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provided by Elsevier - Publisher Connector Neuron, Vol. 29, 85–97, January, 2001, Copyright 2001 by Cell Press Forward Signaling Mediated by -B3 Prevents Contralateral Corticospinal from Recrossing the Spinal Cord Midline

Nobuhiko Yokoyama,*§k Mario I. Romero,*§ shoelace. Although the molecular genetic events that Chad A. Cowan,* Pedro Galvan,* lead to mirror movements in humans have not been Franc¸ oise Helmbacher,† Patrick Charnay,† described, there is an association with various congeni- Luis F. Parada,* and Mark Henkemeyer*‡ tal malformations, including Kallmann, Klippel-Feil, and *Center for Developmental Biology and Wildervanck syndromes (Schott and Wyke, 1981), and Kent Waldrep Foundation Center for familial cases have been identified (Regli et al., 1967). Basic Research on Nerve Growth Mirror movements may be associated with defects in and Regeneration the corticospinal tract (CST), which forms the connec- University of Texas Southwestern Medical Center tions of the motor cortex with the primary motor neurons Dallas, Texas 75390 and interneurons in the spinal cord (Mayston et al., 1997; † Unite´ 368 de l’Institut National de la Sante´ et Krams et al., 1999; Balbi et al., 2000). Normally, cortico- de la Recherche Me´ dicale spinal neurons in the motor cortex send axons toward Ecole Normale Supe´ rieure the ipsilateral side of the ventral hindbrain via the internal Paris capsule/pyramidal tract. The CST growth cones then France decussate across the midline in the caudal medulla and travel down the spinal cord in the contralateral dorsal column (in rodents) forming a thick fasciculated bundle. Summary At the appropriate length down the spinal cord, individ- ual CST axons branch off collateral fibers that enter the To investigate Eph-ephrin bidirectional signaling, a se- contralateral side of the spinal cord to innervate the ries of mutations were generated in the ephrin-B3 lo- appropriate population (Schreyer and cus. The absence of both forward and reverse signal- Jones, 1982; Gribnau et al., 1986; Joosten et al., 1989; ing resulted in mice with mirror movements as typified Akintunde and Buxton, 1992; Stanfield, 1992). The ability by a hopping locomotion. The corticospinal tract was of CST axons to cross the midline only once and then defective as axons failed to respect the midline bound- respect this structure as a barrier afterwards ensures a ary of the spinal cord and bilaterally innervated both high-fidelity connection between the motor cortex and contralateral and ipsilateral motor neuron populations. its contralateral targets in the spinal cord. A second mutation that expresses a truncated ephrin- Growth cones paving the CST encounter a number of B3 lacking its cytoplasmic domain did not lead pathfinding choice points as they extend toward their to hopping, indicating that reverse signaling is not eventual targets in the spinal cord, including at the pyra- required for corticospinal innervation. Ephrin-B3 is midal decussation, where they must cross the midline concentrated at the spinal cord midline, while one of once, along the contralateral dorsal column as they ex- its receptors, EphA4, is expressed in postnatal cortico- tend down into the spinal cord, where they must hug, spinal neurons as their fibers pathfind down the con- but not cross, the midline, and then, at the appropriate tralateral spinal cord. Our data indicate ephrin-B3 distance down the rostral–caudal axis of the dorsal col- functions as a midline-anchored repellent to stimulate umn, where the must make the choice to enter the deeper layers of the contralateral spinal cord to forward signaling in EphA4-expressing axons. innervate the selected motor neuron population. Re- cently, genetic studies in mice have indicated that the Introduction neural cell adhesion molecule L1 plays an important role in the guidance of the CST across the pyramidal Coordinated, asymmetrical movements of limbs and decussation (Cohen et al., 1998). Our studies described digits depend on precise control of motor activities. here and those of Dottori et al. (1998) indicate that Eph- Involuntary movements of one side of the body that ephrin signaling is important for later stages of CST accompany and mirror intentional movements of the pathfinding, those involving the midline barrier that keep other side are frequently present in young children in contralateral corticospinal fibers from crossing back the first decade of life. However, such mirror movements into the ipsilateral side of the spinal cord as they project are rarely observed in the adult population, with the first down the dorsal column. However, in contrast to the description of a pathological condition dating back over report of Dottori et al. (1998), we provide experimental 100 years (Thomayer, 1889; Schott and Wyke, 1981). evidence that points to a different interpretation of the People afflicted with mirror movement