The Transcription Factor LHX1 Regulates the Survival and Directed Migration of POA-derived Cortical Interneurons.

The delicate balance of excitation and inhibition is crucial for proper function of the cerebral cortex, relying on the accurate number and subtype composition of inhibitory gamma-aminobutyric (GABA)-expressing interneurons. Various intrinsic and extrinsic factors precisely orchestrate their multifaceted development including the long-range migration from the basal telencephalon to cortical targets as well as interneuron survival throughout the developmental period. Particularly expressed guidance receptors were described to channel the migration of cortical interneurons deriving from the medial ganglionic eminence (MGE) and the preoptic area (POA) along distinct routes. Hence, unveiling the regulatory genetic networks controlling subtype-specific gene expression profiles is key to understand interneuron-specific developmental programs and to reveal causes for associated disorders. In contrast to MGE-derived interneurons, little is known about the transcriptional networks in interneurons born in the POA. Here, we provide first evidence for the LIM-homeobox transcription factor LHX1 as a crucial key player in the post-mitotic development of POA-derived cortical interneurons. By transcriptional regulation of related genes, LHX1 modulates their survival as well as the subtype-specific expression of guidance receptors of the Eph/ephrin family, thereby affecting directional migration and layer distribution in the adult cortex.

Involvement of EphB/Ephrin-B signaling in axonal survival in mouse experimental glaucoma.

PURPOSE: To examine the functional significance of EphB/ephrin-B upregulation in mouse experimental glaucoma. METHODS: In a loss-of-function approach, mouse mutants lacking EphB2 (EphB2(-/-)) or EphB3 (EphB3(-/-)) protein, and mutants expressing EphB2 truncated in the C-terminus (EphB2(lacZ/lacZ)) were subjected to laser-induced ocular hypertension (LIOH), an experimental mouse model of glaucoma. The number of optic nerve axons was counted in paraphenylenediamine (PPD)-stained sections and compared between EphB mutants and wild type littermates. In a gain-of-function approach, retina/optic nerve explants obtained from LIOH-treated animals were exposed to EphB2-Fc recombinant proteins or Fc control proteins. Tissue sections through the optic nerve head (ONH) were labeled with neuron-specific anti-tubulin ß-III antibody to determine axonal integrity. RESULTS: Both EphB2 and EphB3 null mutant mice exhibited more severe axonal degeneration than wild type littermates after treatment with LIOH. Mutant mice in which the C-terminal portion of EphB2 is truncated had an intermediate phenotype. Application of EphB2-Fc recombinant protein to LIOH-treated optic nerve explants resulted in greater sparing of tubulin ß-III-containing retinal ganglion cell (RGC) axons. CONCLUSIONS: These results provide genetic evidence in mice that both EphB/ephrin-B forward and reverse signaling feed into an endogenous pathway to moderate the effects of glaucomatous insult on RGC axons. LIOH-induced axon loss is maintained in retina/optic nerve explants after removal from an ocular hypertensive environment. Exogenous application of EphB2 protein enhances RGC axon survival in explants, suggesting that modulation of Eph/ephrin signaling may be of therapeutic interest.

Ephrin-B reverse signaling controls septation events at the embryonic midline through separate tyrosine phosphorylation-independent signaling avenues.

We report that the disruption of bidirectional signaling between ephrin-B2 and EphB receptors impairs morphogenetic cell-cell septation and closure events during development of the embryonic midline. A novel role for reverse signaling is identified in tracheoesophageal foregut septation, as animals lacking the cytoplasmic domain of ephrin-B2 present with laryngotracheoesophageal cleft (LTEC), while both EphB2/EphB3 forward signaling and ephrin-B2 reverse signaling are shown to be required for midline fusion of the palate. In a third midline event, EphB2/EphB3 are shown to mediate ventral abdominal wall closure by acting principally as ligands to stimulate ephrin-B reverse signaling. Analysis of new ephrin-B2(6YFΔV) and ephrin-B2(ΔV) mutants that specifically ablate ephrin-B2 tyrosine phosphorylation- and/or PDZ domain-mediated signaling indicates there are at least two distinct phosphorylation-independent components of reverse signaling. These involve both PDZ domain interactions and a non-canonical SH2/PDZ-independent form of reverse signaling that may utilize associations with claudin family tetraspan molecules, as EphB2 and activated ephrin-B2 molecules are specifically co-localized with claudins in epithelia at the point of septation. Finally, the developmental phenotypes described here mirror common human midline birth defects found with the VACTERL association, suggesting a molecular link to bidirectional signaling through B-subclass Ephs and ephrins.

