewal potential and forcing their differentiation. A compelling candidate is one-carbon metabolism shown to play a key role in maintaining stem cell self-renewal in several lineages. Here, we focus on dihydrofolate reductase (DHFR), a key enzyme in one-carbon metabolism, and demonstrate this enzyme's overexpression in several human brain tumors and its expression in human BTIC. We show that DHFR inhibition, either by Methotrexate (MTX) or EphB activation with synthetic ligands, reduces the tumorigenic potential of 4 human BTIC lines, by reducing their self-renewal capacities both in vitro and in a cerebral organoid glioma (GLICO) model. Our data indicate that driving BTIC differentiation by inhibiting DHFR may provide a new therapeutic approach to treating highly refractory aggressive tumors.

Ephrin-B2 paces neuronal production in the developing neocortex.

BACKGROUND: During mammalian cerebral cortex development, different types of projection neurons are produced in a precise temporal order and in stereotypical numbers. The mechanisms regulating timely generation of neocortex projection neurons and ensuring production in sufficient numbers of each neuronal identity are only partially understood. RESULTS: Here, we show that ephrin-B2, a member of the Eph:ephrin cell-to-cell communication pathway, sets the neurogenic tempo in the neocortex. Indeed, conditional mutant embryos for ephrin-B2 exhibit a transient delay in neurogenesis and acute stimulation of Eph signaling by in utero injection of synthetic ephrin-B2 led to a transient increase in neuronal production. Using genetic approaches we show that ephrin-B2 acts on neural progenitors to control their differentiation in a juxtacrine manner. Unexpectedly, we observed that perinatal neuron numbers recovered following both loss and gain of ephrin-B2, highlighting the ability of neural progenitors to adapt their behavior to the state of the system in order to produce stereotypical numbers of neurons. CONCLUSIONS: Altogether, our data uncover a role for ephrin-B2 in embryonic neurogenesis and emphasize the plasticity of neuronal production in the neocortex.

Population Dynamics and Neuronal Polyploidy in the Developing Neocortex.

The mammalian neocortex is composed of different subtypes of projection neurons that are generated sequentially during embryogenesis by differentiation of neural progenitors. While molecular mechanisms that control neuronal production in the developing neocortex have been extensively studied, the dynamics and absolute numbers of the different progenitor and neuronal populations are still poorly characterized. Here, we describe a medium throughput approach based on flow cytometry and well-known identity markers of cortical subpopulations to collect quantitative data over the course of mouse neocortex development. We collected a complete dataset in a physiological developmental context on two progenitor and two neuron populations, including relative proportions and absolute numbers. Our study reveals unexpected total numbers of Tbr2+ progenitors. In addition, we show that polyploid neurons are present throughout neocortex development.

Cross Talk between One-Carbon Metabolism, Eph Signaling, and Histone Methylation Promotes Neural Stem Cell Differentiation.

Metabolic pathways, once seen as a mere consequence of cell states, have emerged as active players in dictating different cellular events such as proliferation, self-renewal, and differentiation. Several studies have reported a role for folate-dependent one-carbon (1C) metabolism in stem cells; however, its exact mode of action and how it interacts with other cues are largely unknown. Here, we report a link between the Eph:ephrin cell-cell communication pathway and 1C metabolism in controlling neural stem cell differentiation. Transcriptional and functional analyses following ephrin stimulation revealed alterations in folate metabolism-related genes and enzymatic activity. In vitro and in vivo data indicate that Eph-B forward signaling alters the methylation state of H3K4 by regulating 1C metabolism and locks neural stem cell in a differentiation-ready state. Our study highlights a functional link between cell-cell communication, metabolism, and epigenomic remodeling in the control of stem cell self-renewal.

Developmental Upregulation of Ephrin-B1 Silences Sema3C/Neuropilin-1 Signaling during Post-crossing Navigation of Corpus Callosum Axons.

The corpus callosum is the largest commissure in the brain, whose main function is to ensure communication between homotopic regions of the cerebral cortex. During fetal development, corpus callosum axons (CCAs) grow toward and across the brain midline and then away on the contralateral hemisphere to their targets. A particular feature of this circuit, which raises a key developmental question, is that the outgoing trajectory of post-crossing CCAs is mirror-symmetric with the incoming trajectory of pre-crossing axons. Here, we show that post-crossing CCAs switch off their response to axon guidance cues, among which the secreted Semaphorin-3C (Sema3C), that act as attractants for pre-crossing axons on their way to the midline. This change is concomitant with an upregulation of the surface protein Ephrin-B1, which acts in CCAs to inhibit Sema3C signaling via interaction with the Neuropilin-1 (Nrp1) receptor. This silencing activity is independent of Eph receptors and involves a N-glycosylation site (N-139) in the extracellular domain of Ephrin-B1. Together, our results reveal a molecular mechanism, involving interaction between the two unrelated guidance receptors Ephrin-B1 and Nrp1, that is used to control the navigation of post-crossing axons in the corpus callosum.

Impact of Metabolic Pathways and Epigenetics on Neural Stem Cells.

