The gut microbiota regulates autism-like behavior by mediating vitamin B6 homeostasis in EphB6-deficient mice.

BACKGROUND: Autism spectrum disorder (ASD) is a developmental disorder, and the effective pharmacological treatments for the core autistic symptoms are currently limited. Increasing evidence, particularly that from clinical studies on ASD patients, suggests a functional link between the gut microbiota and the development of ASD. However, the mechanisms linking the gut microbiota with brain dysfunctions (gut-brain axis) in ASD have not yet been full elucidated. Due to its genetic mutations and downregulated expression in patients with ASD, EPHB6, which also plays important roles in gut homeostasis, is generally considered a candidate gene for ASD. Nonetheless, the role and mechanism of EPHB6 in regulating the gut microbiota and the development of ASD are unclear. RESULTS: Here, we found that the deletion of EphB6 induced autism-like behavior and disturbed the gut microbiota in mice. More importantly, transplantation of the fecal microbiota from EphB6-deficient mice resulted in autism-like behavior in antibiotic-treated C57BL/6J mice, and transplantation of the fecal microbiota from wild-type mice ameliorated the autism-like behavior in EphB6-deficient mice. At the metabolic level, the disturbed gut microbiota in EphB6-deficient mice led to vitamin B6 and dopamine defects. At the cellular level, the excitation/inhibition (E/I) balance in the medial prefrontal cortex was regulated by gut microbiota-mediated vitamin B6 in EphB6-deficient mice. CONCLUSIONS: Our study uncovers a key role for the gut microbiota in the regulation of autism-like social behavior by vitamin B6, dopamine, and the E/I balance in EphB6-deficient mice, and these findings suggest new strategies for understanding and treating ASD. Video abstract.

Loss of the EPH receptor B6 contributes to colorectal cancer metastasis.

Although deregulation of EPHB signaling has been shown to be an important step in colorectal tumorigenesis, the role of EPHB6 in this process has not been investigated. We found here that manipulation of EPHB6 levels in colon cancer cell lines has no effect on their motility and growth on a solid substrate, soft agar or in a xenograft mouse model. We then used an EphB6 knockout mouse model to show that EphB6 inactivation does not efficiently initiate tumorigenesis in the intestinal tract. In addition, when intestinal tumors are initiated genetically or pharmacologically in EphB6+/+ and EphB6-/- mice, no differences were observed in animal survival, tumor multiplicity, size or histology, and proliferation of intestinal epithelial cells or tumor cells. However, reintroduction of EPHB6 into colon cancer cells significantly reduced the number of lung metastasis after tail-vein injection in immunodeficient mice, while EPHB6 knockdown in EPHB6-expressing cells increased their metastatic spread. Consistently, although EPHB6 protein expression in a series of 130 primary colorectal tumors was not associated with patient survival, EPHB6 expression was significantly lower in lymph node metastases compared to primary tumors. Our results indicate that the loss of EPHB6 contributes to the metastatic process of colorectal cancer.

Analysis of local and global topographic order in mouse retinocollicular maps.

We introduce the Lattice Method for the quantitative assessment of the topographic order within the pattern of connections between two structures. We apply this method to published visuocollicular mapping data obtained by Fourier-based intrinsic imaging of mouse colliculus. We find that, in maps from wild types and ß2 knock-outs, at least 150 points on the colliculus are represented in the visual field in the correct relative order. In maps from animals with knock-out of the three ephrinA ligands (TKO), thought to specify the rostrocaudal axis of the map, the projection on the colliculus of each small circular area of visual field is elongated approximately rostrocaudally. Of these projections, 9% are made up of two distinct regions lying along the direction of ingrowth of retinal fibers. These are similar to the ectopic projections found in other ephrinA knock-out data. Coexisting with the ectopic projections, each TKO map contains a submap where neighbor-neighbor relations are preserved, which is ordered along both rostrocaudal and mediolateral axes, in the orientation found in wild-type maps. The submaps vary in size with order well above chance level, which can approach the order in wild-type maps. Knock-out of both ß2 and two of the three ephrinAs yields maps with some order. The ordered TKO maps cannot be produced by correlated neural activity acting alone, as this mechanism is unable to specify map orientation. These results invite reassessment of the role of molecular signaling, particularly that of ephrinAs, in the formation of ordered nerve connections.