EphrinBs regulate D-serine synthesis and release in astrocytes.

There is growing evidence that astrocytes play critical roles in neuron-glial interactions at the synapse. Astrocytes are believed to regulate presynaptic and postsynaptic structures and functions, in part, by the release of gliotransmitters such as glutamate, ATP, and d-serine; however, little is known of how neurons and astrocytes communicate to regulate these processes. Here, we investigated a family of transmembrane proteins called ephrinBs and Eph receptors that are expressed in the synapse and are known to regulate synaptic transmission and plasticity. In addition to their presence on CA1 hippocampal neurons, we determined that ephrins and Eph receptors are also expressed on hippocampal astrocytes. Stimulation of hippocampal astrocytes with soluble ephrinB3, known to be expressed on CA1 postsynaptic dendrites, enhanced d-serine synthesis and release in culture. Conversely, ephrinB3 had no effect on d-serine release from astrocytes deficient in EphB3 and EphA4, which are the primary receptors for ephrinB3. Eph receptors mediate this response through interactions with PICK1 (protein interacting with C-kinase) and by dephosphorylating protein kinase C α to activate the conversion of l-serine to d-serine by serine racemase. These findings are supported in vivo, where reduced d-serine levels and synaptic transmissions are observed in the absence of EphB3 and EphA4. These data support a role for ephrins and Eph receptors in regulating astrocyte gliotransmitters, which may have important implications on synaptic transmission and plasticity.

Cortical control of adaptive locomotion in wild-type mice and mutant mice lacking the ephrin-Eph effector protein alpha2-chimaerin.

In voluntary control, supraspinal motor systems select the appropriate response and plan movement mechanics to match task constraints. Spinal circuits translate supraspinal drive into action. We studied the interplay between motor cortex (M1) and spinal circuits during voluntary movements in wild-type (WT) mice and mice lacking the α2-chimaerin gene (Chn1(-/-)), necessary for ephrinB3-EphA4 signaling. Chn1(-/-) mice have aberrant bilateral corticospinal systems, aberrant bilateral-projecting spinal interneurons, and disordered voluntary control because they express a hopping gait, which may be akin to mirror movements. We addressed three issues. First, we determined the role of the corticospinal system in adaptive control. We trained mice to step over obstacles during treadmill locomotion. We compared performance before and after bilateral M1 ablation. WT mice adaptively modified their trajectory to step over obstacles, and M1 ablation increased substantially the incidence of errant steps over the obstacle. Chn1(-/-) mice randomly stepped or hopped during unobstructed locomotion but hopped over the obstacle. Bilateral M1 ablation eliminated this obstacle-dependent hop selection and increased forelimb obstacle contact errors. Second, we characterized the laterality of corticospinal action in Chn1(-/-) mice using pseudorabies virus retrograde transneuronal transport and intracortical microstimulation. We showed bilateral connections between M1 and forelimb muscles in Chn1(-/-) and unilateral connections in WT mice. Third, in Chn1(-/-) mice, we studied adaptive responses before and after unilateral M1 ablation. We identified a more important role for contralateral than ipsilateral M1 in hopping over the obstacle. Our findings suggest an important role for M1 in the mouse in moment-to-moment adaptive control, and further, using Chn1(-/-) mice, a role in mediating task-dependent selection of mirror-like hopping movements over the obstacle. Our findings also stress the importance of subcortical control during adaptive locomotion because key features of the trajectory remained largely intact after M1 ablation.

Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning.