Balancing self-renewal with differentiation is crucial for neural stem cells (NSC) functions to ensure tissue development and homeostasis. Over the last years, multiple studies have highlighted the coupling of either metabolic or epigenetic reprogramming to NSC fate decisions. Metabolites are essential as they provide the energy and building blocks for proper cell function. Moreover, metabolites can also function as substrates and/or cofactors for epigenetic modifiers. It is becoming more evident that metabolic alterations and epigenetics rewiring are highly intertwined; however, their relation regarding determining NSC fate is not well understood. In this review, we summarize the major metabolic pathways and epigenetic modifications that play a role in NSC. We then focus on the notion that nutrients availability can function as a switch to modify the epigenetic machinery and drive NSC sequential differentiation during embryonic neurogenesis.

Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord.

BACKGROUND: In the vertebrate spinal cord, motor neurons (MN) are generated in stereotypical numbers from a pool of dedicated progenitors (pMN) whose number depends on signals that control their specification but also their proliferation and differentiation rates. Although the initial steps of pMN specification have been extensively studied, how pMN numbers are regulated over time is less well characterized. RESULTS: Here, we show that ephrinB2 and ephrinB3 are differentially expressed in progenitor domains in the ventral spinal cord with several Eph receptors more broadly expressed. Genetic loss-of-function analyses show that ephrinB2 and ephrinB3 inversely control pMN numbers and that these changes in progenitor numbers correlate with changes in motor neuron numbers. Detailed phenotypic analyses by immunostaining and genetic interaction studies between ephrinB2 and Shh indicate that changes in pMN numbers in ephrin mutants are due to alteration in progenitor identity at late stages of development. CONCLUSIONS: Altogether our data reveal that Eph:ephrin signaling is required to control progenitor identities in the ventral spinal cord.

Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis.

p57(Kip2) knock-in mouse reveals CDK-independent contribution in the development of Beckwith-Wiedemann syndrome.

CDKN1C encodes the cyclin-CDK inhibitor p57(Kip2) (p57), a negative regulator of the cell cycle and putative tumour suppressor. Genetic and epigenetic alterations causing loss of p57 function are the most frequent cause of Beckwith-Wiedemann syndrome (BWS), a genetic disorder characterized by multiple developmental anomalies and increased susceptibility to tumour development during childhood. So far, BWS development has been attributed entirely to the deregulation of proliferation caused by loss of p57-mediated CDK inhibition. However, a fraction of BWS patients have point mutations in CDKN1C located outside of the CDK inhibitory region, suggesting the involvement of other parts of the protein in the disease. To test this possibility, we generated knock-in mice deficient for p57-mediated cyclin-CDK inhibition (p57(CK) (-) ), the only clearly defined function of p57. Comparative analysis of p57(CK) (-) and p57(KO) mice provided clear evidence for CDK-independent roles of p57 and revealed that BWS is not caused entirely by CDK deregulation, as several features of BWS are caused by the loss of CDK-independent roles of p57. Thus, while the genetic origin of BWS is well understood, our results underscore that the underlying molecular mechanisms remain largely unclear. To probe these mechanisms further, we determined the p57 interactome. Several partners identified are involved in genetic disorders with features resembling those caused by CDKN1C mutation, suggesting that they could be involved in BWS pathogenesis and revealing a possible connection between seemingly distinct syndromes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

EphrinB2 sharpens lateral motor column division in the developing spinal cord.

BACKGROUND: During sensori-motor circuit development, the somas of motoneurons (MN) are distributed in a topographic manner in the ventral horn of the neural tube. Indeed, their position within the lateral motor columns (LMC) correlates with axonal trajectories and identity of target limb muscles. The mechanisms by which this topographic distribution is established remains poorly understood. To address this issue, we assessed the role of ephrinB2 in MN topographic organization in the developing mouse spinal cord. RESULTS: First, we used a reporter mouse line to establish the spatio-temporal expression pattern of EfnB2 in the developing LMC. We show that early in LMC development, ephrinB2 is differentially expressed in MN of the lateral versus medial LMC, suggesting a possible role in MN sorting and/or migration. We demonstrate that while MN-specific excision of EfnB2 did not perturb specification or migration of MN, conditional loss of ephrinB2 led to the blurring of the LMC divisional boundary and to errors in the selection of LMC axon trajectory in the limb. CONCLUSIONS: Altogether, our study uncovered a novel cell autonomous role for ephrinB2 in LMC MN thus emphasizing the prevalent role of this ephrin member in maintaining cell population boundaries.

Cochlear supporting cell transdifferentiation and integration into hair cell layers by inhibition of ephrin-B2 signalling.

In mammals, cochlear sensory hair cells that are responsible for hearing are postmitotic and are not replaced after loss. One of the most promising strategies to regenerate hair cells is to identify and inhibit the factors preventing the conversion of adjacent non-sensory supporting cells into hair cells. Here we demonstrate that mammalian hair cells can be directly generated from supporting cells by inhibition of ephrin-B2 signalling. Using either ephrin-B2 conditional knockout mice, shRNA-mediated gene silencing or soluble inhibitors, we found that downregulation of ephrin-B2 signalling at embryonic stages results in supporting cell translocation into hair cell layers and subsequent switch in cell identity from supporting cell to hair cell fate. As transdifferentiation is here a result of displacement across boundary, this original finding presents the interest that newly generated hair cells directly integrate either hair cell layer, then would be likely more rapidly able to fit into functional circuitry.

