The Role of the Eph/Ephrin Family During Cortical Development and Cerebral Malformations Katrin Gerstmann1 and Geraldine Zimmer2*

Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 1 of 26

RESEARCH ARTICLE

The role of the Eph/ephrin family during cortical development and cerebral malformations Katrin Gerstmann1 and Geraldine Zimmer2*

Authors’ affiliations:

1 Institute NeuroMyoGène, CNRS UMR 5310, INSERM U1217, Université Lyon 1, Villeurbanne, France, E-mail: ([email protected]) 2 University Hospital Jena, Institute of Human Genetics, Am Klinikum 1, 07747 Jena, Germany, E-mail: ([email protected])

* Corresponding Author

Abstract Neuronal numbers and the associated size of the cerebral cortex, surface folding and laminar organisation are determined by precise developmental mechanisms that are orchestrated by several intrinsic and extrinsic molecules. Abnormalities during development can cause manifold microscopic and macroscopic cortical malformations, mostly accompanied by clinical consequences such as mental disorders, intellectual disabilities, or epileptic seizures. Most cortical malformations and associated neurological disorders result from genetic defects, however the cellular mechanisms remain complex and poorly understood. Eph receptor tyrosine kinases and their ligands, the ephrins, are abundantly expressed in the developing brain where they regulate several developmental processes that are crucial for correct brain formation. Ephrin family members represent membrane-bound proteins that are key players in complex short-range cell-cell communication. In addition, mechanisms for long-range interactions have been described recently. Several ephrins have already been shown to control cell cycle dynamics of cortical stem cells during corticogenesis and the positioning of postmitotic neurons. In addition, mutations in genes encoding for members of the Eph/ephrin family are implicated in mental disorders, although the underlying mechanisms remain to be elucidated. A deeper understanding of Eph/ephrin interactions during cerebral cortex development will be beneficial to shed light on developmental disabilities. Here, we discuss the function of Eph/ephrin system during the different processes of corticogenesis and the impact on cerebral malformations.

Keywords: ephrins, corticogenesis, malformation

Introduction amplifying progenitor cells. These intermediate progenitors translocate their The formation of the cerebral cortex, the cell bodies more basally, forming the seat of higher cognitive functions in the subventricular zone and dividing mammalian brain, is a highly sophisticated symmetrically to indirectly generate the process requiring the precise interplay of majority of neurons (1, 4, 5). The transient several developmental steps. Perturbation amplifying progenitors are already present of neural development can result in at early stages of neurogenesis and are manifold cortical malformations, often suggested to contribute to the neuronal associated with psychological disorders and production of all cortical layers (1, 4, 6). mental disabilities. The human cerebral cortex is generated during the first two The precise regulation of the progenitor trimesters of gestation. Within this period, pool is crucial for the correct development neuronal stem cells residing in the of the cerebral cortex. Overproduction of epithelium of the neural tube generate stem cells can lead to megalencephaly, diverse subtypes of progenitor, neuronal whereas the loss of neuronal stem cells and glial cells. These stem cells display a caused by precocious differentiation or polarized morphology with a basal process increased apoptosis results in anchored to the pial surface and an apical microencephaly (7). The cerebral cortex is process that is in contact with the formed in a temporally regulated inside-out cerebrospinal fluid. During mitotic cell fashion. Neurons destined for deep layers division, they perform characteristic are generated first, whereas those born later interkinetic nuclear migration synchronized migrate through the already existing deeper with cell cycle progression. The S-phase layers to form the superficial ones (8). takes place in the basal part of the Thereby, the radial processes of the ventricular zone, whereas G2 nuclei progenitor cells serve as a scaffold guiding translocate apically towards the ventricular the migrating post-mitotic neurons to their surface, where the M-phase occurs (1). target layers. Impaired neuronal migration However, the relevance of this nuclear is implicated in cortical malformations like translocation along the vertical axis is not lissencephaly, polymicrogyria, or hetero- clear and until now no human cortical topia (9). Interestingly, defects in cellular malformations are associated with defects adhesion result in equal phenotypes (9), as in these nuclear movements. For this adhesion is critical for neuronal migration. reason, we will not discuss it further. Eph (Erythropoietin-producing hepato- Before the first neurons are generated, cellular) receptor tyrosine kinases, and their neuronal stem cells divide symmetrically to membrane-bound ligands, the ephrins (Eph expand the pool of progenitor cells (2, 3). receptor interacting proteins), are critically At the onset of neurogenesis, stem cells involved in the regulation of developmental divide asymmetrically to generate post- processes underlying the formation of the mitotic neurons or intermediate, transient cerebral cortex including proliferation,