EphB controls NMDA receptor function and synaptic targeting in a subunit-specific manner.

Dynamic regulation of the localization and function of NMDA receptors (NMDARs) is critical for synaptic development and function. The composition and localization of NMDAR subunits at synapses are tightly regulated and can influence the ability of individual synapses to undergo long-lasting changes in response to stimuli. Here, we examine mechanisms by which EphB2, a receptor tyrosine kinase that binds and phosphorylates NMDARs, controls NMDAR subunit localization and function at synapses. We find that, in mature neurons, EphB2 expression levels regulate the amount of NMDARs at synapses, and EphB activation decreases Ca(2+)-dependent desensitization of NR2B-containing NMDARs. EphBs are required for enhanced localization of NR2B-containing NMDARs at synapses of mature neurons; triple EphB knock-out mice lacking EphB1-3 exhibit homeostatic upregulation of NMDAR surface expression and loss of proper targeting to synaptic sites. These findings demonstrate that, in the mature nervous system, EphBs are key regulators of the synaptic localization of NMDARs.

The Eph/ephrinB signal balance determines the pattern of T-cell maturation in the thymus.

In order to carry out an in-depth study of the roles of EphB receptors in T-cell development and to determine the specific relevance of forward and reverse signals in the process, we established severe combined immunodeficient (SCID) mice chimeras with wild-type (WT) or EphB-deficient bone marrow cells. The obtained results demonstrate that EphB2 contributes more significantly than EphB3 in the control of CD4(-)CD8(-) (DN)-CD4(+)CD8(+) (DP) progression, and that reverse signals generated in SCID mice receiving EphB2LacZ precursors, which express the EphB2 extracellular domain, partially rescue the blockade of DN cell maturation observed in EphB2-null chimeras. In addition, increased apoptotic DP thymocytes occurring in EphB2 and/or EphB3 SCID chimeras also contribute to the reduced proportions of DP cells. However, EphB2LacZ chimeras do not show any changes in the proportions of apoptotic DP cells, thus suggesting that there is a role for ephrinB reverse signaling in thymocyte survival. The maturation of DP to CD4(+)CD8(-) or CD4(-)CD8(+) seems to need EphB2 forward signaling and EphB3; a fact that was confirmed in reaggregates formed with either EphB2- or EphB3-deficient DP thymocytes and WT thymic epithelial cells (TECs). The DP thymocyte-TEC conjugate formation was also affected by the absence of EphB receptors. Finally, EphB-deficient SCID chimeras show profoundly altered thymic epithelial organization that confirms a significant role for EphB2 and EphB3 receptors in the thymocyte-TEC crosstalk.

Integration of neuronal clones in the radial cortical columns by EphA and ephrin-A signalling.

The cerebral cortex is a laminated sheet of neurons composed of the arrays of intersecting radial columns. During development, excitatory projection neurons originating from the proliferative units at the ventricular surface of the embryonic cerebral vesicles migrate along elongated radial glial fibres to form a cellular infrastructure of radial (vertical) ontogenetic columns in the overlaying cortical plate. However, a subpopulation of these clonally related neurons also undergoes a short lateral shift and transfers from their parental to the neighbouring radial glial fibres, and intermixes with neurons originating from neighbouring proliferative units. This columnar organization acts as the primary information processing unit in the cortex. The molecular mechanisms, role and significance of this lateral dispersion for cortical development are not understood. Here we show that an Eph receptor A (EphA) and ephrin A (Efna) signalling-dependent shift in the allocation of clonally related neurons is essential for the proper assembly of cortical columns. In contrast to the relatively uniform labelling of the developing cortical plate by various molecular markers and retrograde tracers in wild-type mice, we found alternating labelling of columnar compartments in Efna knockout mice that are caused by impaired lateral dispersion of migrating neurons rather than by altered cell production or death. Furthermore, in utero electroporation showed that lateral dispersion depends on the expression levels of EphAs and ephrin-As during neuronal migration. This so far unrecognized mechanism for lateral neuronal dispersion seems to be essential for the proper intermixing of neuronal types in the cortical columns, which, when disrupted, might contribute to neuropsychiatric disorders associated with abnormal columnar organization.