It has been suggested that ephrin-B proteins have receptor-like roles in the control of axon pathfinding by repulsion, although it is largely unknown how the reverse signals are coupled to downstream intracellular molecules and how they induce cytoskeletal reorganization at the axon terminal. We found that ephrin-B3 (EB3) was able to function as a repulsive guidance receptor and mediate stereotyped pruning of murine hippocampal mossy fiber axons during postnatal development. Targeted intracellular point mutants showed that axon pruning requires tyrosine phosphorylation-dependent reverse signaling and coupling to the SH2/SH3 adaptor protein Grb4 (also known as Nckbeta/Nck2). Furthermore, we found that the second SH3 domain of Grb4 is required and sufficient for axon pruning/retraction by mediating interactions with Dock180 and PAK to bring about guanine nucleotide exchange and signaling downstream of Rac, respectively. Our results reveal a previously unknown pathway that controls axon pruning and elucidate the biochemical mechanism by which ephrin-B reverse signals regulate actin dynamics to bring about the retraction of growth cones.

Postsynaptic ephrinB3 promotes shaft glutamatergic synapse formation.

Excitatory synapses in the CNS are formed on both dendritic spines and shafts. Recent studies show that the density of shaft synapses may be independently regulated by behavioral learning and the induction of synaptic plasticity, suggesting that distinct mechanisms are involved in regulating these two types of synapses. Although the molecular mechanisms underlying spinogenesis and spine synapse formation are being delineated, those regulating shaft synapses are still unknown. Here, we show that postsynaptic ephrinB3 expression promotes the formation of glutamatergic synapses specifically on the shafts, not on spines. Reducing or increasing postsynaptic ephrinB3 expression selectively decreases or increases shaft synapse density, respectively. In the ephrinB3 knock-out mouse, although spine synapses are normal, shaft synapse formation is reduced in the hippocampus. Overexpression of glutamate receptor-interacting protein 1 (GRIP1) rescues ephrinB3 knockdown phenotype by restoring shaft synapse density. GRIP1 knockdown prevents the increase in shaft synapse density induced by ephrinB3 overexpression. Together, our results reveal a novel mechanism for independent modulation of shaft synapses through ephrinB3 reverse signaling.

Envelope-receptor interactions in Nipah virus pathobiology.

Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope-receptor interactions in NiV pathobiology. Finally, we also describe how galectin-1 (an innate immune defense lectin) can interact with specific N-glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens.

Ephrin-B3 ligand promotes glioma invasion through activation of Rac1.

Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion.

EphB receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord.

EphB receptors and their ephrin-B ligands are required for midline guidance decisions at several rostrocaudal levels of the developing CNS. In the embryonic vertebrate spinal cord, ephrin-B3 is localized to the floor plate (FP) at the ventral midline (VM), ephrin-B1 and ephrin-B2 are expressed in the dorsal spinal cord, and decussated EphB receptor-bearing commissural axons navigate between these ventral and dorsal ephrin-B domains. Despite these compelling expression patterns, the in vivo role(s) for EphB and ephrin-B proteins in regulating the guidance of spinal commissural axons has not been established. Here, we use DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeling to assess the pathfinding of commissural axons in the spinal cords of ephrin-B and EphB mutant mouse embryos. In mice lacking ephrin-B3 or multiple EphB receptors, a significant number of axons followed aberrant trajectories in the immediate vicinity of the VM. Furthermore, forked transverse commissural (FTC) axons, a unique class of commissural axons that continues to project in the transverse plane on the contralateral side of the FP, were present at a markedly higher frequency in ephrin-B3 and EphB mutants, compared with wild-type embryos. Neither the midline guidance errors nor excessive numbers of FTC axons were observed in the spinal cords of ephrin-B3(lacz) mice that express a truncated form of ephrin-B3, which is capable of forward but not reverse signaling. In contrast to the midline guidance defects observed in EphB and ephrin-B3 mutant embryos, wild-type-like contralateral projections were observed in mice lacking ephrin-B1 and/or ephrin-B2.

Distinct roles for ephrinB3 in the formation and function of hippocampal synapses.