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 3 of 26 apoptosis, cellular adhesion, division interact with ephrinA1 (14, 19, 20) and orientation, and cell fate determination. EphrinB2 (21), respectively. Furthermore, Here we discuss the Eph/ephrin-dependent ephrins display different affinities to regulation of developmental processes and distinct receptors. For example, ephrinA5 potential implications in cortical has a stronger affinity to EphA4 and EphA5 malformations and neurological disorders. than to EphA3 (22).

A special feature of the Eph/ephrin family Structure, interactions and signalling of is the potential of bidirectional signalling the Eph/ephrin family (Figure 1). Forward signalling describes the induction of intracellular signalling in Eph receptor tyrosine kinases and their the receptor-bearing cell upon ligand ephrin ligands are widely expressed in the binding inducing receptor auto- developing brain (10-13) and numerous phosphorylation and subsequent activation studies have demonstrated their function in of downstream targets. Although ephrins do various developmental processes. The 15 not exhibit a catalytic activity, they can Eph receptor tyrosine kinases and 9 ephrin trigger a reverse signalling in ligand- ligands are classified into two groups, expressing cells after binding to cognate according to their structural similarities and receptors (17, 23-25). For class B-ephrins, binding affinities (Figure 1; 14). Class-A the reverse signalling results in the ephrins are tethered to the membrane by a phosphorylation of their cytoplasmic glycosylphosphatidyl-inositol anchor, domain, which triggers signal transduction. whereas class-B ephrins are trans- Although class A-ephrins do not possess an membrane proteins with a cytoplasmic intracellular domain, there is some evidence domain and a PDZ binding motif (15, 16). for the initiation of reverse signalling Eph receptor tyrosine kinases are through interactions with adapter proteins monomeric receptors that homodimerize (26, 27). and phosphorylate each other upon ligand binding. Furthermore, the receptors contain Another unique feature of Eph receptors a C-terminal PDZ-domain that potentially among tyrosine kinases is the capability to modulates intracellular signalling (15, 17, form higher-order clusters (28, 29). Such 18). EphA receptors bind promiscuously to protein assemblies can include more than A-ligands and EphB receptors to B-ephrins one Eph receptor, and the size as well as the (Figure 2). However, class-crossing composition of the cluster determines the interactions as well as exclusive binding cellular response. Various factors regulate affinities have also been described (14, 19, cluster size and several Eph receptors have 20). For instance, EphA4 shows class- different capacities to cluster (30). Thus, the crossing interactions with ephrinB2 and nature and specificity of a biological ephrinB3, whereas EphB2 binds ephrinA5. response arises from the particular In turn, EphA1 and EphB4 exclusively clustering of a receptor.

Figure 1: Schematic structure of Eph-receptors and their membrane-bound ephrin ligands. Class A-ephrins are tethered to the membrane by a glycosylphosphatidyl-inositol anchor, whereas class B-ephrins are trans-membrane proteins with a cytoplasmic domain and a PDZ binding motif. Upon ligand binding, forward signalling is induced in the receptor-bearing cell. Although ephrins do not exhibit a catalytic activity, they can trigger a reverse signalling after receptor binding.

Figure 2: Illustration of structural similarities and binding affinities of Eph receptors and their ephrin ligands. EphA receptors bind promiscuously to A-ligands and EphB receptors to B-ephrins (black line). However class-crossing interactions (brown arrows) and exclusive binding affinities (violet arrows) have been described.