Eph and ephrin: Key molecules for the organization and function of the thymus gland

Las Eph son la mayor familia de receptores tirosina quinasa presentesen la mayoría de tipos celulares. Junto con sus ligandos, las ephrinas, lasEph participan en la organogénesis de muchos tejidos regulando numerosos procesos, como el posicionamiento y la migración celular, los cualesson claves para el correcto funcionamiento del timo, un órgano linfoide primario implicado en la maduración de las células T. En el presente trabajo,revisamos diferentes resultados sobre el papel que estas moléculas juegan en la biología del timo. La mayoría de las Eph y ephrinas se expresanen el timo adulto y fetal, tanto en los timocitos como en las células del estroma. Estas moléculas tienen un papel esencial regulando el tamaño del timo,a través del control de la supervivencia de los timocitos y de las células epiteliales tímicas (TEC). Además, estudios in vivo e in vitro demuestran quemodificaciones en la señalización de Eph y ephrinas resultan en fenotipostímicos específicos, concluyendo que dicha señalización determina finalmente tanto el patrón de maduración y diferenciación de los timocitos comoel de las TEC. El papel de Eph y ephrinas en la función del timo aparecepronto en la ontogenia. En este sentido, varios resultados apoyan su relevancia en procesos claves para la organización del órgano, tales como elreclutamiento de los progenitores linfoides al primordio tímico, el patrónde ramificación del epitelio tímico y el posicionamiento de los timocitos ylas TEC en el timo en crecimiento. Algunas de las alteraciones fenotípicasobservadas en el timo de ratones deficientes en Eph y ephrinas se observantambién en los órganos linfoides periféricos, pero no hay evidencias de unaalteración funcional en sus sistemas inmunes (AU)

Eph are the largest family of protein tyrosine kinases, which are described in most cell types. Together with their ligands, ephrins, Eph participate in the organogenesis of many tissues mediating numerous processes, such as cell positioning and cell migration, which are key for thefunctioning of the thymus: a primary lymphoid organ involved in T-cellmaturation. In the present study, we review available data on the role played by these families of molecules in the biology of the thymus gland.Most Eph and ephrin are expressed in adult and fetal thymus, frequentlyin both thymocytes and thymic stromal cells. They appear to play an essential role in governing thymus size through the control of survival of thymocytes and thymic epithelial cells (TEC). Furthermore, studies in vivoand in vitro demonstrate that altered Eph/ephrin signalling results in specific thymus phenotypes and conclude that the balance of Eph/ephrinsignals finally determine the pattern of maturation/differentiation of thymocytes and TEC. Indeed, the role of Eph/ephrin in thymus functionappears early in ontogeny. In this regard, several results emphasize theirrelevance in key processes for thymus organization, such as the recruitment of lymphoid progenitors to the thymic primordium, the branchingpattern of thymus epithelium and the positioning of thymocytes and TECin the growing gland. Some of the phenotypic alterations observed in thethymus of Eph/ephrin deficient mice are reflected in the peripheral lymphoid organs, but there is no evidence for alterations in the function of theirimmune systems (AU)

EphB receptors couple dendritic filopodia motility to synapse formation.

Motile dendritic filopodial processes are thought to be precursors of spine synapses, but how motility relates to cell-surface cues required for axon-dendrite recognition and synaptogenesis remains unclear. We demonstrate with dynamic imaging that loss of EphBs results in reduced motility of filopodia in cultured cortical neurons and brain slice. EphB knockdown and rescue experiments during different developmental time windows show that EphBs are required for synaptogenesis only when filopodia are most abundant and motile. In the context of EphB knockdown and reduced filopodia motility, independent rescue of either motility with PAK or of Eph-ephrin binding with an EphB2 kinase mutant is not sufficient to restore synapse formation. Strikingly, the combination of PAK and kinase-inactive EphB2 rescues synaptogenesis. Deletion of the ephrin-binding domain from EphB2 precludes rescue, indicating that both motility and trans-cellular interactions are required. Our findings provide a mechanistic link between dendritic filopodia motility and synapse differentiation.

Alterations in the thymocyte phenotype of EphB-deficient mice largely affect the double negative cell compartment.

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4- CD8-) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44- CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25-) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases.

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development.