disorders exhibit biochemical basis for this repellent activity, implicating a life-long deficit in intermanual coordination needed receptor forward signaling within the CST axons as the when both limbs are required to perform an asymmetri- mechanism for repulsion. cal task, such as in typing, buttoning a shirt, or tying a The Eph receptors form the largest subfamily of recep- tor tyrosine kinases and play pivotal roles in axonal pathfinding, neural crest cell migration, vascular devel- ‡ To whom correspondence should be addressed (e-mail: henk@utsw. opment, and other cell–cell recognition events (Flana- swmed.edu). § These authors contributed equally to this work. gan and Vanderhaeghen, 1998; Frisen et al., 1999; Mel- k Present address: Department of Pharmacology, College of Medi- litzer et al., 2000). These functions are achieved through cine, University of Illinois at Chicago, Chicago, Illinois 60612. interactions with specific membrane-bound ligands, Neuron 86

known as . In vertebrates, 14 different Eph recep- which encodes the C-terminal 20 residues of the extra- tors and 8 different ephrins are known, being divided cellular domain and the entire transmembrane and cyto- into two subclasses, A and B. In general, EphA receptors plasmic domains. have affinity for GPI-anchored ephrin-A ligands, while The second mutation, ephrin-B3lacZ, was designed to EphB receptors and EphA4 have affinity for ephrin-B modify the last (fifth) exon by replacing the sequence ligands, which are anchored to the membrane by a encoding the bulk of the cytoplasmic domain (amino hydrophobic transmembrane domain and a short cyto- acids 264–340) with a bacterial lacZ cassette that was plasmic tail. In addition to forward signaling elicited flanked on both ends with loxP site-specific recombination in the Eph receptor-bearing cells after encountering sequences (Figure 1A). The fusion of this loxP-lacZ-loxP ephrin-expressing cells, there is strong evidence indi- cassette is in-frame with the ephrin-B3 open reading frame cating that the ephrin-bearing cells themselves are able and, after Flp-mediated recombination to remove the to transduce intracellular signals (reverse signaling) frt-flanked neo cassette, was designed to express a upon binding to their cognate receptors (Henkemeyer membrane-bound ephrin-B3-␤-galactosidase (ephrin-B3- et al., 1996; Holland et al., 1996; Bruckner et al., 1997; ␤-gal) fusion protein in which the ephrin-B3 cytoplasmic Davy et al., 1999; Mellitzer et al., 1999; Xu et al., 1999; domain is deleted and replaced with ␤-gal. As designed, Birgbauer et al., 2000; Davy and Robbins, 2000). Thus, the ephrin-B3neo mutation interferes with both the for- upon cell–cell contact, Ephs and ephrins activate bidi- ward and reverse signals, while the ephrin-B3lacZ muta- rectional signals that are transduced into both cells, and tion selectively disrupts only the reverse component of each molecule can be considered to function as a bidirectional signaling (Figure 1B). Moreover, the cova- capable of sending non-cell-autonomous signals to ad- lent attachment of ␤-gal to the truncated tail of ephrin- jacent cells and as receptors capable of transducing B3 serves as a sensitive marker to visualize the spatial, cell-autonomous signals into their own cell. developmental, and subcellular distribution pattern of Among the three transmembrane ephrins, ephrin-B3 this protein (see below). is notable for its remarkably restricted expression pat- The third allele, ephrin-B3T, was generated after Cre- tern down the midline of the neural tube (Gale et al., mediated excision of the loxP-flanked lacZ sequences 1996; Bergemann et al., 1998; Imondi et al., 2000). This (Figure 1A). This truncates the ephrin-B3 protein by jux- suggests ephrin-B3 may have important roles in regulat- taposing a stop of translation incorporated at the end ing the midline guidance of axons in the developing of the 3Ј loxP site that remains after removal of the lacZ spinal cord. In order to better understand the biological cassette. Similar to the ephrin-B3lacZ allele, the ephrin- functions of ephrin-B3 and to discriminate between the B3T mutation truncates the cytoplasmic domain of forward and reverse signals it may propagate, we gener- ephrin-B3 at the same residue, 264; however, it does ated an allelic series of mutations in the mouse ephrin- not have ␤-gal attached to it (Figure 1B). B3 locus. The first mutation was designed to provide a Using the initial ephrin-B3neo/ϩ targeted ES cells, chi- loss-of-function or hypomorphic allele that interrupts meric mice were generated and used to recover germ- both forward and reverse signaling, while a second muta- line transmission of this allele. Subsequent alleles were tion was created to truncate the ephrin-B3 cytoplasmic obtained by successive genetic crosses to a Flp-express- domain and disrupt only reverse signaling. Our genetic ing transgene (Dymecki, 1996) to generate the ephrin- studies indicate that