Receptor-ligand interactions between usually involves paracrine interactions different cells are named trans-interactions. between neighbouring cells. However, this When cells express both, receptors as well established model of short-range interaction as corresponding ligands, cis-interactions of has been extended to long-range neighboured receptors and ligands are communication by recent work. For possible, which often diminishes forward instance, the developing thalamus was signalling responses, resulting from trans- suggested to exert inter-regional control interactions (31). Thus, co-expression of over the proliferation of cortical progenitor ligands and Eph receptors can modulate cells through axonal fibres growing into the cell-cell signalling and further increases the cerebral cortex during development, spectrum of physiological responses. For thereby modulating cell cycle kinetics of trans-binding of ephrins and Eph receptors, apical progenitor cells through interactions direct cell-cell contacts are required for with the radial processes (6). Besides this short-range communication (32) that cell contact-dependent mechanism, class A-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 6 of 26 ephrins can also be cleaved and released by appropriate numbers of neurons and proteases activating EphA receptors as intermediate precursor cells. The diffusible molecules (33). This emphasizes modulation of the balance between that soluble forms of ephrins can also act as proliferation, differentiation, adhesion and long-range cues (34, 35). In addition, it has apoptosis is crucial to generate a functional been described that guidance proteins from cortex and defects are associated with the cerebrospinal fluid influence the cell severe brain malformations accompanied by cycle kinetics of neuronal progenitor cells mental disorders. Several ephrins are that are lining the cerebral lumen (36, 37). expressed in the neurogenic niches and they Recent studies reported the existence of have been identified to influence various several ephrin members in the embryonic aspects of cortical proliferation. cavities (38), which provides the exciting Paracrine activation of EphA4 by ephrinB1 possibility for an extrinsic regulation of binding promotes cortical stem cell neuronal stem cells by the cerebrospinal proliferation and the expansion of the fluid. More recently it has been published progenitor cell pool (41). In turn, ephrinB1 that ephrins were also detected in reverse signalling induced by EphB1 extracellular vesicles that are released by binding maintains the cortical progenitor different cell types in the brain (39). The cell state (Figure 3; 42). Genetic depletion authors have shown that EphB2 present in of ephrinB1 promotes premature cell cycle exosomes can induce a reverse signalling in exit accompanied by a concomitant loss of ephrinB1-expressing cells, whereby the cortical progenitor cells (42). It has been initiated signalling cascade results in axonal suggested that ephrinB1 serves as a retraction. These findings demonstrate that molecular switch during development, Eph/ephrin signalling exhibits a wide range controlling the balance between progenitor of interactions in addition to the classical cell maintenance and neuronal binding, opening new perspectives in regard differentiation (43). Other proteins that to their implication in developmental interact with ephrinB1 potentially mediate mechanisms. this function. For instance, the zinc-finger and homeodomain protein 2 (ZHX2) is The Eph/ephrin family is involved in the expressed in cortical progenitor cells and control of brain size through the acts as transcriptional repressor whose regulation of cortical proliferation activity is enhanced by co-expression of the ephrinB1 intracellular domain. ZHX2 binds The size of the cerebral cortex depends on directly to the cytoplasmatic domain of the multiplication of neuronal stem cells in ephrinB1, which prevents the differentia- the neuroepithelium that generate diverse tion of neuronal progenitor cells (44). More progenitor, neuronal and glial subtypes recently it was shown that extrinsic (40). Several mechanisms control the size ephrinA5 ligands expressed by thalamic and composition of the cortical stem cell fibres activate EphA4 receptors of apical pool, orchestrating the production of progenitors, thereby controlling their

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 7 of 26 proliferation and cell fate decision activated protein kinase pathway that (Figure 3). Deletion of EFNA5 results in an controls neuronal differentiation in the increased production of basal precursors at developing nervous system (45). Further, it the expense of apical progenitors (6). has been published recently that ephrinA2 Moreover, EphA receptors have been seems crucial for the production of identified as positive regulators of mitogen- excitatory neurons (Figure 3; 46).