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2-Fc or ephrinB1-Fc fusion proteins to fetal thymus organ cultures established from 17-day-old fetal mice decreases the numbers of both double-positive (CD4(+)CD8(+)) and single-positive (both CD4(+)CD8(-) and CD4(-)CD8(+)) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4(+)CD8(+) thymocytes, we have also demonstrated that ephrinB1-Fc proteins are able to disorganize the three-dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB-Fc treatment also decreases the formation of cell conjugates by CD4(+)CD8(+) thymocytes and TEC as well as the TCR-dependent signaling between both cell types. Finally, immobilized EphB2-Fc and ephrinB1-Fc modulate the anti-CD3 antibody-induced apoptosis of CD4(+)CD8(+) thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three-dimensional organization of TEC.

Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors.

The majority of mature excitatory synapses in the CNS are found on dendritic spines and contain AMPA- and NMDA-type glutamate receptors apposed to presynaptic specializations. EphB receptor tyrosine kinase signaling has been implicated in both NMDA-type glutamate receptor clustering and dendritic spine formation, but it remains unclear whether EphB plays a broader role in presynaptic and postsynaptic development. Here, we find that EphB2 is involved in organizing excitatory synapses through the independent activities of particular EphB2 protein domains. We demonstrate that EphB2 controls AMPA-type glutamate receptor localization through PDZ (postsynaptic density-95/Discs large/zona occludens-1) binding domain interactions and triggers presynaptic differentiation via its ephrin binding domain. Knockdown of EphB2 in dissociated neurons results in decreased functional synaptic inputs, spines, and presynaptic specializations. Mice lacking EphB1-EphB3 have reduced numbers of synapses, and defects are rescued with postnatal reexpression of EphB2 in single neurons in brain slice. These results demonstrate that EphB2 acts to control the organization of specific classes of mature glutamatergic synapses.

A unifying model for activity-dependent and activity-independent mechanisms predicts complete structure of topographic maps in ephrin-A deficient mice.

Axons of retinal ganglion cells establish orderly projections to the superior colliculus of the midbrain. Axons of neighboring cells terminate proximally in the superior colliculus thus forming a topographically precise representation of the visual world. Coordinate axes are encoded in retina and in the target through graded expression of chemical labels. Additional sharpening of projections is provided by electric activity, which is correlated between neighboring axons. Here we propose a quantitative model, which allows combining the effects of chemical labels and correlated activity in a single approach. Using this model we study a complete structure of two-dimensional topographic maps in mutant mice, in which the label encoding the horizontal retinal coordinate ephrin-A is reduced/eliminated. We show that topographic maps in ephrin-A deficient mice display a granular structure, with the regions of smooth mapping separated by linear discontinuities reminiscent of fractures observed in the maps of preferred orientation.

Multiple Eph receptors and B-class ephrins regulate midline crossing of corpus callosum fibers in the developing mouse forebrain.

Agenesis of the corpus callosum (CC) is a rare birth defect that occurs in isolated conditions and in combination with other developmental cerebral abnormalities. Recent identification of families of growth and guidance molecules has generated interest in the mechanisms that regulate callosal growth. One family, ephrins and Eph receptors, has been implicated in mediating midline pathfinding decisions; however, the complexity of these interactions has yet to be unraveled. Our studies shed light on which B-class ephrins and Eph receptors function to regulate CC midline growth and how these molecules interact with important guideposts during development. We show that multiple Eph receptors (B1, B2, B3, and A4) and B-class ephrins (B1, B2, and B3) are present and function in developing forebrain callosal fibers based on both spatial and temporal expression patterns and analysis of gene-targeted knock-out mice. Defects are most pronounced in the combination double knock-out mice, suggesting that compensatory mechanisms exist for several of these family members. Furthermore, these CC defects range from mild hypoplasia to complete agenesis and Probst's bundle formation. Further analysis revealed that Probst's bundle formation may reflect aberrant glial formations and/or altered sensitivity of CC axons to other guidance cues. Our results support a significant role for ephrins and Eph receptors in CC development and may provide insight to possible mechanisms involved in axon midline crossing and human disorder.

EphB receptor activity suppresses colorectal cancer progression.