The transmembrane ephrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors are presumed to be pre-synaptic. Interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), which has been reported to be mediated by reverse signaling into the post-synaptic membrane. Here, we demonstrate that the cytoplasmic domain of ephrinB3 and hence reverse signaling is not required for ephrinB dependent learning and memory tasks or for LTP of these synapses. Consistent with previous reports, we find that ephrinB3(KO) null mutant mice exhibit a striking reduction in CA3-CA1 LTP that is associated with defective learning and memory tasks. We find the null mutants also show changes in both pre- and post-synaptic proteins including increased levels of synapsin and synaptobrevin and reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3(lacZ) reverse signaling mutants that specifically delete the ephrinB3 intracellular region, supporting a cytoplasmic domain-independent forward signaling role for ephrinB3 in these processes. We also find that both ephrinB3(KO) and ephrinB3(lacZ) mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling to modulate a number of other proteins involved in synaptic activity and learning/memory.

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.

Physiological, anatomical and genetic identification of CPG neurons in the developing mammalian spinal cord.

The basic motor patterns underlying rhythmic limb movements during locomotion are generated by neuronal networks located within the spinal cord. These networks are called Central Pattern Generators (CPGs). Isolated spinal cord preparations from newborn rats and mice have become increasingly important for understanding the organization of the CPG in the mammalian spinal cord. Early studies using these preparations have focused on the overall network structure and the localization of the CPG. In this review we concentrate on recent experiments aimed at identifying and characterizing CPG-interneurons in the rodent. These experiments include the organization and function of descending commissural interneurons (dCINs) in the hindlimb CPG of the neonatal rat, as well as the role of Ephrin receptor A4 (EphA4) and its Ephrin ligand B3 (EphrinB3), in the construction of the mammalian locomotor network. These latter experiments have defined EphA4 as a molecular marker for mammalian excitatory hindlimb CPG neurons. We also review genetic approaches that can be applied to the mouse spinal cord. These include methods for identifying sub-populations of neurons by genetically encoded reporters, techniques to trace network connectivity with cell-specific genetically encoded tracers, and ways to selectively ablate or eliminate neuron populations from the CPG. We propose that by applying a multidisciplinary approach it will be possible to understand the network structure of the mammalian locomotor CPG. Such an understanding will be instrumental in devising new therapeutic strategies for patients with spinal cord injury.

Eph receptor deficiencies lead to altered cochlear function.

Ephrins and Eph receptors are a family of molecules that have been implicated in many developmental processes including neuronal network formation, guidance of cell migration, and axonal pathfinding. These molecules exhibit the ability to send bidirectional signals following ligand-receptor interactions resulting from cell-cell contacts. Gene-targeted knockout mice of B-class ephrins and Eph receptors have been shown to display phenotypic responses that correlate with anatomical defects. For example, disruption of the EphB2 receptor leads to defects of the vestibular system, including pathfinding abnormalities in efferent axons and reduced endolymph production. Such developmental distortions lead to deficiencies in ionic homeostasis and repetitive circling behaviors. The present study demonstrates that B-class ephrins and Eph receptors are expressed in cochlear tissues, suggesting that they may play some role in auditory function. To determine whether ephrins and Eph receptors have a functional role in the peripheral auditory system, distortion-product otoacoustic emission (DPOAE) levels, collected across a broad frequency range, were compared between groups of mice expressing different Eph receptor genotypes. In particular, EphB1 and EphB3 receptor knockout mice exhibited significantly diminished DPOAE levels as compared to wild-type littermates, indicating that these specific Eph receptors are necessary for normal cochlear function.

Role of EphA4 and EphrinB3 in local neuronal circuits that control walking.

Local circuits in the spinal cord that generate locomotion are termed central pattern generators (CPGs). These provide coordinated bilateral control over the normal limb alternation that underlies walking. The molecules that organize the mammalian CPG are unknown. Isolated spinal cords from mice lacking either the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation and instead exhibit synchrony. We identified EphA4-positive neurons as an excitatory component of the locomotor CPG. Our study shows that dramatic locomotor changes can occur as a consequence of local genetic rewiring and identifies genes required for the development of normal locomotor behavior.