Figure 3: Eph/ephrin family members regulate essential cellular processes during cortical development. EphB2-induced reverse signalling of ephrinB1 promotes integrin-based apical adhesion. Activation of EphA7 by ephrinA5 causes cell death of cortical stem cells and regulates the size of the progenitor pool. Paracrine activation of EphA4 by ephrinB1 or due to binding to ephrinA5 on thalamic axons promotes symmetric divisions and cortical progenitor maintenance. EphB1-induced ephrinB1 reverse signalling maintains the cortical progenitor cell state. EphrinA2 reverse signalling promotes neurogenic divisions and neuronal maturation. EphrinB2-induced EphA4 activation inhibits migrating neurons. Furthermore EphA/ephrinA signalling controls lateral dispersion of postmitotic neurons. EphrinA5 is required to generate cortical columns by restricting lateral dispersion, whereas ephrinB1 promotes the lateral distribution of post-mitotic cells.

Apical progenitor cells give rise to were suggested to perform just one terminal intermediate progenitor cells, a more neurogenic division to produce two post- restricted type of transient amplifying mitotic neurons (47). In contrast, in precursor that generates the majority of primates and humans there are many types excitatory neurons in the developing cortex. of transient amplifying precursor In mice, these intermediate progenitors characterized by a remarkably increased

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 8 of 26 potential to proliferate and to produce more The Eph/ephrin system affects the diverse progeny (48). These transient survival and apoptosis of neuronal amplifying precursor cells are thought to progenitor cells promote the tangential expansion during The size of the cortical progenitor pool also cortical evolution (49, 50) and are depends on the regulation of programmed correlated with the appearance and degree cell death. Inhibiting Caspase-dependent of gyrification (50-52). Therefore, cortical apoptosis results in developmental generating correct numbers of transient abnormalities. CASP3 and CASP9-deficient amplifying precursors is crucial for correct embryos exhibit reduced apoptosis of cerebral cortex development and defects neuroepithelial stem cells, which is result in cortical malformations such as accompanied by an abnormal expansion of lissencephaly, polymicrogyria, or autism the progenitor pool, severe cortical spectrum disorders in which the patients malformations and excessive hyperplasia exhibit an overall higher degree of (59, 60). Ephrin family members are gyrification (53-55). involved in the control of cortical apoptosis Just a few genes are known to induce with consequential impact on cortical cortical expansion and to increase folding expansion. For example, EphA4 promotes (52, 56). There is emerging evidence that Caspase-dependent cell death in the Eph/ephrin family members are implicated ventricular wall with relevance for adult in the regulation of the transient amplifying neurogenesis. In turn, depletion of EphA4 progenitor pool. EphA4/ephrinA5- or infusion of soluble ephrinB3 into the interactions influence the generation of lateral ventricle inhibits apoptosis (61). intermediate progenitor cells during Moreover, expression of EphA7 and neurogenesis (6). Moreover, the ephrinB2 is mutually exclusive in the cells transcription factor PAX6 is expressed in of the ventricular wall, and EphA7-induced apical progenitor cells and critical for the ephrinB2 reverse signalling causes generation of intermediate progenitors (57). apoptosis and thereby negatively regulates Interestingly, it has already been shown that neural progenitor proliferation and EFNA5-deficient mice, which show altered neurogenesis in adult mice (62). In addition, numbers of intermediate progenitor cells EphB3/ephrinB3-interactions in the adult (6), display decreased PAX6 expression SVZ are transiently inhibited after levels (11). Furthermore, EphA3 and traumatic brain injury, probably to enhance EphA5 are expressed in the primate SVZ, the expansion and survival of neuronal suggesting that they play a role in transient precursors (63). During development, amplifying precursors in higher mammals overexpression of EphA8 in the dorsal (58). Together, these studies suggest that midline of the diencephalon and the Eph/ephrin system is implicated in the mesencephalon causes apoptosis of generation of transient amplifying precursor neuroepithelial cells (64). Another study cells and potentially plays a role during has shown that the activation of EphA7 by cerebral cortex expansion and gyrification. ectopic overexpression of ephrinA5 induces