Most sporadic colorectal cancers are initiated by activating Wnt pathway mutations, characterized by the stabilization of beta-catenin and constitutive transcription by the beta-catenin/T cell factor-4 (Tcf-4) complex. EphB guidance receptors are Tcf4 target genes that control intestinal epithelial architecture through repulsive interactions with Ephrin-B ligands. Here we show that, although Wnt signalling remains constitutively active, most human colorectal cancers lose expression of EphB at the adenoma-carcinoma transition. Loss of EphB expression strongly correlates with degree of malignancy. Furthermore, reduction of EphB activity accelerates tumorigenesis in the colon and rectum of Apc(Min/+) mice, and results in the formation of aggressive adenocarcinomas. Our data demonstrate that loss of EphB expression represents a critical step in colorectal cancer progression.

Ephrin signalling controls brain size by regulating apoptosis of neural progenitors.

Mechanisms controlling brain size include the regulation of neural progenitor cell proliferation, differentiation, survival and migration. Here we show that ephrin-A/EphA receptor signalling plays a key role in controlling the size of the mouse cerebral cortex by regulating cortical progenitor cell apoptosis. In vivo gain of EphA receptor function, achieved through ectopic expression of ephrin-A5 in early cortical progenitors expressing EphA7, caused a transient wave of neural progenitor cell apoptosis, resulting in premature depletion of progenitors and a subsequent dramatic decrease in cortical size. In vitro treatment with soluble ephrin-A ligands similarly induced the rapid death of cultured dissociated cortical progenitors in a caspase-3-dependent manner, thereby confirming a direct effect of ephrin/Eph signalling on apoptotic cascades. Conversely, in vivo loss of EphA function, achieved through EphA7 gene disruption, caused a reduction in apoptosis occurring normally in forebrain neural progenitors, resulting in an increase in cortical size and, in extreme cases, exencephalic forebrain overgrowth. Together, these results identify ephrin/Eph signalling as a physiological trigger for apoptosis that can alter brain size and shape by regulating the number of neural progenitors.

A relative signalling model for the formation of a topographic neural map.

The highly ordered wiring of retinal ganglion cell (RGC) neurons in the eye to their synaptic targets in the superior colliculus of the midbrain has long served as the dominant experimental system for the analysis of topographic neural maps. Here we describe a quantitative model for the development of one arm of this map--the wiring of the nasal-temporal axis of the retina to the caudal-rostral axis of the superior colliculus. The model is based on RGC-RGC competition that is governed by comparisons of EphA receptor signalling intensity, which are made using ratios of, rather than absolute differences in, EphA signalling between RGCs. Molecular genetic experiments, exploiting a combinatorial series of EphA receptor knock-in and knockout mice, confirm the salient predictions of the model, and show that it both describes and predicts topographic mapping.

Loss-of-function analysis of EphA receptors in retinotectal mapping.

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo. A gene disruption of mouse EphA5, replacing the intracellular domain with beta-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo, with temporal axons shifted posteriorly and nasal axons anteriorly, but with the entire target still filled by retinal axons. The anterior shift of nasal axons was not accompanied by increased responsiveness to tectal repellent activity, in contrast to the comparable anterior shift in ephrin-A knock-outs, helping to resolve a previous ambiguity in interpreting the ephrin gene knock-outs. The results show the functional requirement for endogenous EphA receptors in retinotectal mapping, show that the receptor intracellular domain is required for a forward signaling response to topographic cues, and provide new evidence for a role of axon competition in topographic mapping.

Hippocampal plasticity requires postsynaptic ephrinBs.

Chemical synapses contain specialized pre- and postsynaptic structures that regulate synaptic transmission and plasticity. EphB receptor tyrosine kinases are important molecular components in this process. Previously, EphB receptors were shown to act postsynaptically, whereas their transmembrane ligands, the ephrinBs, were presumed to act presynaptically. Here we show that in mouse hippocampal CA1 neurons, the Eph/ephrin system is used in an inverted manner: ephrinBs are predominantly localized postsynaptically and are required for synaptic plasticity. We further demonstrate that EphA4, a candidate receptor, is also critically involved in long-term plasticity independent of its cytoplasmic domain, suggesting that ephrinBs are the active signaling partner. This work raises the intriguing possibility that depending on the type of synapse, Eph/ephrins can be involved in activity-dependent plasticity in converse ways, with ephrinBs on the pre- or the postsynaptic side.

Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus.

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.