Cortical abnormalities and non-spatial learning deficits in a mouse model of CranioFrontoNasal syndrome.

Eph receptors and their ephrin ligands play critical roles in the development of the nervous system, however, less is known about their functions in the adult brain. Here, we investigated the function of ephrinB1, an ephrinB family member that is mutated in CranioFrontoNasal Syndrome. We show that ephrinB1 deficient mice (EfnB1(Y/-)) demonstrate spared spatial learning and memory but exhibit exclusive impairment in non-spatial learning and memory tasks. We established that ephrinB1 does not control learning and memory through direct modulation of synaptic plasticity in adults, since it is not expressed in the adult brain. Rather we show that the cortex of EfnB1(Y/-) mice displayed supernumerary neurons, with a particular increase in calretinin-positive interneurons. Further, the increased neuron number in EfnB1(Y/-) mutants correlated with shorter dendritic arborization and decreased spine densities of cortical pyramidal neurons. Our findings indicate that ephrinB1 plays an important role in cortical maturation and that its loss has deleterious consequences on selective cognitive functions in the adult.

Eph:ephrin-B1 forward signaling controls fasciculation of sensory and motor axons.

Axon fasciculation is one of the processes controlling topographic innervation during embryonic development. While axon guidance steers extending axons in the accurate direction, axon fasciculation allows sets of co-extending axons to grow in tight bundles. The Eph:ephrin family has been involved both in axon guidance and fasciculation, yet it remains unclear how these two distinct types of responses are elicited. Herein we have characterized the role of ephrin-B1, a member of the ephrinB family in sensory and motor innervation of the limb. We show that ephrin-B1 is expressed in sensory axons and in the limb bud mesenchyme while EphB2 is expressed in motor and sensory axons. Loss of ephrin-B1 had no impact on the accurate dorso-ventral innervation of the limb by motor axons, yet EfnB1 mutants exhibited decreased fasciculation of peripheral motor and sensory nerves. Using tissue-specific excision of EfnB1 and in vitro experiments, we demonstrate that ephrin-B1 controls fasciculation of axons via a surround repulsion mechanism involving growth cone collapse of EphB2-expressing axons. Altogether, our results highlight the complex role of Eph:ephrin signaling in the development of the sensory-motor circuit innervating the limb.

Ephrin B1 maintains apical adhesion of neural progenitors.

Apical neural progenitors are polarized cells for which the apical membrane is the site of cell-cell and cell-extracellular matrix adhesion events that are essential for maintaining the integrity of the developing neuroepithelium. Apical adhesion is important for several aspects of the nervous system development, including morphogenesis and neurogenesis, yet the mechanisms underlying its regulation remain poorly understood. Here, we show that ephrin B1, a cell surface protein that engages in cell signaling upon binding cognate Eph receptors, controls normal morphogenesis of the developing cortex. Efnb1-deficient embryos exhibit morphological alterations of the neuroepithelium that correlate with neural tube closure defects. Using loss-of-function experiments by ex vivo electroporation, we demonstrate that ephrin B1 is required in apical progenitors (APs) to maintain their apical adhesion. Mechanistically, we show that ephrin B1 controls cell-ECM adhesion by promoting apical localization of integrin ß1 and we identify ADP-ribosylation factor 6 (Arf6) as an important effector of ephrin B1 reverse signaling in apical adhesion of APs. Our results provide evidence for an important role for ephrin B1 in maintaining the structural integrity of the developing cortex and highlight the importance of tightly controlling apical cell-ECM adhesion for neuroepithelial development.

Regulation and misregulation of Eph/ephrin expression.

The erythropoietin-producing hepatocellular (Eph) receptors form the largest family of receptor tyrosine kinases. Upon interaction of the Eph receptors with their ligands the ephrins, signaling cascades are initiated downstream of both receptor and ligand, a feature known as bidirectional signaling. The Eph receptors and ephrin ligands mediate important roles in embryonic development, particularly in establishing tissue organization by mediating cell adhesion or cell repulsion. In several adult tissues, at least one Eph/ephrin pair is found to play critical roles in tissue physiology and homeostasis. In recent years numerous members of this family have gained considerable attention since changes in their expression levels are a typical feature in cancer cells. Despite the fact that Eph/ephrin developmental expression profiles are well documented, little is known on transcriptional and post-transcriptional mechanisms that permits their highly specific, graded, complementary or overlapping expression patterns. Therefore understanding the transcriptional and post-transcriptional mechanisms regulating Eph/ephrin expression has far-reaching significance in biology. This review provides an overview of the mechanisms regulating Eph/ephrin expression. We highlight important emerging mechanisms of Eph/ephrin regulation or misregulation such as epigenetics and miRNAs.