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 9 of 26 death of cortical stem cells (Figure 3) mental retardation (66). Microencephaly is accompanied by reduced numbers of associated to defects in progenitor cell cortical progenitor cells and dramatic division; however, the exact cellular decrease of brain size. In turn, depletion of mechanisms remain to be investigated. EphA7 results in reduced cell death and During early corticogenesis, the neuronal exencephalic overgrowth of the forebrain stem cells divide symmetrically with a (65). However, analyses of EFNA5- planar division angle that expands the stem deficient embryos reveal no differences in cell pool. However, at the onset of the numbers of apoptotic cells in the neurogenesis, cells divide asymmetrically ventricular zone and therefore do not with more deviated angles to produce support a direct pro-apoptotic effect of various daughter cells with different fates ephrinA5 on cortical progenitor cells (6). (4, 5), presumably as a result of asymmetric Overexpression of ephrinA5 might induce inheritance of cell fate determining factors apoptosis, whereas physiological levels of (67, 68). The orientation of cell division is the ligand may not reach the threshold precisely controlled by several intrinsic and required to induce cell death. Thus, extrinsic signals and spindle orientation Eph/ephrin-bidirectional signalling seems defects are associated with cortical to generally promote physiological malformations like microencephaly as well apoptosis, whereas overstimulation of the as macroencephaly. Moreover, alterations system results in massive cell death (64). in relative numbers of symmetric and The spatially restricted mutual expression asymmetric cell divisions result in changes patterns of ephrin family members was of the cortical surface area. For instance, already suggested to play a role during the delayed onset of asymmetric divisions tissue boundary formation and may prevent results in an increased cortical surface area excessive growth through induction of (69). apoptosis as shown for the overstimulation of the Eph/ephrin-system in neuroepithelial There is a link between mechanical cues cells, thereby affecting brain size and tissue from the environment and the alignment of morphogenesis. the mitotic spindle (70). The distribution of adhesive contacts during interphase Functional implications of ephrins in precisely predicts the future orientation of progenitor cell division the mitotic spindle (71). As the Eph/ephrin system is known to modulate cellular Recent studies suggest that ephrin family adhesion, it is feasible that they do not just members are implicated in the modulation regulate apical attachment, but may also of cell division orientation. Patients with a exert mechanical tension on dividing deletion of a gene cluster of ephrin family progenitor cells to influence fate members, including EFNA1, EFNA3 and determination. In ascidian embryos EFNA4, exhibit microencephaly and extracellular FGF and ephrin signals developmental delay accompanied by polarize the mitotic mother cell via

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 10 of 26 interactions with the adjacent ectoderm, provide an explanation for the thereby promoting asymmetric cell division microcephaly observed in patients with and inducing distinct cell fates in the deletions in the EPH/EFN gene cluster (66). resultant daughter pair (72). Moreover, it In addition, ephrins may also influence has been shown that ephrinA5 is implicated cortical progenitor divisions through the in the regulation of asymmetric divisions in modulation of the canonical Wnt pathway. the neural tube and notochord of Ciona Wnt is essential for the maintenance of intestinalis by functionally polarizing the cortical progenitor cells and self-renewal mother cell and thereby promoting the divisions (78), and defects in canonical Wnt generation of neuronal daughters (73). signalling result in disturbed laminar Defects in the ephrin signalling results in organisation of the cortex (79). EphB symmetric divisions and generated cells receptors are Wnt-target genes in the adapt the notochord fate. intestinal epithelium (80) and it has been In the developing cortex, ephrinB1 and shown that ephrinA3 suppresses Wnt- ephrinB2 regulate the activity of the Par- signalling in retinal stem cells through the protein complex (74-76) that is located in activation of EphA4, thereby inhibiting the apical end feet of neuronal stem cells proliferation and increasing differentiation (1). This complex plays a key role for (81). Additionally, in the developing cortex apico-basal polarity, mitosis and promotes canonical Wnt-signalling was reported to proliferative divisions as well as the negatively regulate EFNB3-expression (82). generation of basal precursors (77). Ephrin family members are also implicated EphrinB1 reverse signalling promotes the in cytokinesis, the final phase of cell transition from proliferative symmetric division, during which the daughter cells division towards neurogenic asymmetric become separated. The completion of divisions (42). In contrast, it has been cytokinesis is controlled by factors of the shown that ephrinA5 promotes symmetric, environment. The function of citron kinase proliferative divisions and the maintenance is crucial for the cytokinesis (83) and of apical progenitors during early mutations in the coding gene affect brain neurogenesis (6). Loss of ephrinA5 function development (84) and cause primary results in asymmetric divisions microcephaly in humans (85). It has been accompanied by increased numbers of shown that Eph-mediated tyrosine intermediate progenitors and post-mitotic phosphorylation of citron kinase regulates neurons. More recently it has been abscission and EphB2 forward signalling published that ephrinA2 reverse signalling causes midbody forming failures, thereby promotes neurogenic divisions and neuronal preventing the completion of cytokinesis differentiation (Figure 3; 46). Together, (86). Hence, the Eph/ephrin family seems these studies strongly support a role for relevant for various subcellular regulatory ephrins in the regulation of division processes underlying progenitor division. orientation and fate determination in the developing neuroepithelium, which would

Eph/ephrins and cellular adhesion Cadherin to the adherent junctions during lens development and thereby promotes the Regulation of cellular adhesion is crucial formation of cell-cell contacts (90). Eph for the appropriate formation of the cerebral receptor stimulation with pre-clustered cortex and defects in adhesion are ephrins induces aggregation of endothelial associated with cortical malformations. and embryonic cortical cells in culture and Cortical stem cells are highly polarized Eph/ephrin-interactions can regulate the cells that are basally anchored to the pial function of other cell adhesion molecules surface and apically to the ventricular (91-93). It has been further shown that the border. Adhesion is intimately linked to activation of Eph receptors potentially proliferation, the precise generation of the increases the adhesiveness to the neuronal numbers and cortical precursors as extracellular matrix via integrins (94). well as to neuronal migration. Thus, the Moreover, ephrinA5 regulates integrin disruption of either basal or apical function and thereby modulates cellular attachment can cause proliferation and adhesion (95). Paracrine interactions migration disorders (9). between ephrinB1 and EphB2 on adjacent Cortical stem cells are tightly attached to cells promote apical integrin-based adjacent neighbours and receive pro-mitotic adhesion (Figure 3) and deletion of EFNB1 signals from surrounding cells and the results in abnormal neuroepithelia and cerebrospinal fluid (36, 41). Attachment to exencephaly (88). the ventricular surface is associated with The basal process is anchored to the basal symmetric division and loss of apical membrane adjacent to the pial surface and adhesion during asymmetric division causes serves as a scaffold for radially migrating changes of cellular fate (87). Delamination neurons. Thus, incorrect basal attachment of neuronal precursors from the apical can cause migratory defects such as surface could be responsible for the heterotopia or lissencephaly. The basal emergence of intermediate progenitor cells membrane represents an anchor for the during evolution (88). Apical attachment basal end feet and a physical barrier, defects result in precocious delamination, respectively. Basal attachment of the radial accompanied by concomitant differentiation processes regulates cortical proliferation via and loss of cortical stem cells (87, 89). integrin-signalling (96, 97) and integrin- However, cells that are committed to deficiency causes defects in the cortical differentiate reduce adhesiveness and organisation (9). It has been published that disengage from the neuroepithelium. ephrin family members can directly or A key component of the adherent junction indirectly modulate integrin signalling (98, complex is N-Cadherin and loss of function 99). However, an implication in the results in precocious delamination and regulation of basal attachment has not yet accumulation of ectopic neurons in the been shown. Another adhesion protein that ventricular zone (87, 89). It has already is secreted in the basal membrane is laminin been described, that ephrinA5 recruits N- and deficiency of this glycoprotein is

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 12 of 26 associated with cobblestone lissencephaly developing cortical layers. Thus, radial (100). Stimulation of EphA2 forward migration is the major mechanism that signalling increases the secretion of laminin controls neuronal positioning (104, 105) in proximal tubular cell lines (101) and B- and mis-regulation results in cortical ephrins modulate laminin-mediated malformations like lissencephaly and interactions of thymocytes (102). However, heterotopia. Ephrin family members are it remains to be determined whether ephrins already known to control tangential regulate laminin-mediated adhesion in the migration of cortical interneurons (106- developing cortex. 110), but they also influence radial migration. Another important developmental step in which ephrins are intimately involved The initiating step after delamination is the through regulation of adhesion is the multipolar bipolar transition in which new- closure of the neuronal tube. In human born neurons acquire neuronal polarity by embryos, the neural plate arises 18 days converting from a multipolar to a bipolar after fertilisation and the neural tube is morphology with a leading and trailing formed between the weeks 3 -11 of process (111). During early neurogenesis, gastrulation. Neural tube closure defects post-mitotic neurons mainly migrate in a result in spina bifida or anencephaly. The glia-independent manner by phasic edges of the neural folds co-express EPHA7 translocation (112). At later stages of and EFNA5 (103). EphA7 is spliced in three corticogenesis gliophil migration different forms, one full length and two dominates, in which the basal processes of truncated versions. The expression of the radial glia cells build a scaffold along truncated proteins suppresses the tyrosine which neurons migrate to their cortical phosphorylation of the full length EphA7 target layers (113, 114). The morphology thereby changing the cellular response from and integrity of the glial fibres influences repulsion to adhesion (103). It was the radial migration (115) and disruption of proposed that EphA7 in combination with the processes causes cortical malformations ephrinA5 serves as a molecular switch similar to migration defects (9). Hence, one during neural tube formation. This mechanism through which ephrins regulate assumption leads to the intriguing migration seems to rely on the regulation of possibility that below the threshold level for adhesive complexes, enabling coordinated repulsion, persistent Eph receptor activation interactions between the glial fibres and the leads to an adhesive response (94). migrating neurons required for proper migration.

Ephrins regulate the radial migration of During locomotion, the leading process excitatory neurons extends to the pial surface followed by the nucleokinesis, the movement of the nucleus After exiting the cell cycle, post-mitotic (116). Thereby, the centrosome serves as a cells migrate radially towards the pial microtubule organising centre that is surface to reach their final position in the

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 13 of 26 proximal to the leading process. From there that mutations in the gene coding for Reelin microtubules extend towards the anterior result in severe abnormalities of the cellular leading process and posterior towards the organisation of the cortex. The function of nucleus to form a perinuclear cage and to Reelin in establishing the cortical layers exert pulling forces (117). In contrast, seems associated to EphB/ephrinB posterior contraction of myosin II generates signalling as EFNB1/B2/B3 triple mutant pushing forces (118). During locomotion, mice mimic the Reeler phenotype with the the leading process defines the direction of disrupted laminar organisation (122), the movement and extracellular cues suggesting a link between the canonial instruct the migration. Therefore, the Reelin pathway and ephrinB signalling. growth cone exhibits a miscellaneous However, other studies did not confirm this assortment of guidance molecules to extend crosstalk (123). towards the correct direction. The Besides its laminar architecture, the cortex expression of EFNB2, EPHA4 and EPHB1 is further organised in ontogenetic columns is complementary in the developing cortex of clonally related neurons that are and regulate the radial migration (119). generated by a single stem cell and are EphA4 activation by ephrinB2 inhibits stereotypically interconnected (124). migrating neurons and therefore acts as a Thereby the glial mother cell serves as a stop signal in the intermediate zone scaffold during migration, ensuring the (Figure 3). Neurons with a low expression precise radial location of the generated levels of EphA4 can pass, whereas cells daughter cells within the ontogenetic with a high EphA4-expression are repelled. column. However, some clonally-related This may contribute to the ordered neurons undergo a lateral shift from their formation of the cortical layers. original glial cell to an adjacent basal Consequently, EPHA4-deficiency disrupts process (125) that might represent an early the barrier constituted by ephrinB2 and information processing mechanism in the results in aberrant migration of cortical developing cortex. It has been shown that neurons (119). ephrin family members control this lateral Locomotion terminates when the neurons dispersion. EphA/ephrinA signalling is reach the final destination and detach from required to generate cortical columns the glial fibres. This requires anti-adhesive (Figure 3) and deletion of EFNA5 results in signals to allow detachment from the radial impaired lateral distribution (125). processes (120) or decreased levels of cell Furthermore, ephrinB1 regulates the lateral adhesion molecules in the membrane as dispersion of post-mitotic cells by limiting shown for N-Cadherin (121). Impairments the neurite extension during the multipolar in terminal translocation can lead to over- phase and gain of ephrinB1 function leads migration of neurons into the meninges to abnormal clustering of neurons (126). resulting in neocortical dysplasia, Even though defects in the lateral polymicrogyria, or cobblestone distribution are not verifiable yet in the lissencephaly (9). It has been demonstrated adult human brain, it is conceivable that this

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra Gerstmann K. et al. Medical Research Archives, vol. 6, issue 3, March 2018 issue Page 14 of 26 can result in mental disorders and cognitive signalling, their interactions with potential deficits. In summary, the Eph/ephrin family effector proteins and crosstalk with other is implicated in the regulation of various pathways during fundamental aspects of cortical migration. neurodevelopmental processes will help to gain valuable insights in the pathophysiology of human cortical Conclusions malformations. Relating human mental diseases to failures in developmental processes is challenging. Acknowledgement Defects in different mechanisms can result in similar phenotypes as most of the We are grateful to Edmund Derrington for developmental and subcellular processes critical reading of the manuscript. We are intimately linked to each other. For apologize to all colleagues whose instance, adhesion plays a role during publications we were not able to cite due to proliferation, differentiation and migration. reasons of space limitations. This work Although, the causative genes for a variety received support from the Deutsche of human syndromes have already been Forschungsgemeinschaft (DFG) [ZI identified, the defects in the cellular 1178 1224/2-1] and the IZKF Jena. processes causing the cortical malformations still remain to be elucidated. Conflict of Interest Therefore, it is essential to analyse the role of the respective gene in a particular The authors declare no competing interests developmental context.

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Sunkin SM, Mouton PR, Roy S, Wynshaw-Boris A, Colamarino SA, Lein ES, Courchesne E. Patches of

Table 1: Expression of ephrin family members in the cerebral cortex during development, their cellular role during cortical development and their described implications in mental diseases.

Cellular function during brain Molecul Expression Related brain disease development CP, IMZ (postmitotic neurons) EphA3 (6, 41) Cortex expansion (58) Autism spectrum disorders (129) Primate SVZ (58) Cortical stem cell expansion VZ, CP, IMZ EphA4 Promotes symmetric divisions Mental disorders (131) (6, 41) (6, 41) Mild neurological disorders and structural symptoms Cortical progenitor cells (65) (130) EphA7 Pro-apoptotic (65) CP, IMZ, VZ (41) Autism spectrum disorders (129) Exencephalic overgrowth of forebrain (65) Primate SVZ (58) EphA5 Cortex expansion (58) CP (41) Activation of ephrinB1 by EphB1 maintains Schizophrenia EphB1 IMZ (41) cortical progenitor state (42) (132) Completion of cytokinesis (86) Mental disorders (131) EphB2 CP,IMZ (41) Apical Integrin-based adhesion (88) Microencephaly, developmental ephrinA1 CP+IMZ (41) delay (66) Microencephaly, developmental Excitatory neuron differentiation ephrinA2 CP+IMZ+proliferative zone (41) delay (66) (128) Autism spectrum disorder (133) Microencephaly, developmental ephrinA4 CP+IMZ (41) delay (66) Epilepsy (134) Maintainance of apical progenitor cells (Symmetric division); control production of IPCs IMZ (thalamic fibres) Schizophrenia EphrinA5 (6) (6) (135) Proapoptotic (65) Lateral dispersion of postmitotic neurons (125) Apical attachment (88) Apical Integrin-based adhesion (88) Proliferative zone Exencephaly EphrinB1 Maintanance of cortical stem cells (42) (41, 42) (88) Lateral dispersion of postmitotic neurons (126) Promotes neurogenic division and neuronal Schizophrenia (87) EphrinB2 VZ, IMZ ,CP (127; 128) maturation (128)

Stop signal for migrating neurons (119)