ephrin-B3 functions as a membrane- B3lacZ allele, and then to a Cre-expressing transgene bound midline-expressed repellent to prevent EphA4- (Voiculescu et al., 2000) to generate the ephrin-B3T allele. expressing corticospinal axons from branching back Studies of the ephrin-B3neo and ephrin-B3lacZ mutations into the ipsilateral side of the spinal cord. are presented here, while a full description of the ephrin- B3T allele awaits further analysis. Results Characterization of ephrin-B3neo Generation of an Allelic Series of Mutations and ephrin-B3lacZ Alleles in ephrin-B3 Both the ephrin-B3neo and ephrin-B3lacZ mutations were In order to genetically dissect cell-autonomous and non- found to be homozygous viable at the expected Mende- cell-autonomous functions of the ephrin-B3 prod- lian ratios and the adults appeared healthy, fertile, and uct, we engineered an allelic series of mutations in the long lived. In order to examine the expression of ephrin- murine ephrin-B3 germline. The ephrin-B3 targeting B3 derived from the ephrin-B3neo allele, total RNA was vector used incorporated both loxP and frt site-specific extracted from the brains of a set of littermates obtained recombination sequences and was designed to gener- from an intercross of ϩ/Ϫ males and females and sub- ate a series of three mutations from a single gene tar- jected to Northern blot analysis (Figure 2A). The probe geting event in murine embryonic stem (ES) cells (Figure used was a cDNA fragment that corresponds to the 1A). The first mutation, the result of homologous recom- extracellular domain of murine ephrin-B3 (amino acids bination following electroporation of the targeting vector 16–182), which was not deleted in the targeted allele. An into ES cells, was designed to cause a hypomorphic, expected 3.2 kb transcript was detected in ϩ/ϩ brains potentially loss-of-function mutation, called ephrin- (Figure 2A, lanes 8, 9, and 11) and in ϩ/Ϫ brains (Figure B3neo. This mutation inserts into the ephrin-B3 fourth 2A, lanes 2–5, 7, and 10). In addition, the ϩ/Ϫ samples intron a neomycin resistance (neo) cassette that was presented a 2.0 kb aberrant transcript that was reduced flanked with frt site-specific recombination sequences. to about 30% of the normal 3.2 kb message. In Ϫ/Ϫ This separates the 5Ј exons encoding the majority of brains, only the 2.0 kb aberrant transcript was detected the extracellular domain of ephrin-B3 from the last exon, with double the intensity of that observed in the ϩ/Ϫ Mirror Movement Disorder in ephrin-B3 Mutant Mice 87

Figure 1. Generation of a Three Allele Series of Mutations in the ephrin-B3 Gene (A) Genomic map of the wild-type ephrin-B3 locus, gene targeting vector, and map of the three alleles produced. The exons are shown as boxes, introns and 5Ј and 3Ј nontranscribed regions as lines, and BamHI (B), EcoRI (E), HindIII (H), KpnI (K), and SalI (S) restriction sites are indicated. The vector placed an frt-flanked cassette containing PGK-neo with the SV40 poly(A) in the fourth intron. The last (fifth) exon that encodes ephrin-B3 amino acids 205–340 was modified to replace the majority of the cytoplasmic domain (codons 264–340) with an in-frame fusion to a loxP-flanked lacZ cassette. The 5Ј arm of the targeting vector contained 2.3 kb of homology, the 3Ј arm 7.4 kb, and for negative selection, the PGK-tk cassette was inserted at the 5Ј end of the vector in the opposite orientation. Following homologous recombination in ES cells, the initial targeted insertion was expected to act as a loss-of-function or hypomorphic allele (ephrin-B3neo) by insertion of the neo cassette into the fourth intron. Following germline transmission of the ephrin-B3neo mutation, the neo cassette was excised in vivo by crossing to a germline Flp-expressing transgenic mouse. This converted the locus to generate the second mutant allele (ephrin-B3lacZ) that expresses a truncated, membrane-bound, ephrin-B3 protein comprised of the extracellular and transmembrane domains covalently attached to ␤-gal in the cytoplasm. The third allele (ephrin-B3T) was obtained by crossing to a germline Cre-expressing mouse to catalyze removal of the loxP- flanked lacZ sequences. The location of 5Ј and 3Ј external probes used to confirm the various recombination events by Southern blot are shown with the digests used and expected sizes indicated. (B) Schematic outlining the principles of bidirectional signaling and how the mutations generated in this analysis were designed to selectively perturb these signals. Neuron 88

Figure 2. Characterization of ephrin-B3neo and ephrin-B3lacZ Mutations (A) Northern blot analysis of ephrin-B3neo mu- tants. Total RNA extracted from the brains of adult mice was subjected to Northern blot analysis with an ephrin-B3 cDNA probe cor- responding to the extracellular domain (co- dons 16–182). A 3.2 kb wild-type band was detected in all the ϩ/ϩ (lanes 8, 9, and 11) and ϩ/Ϫ (lanes 2–5, 7, and 10) littermates, but not in Ϫ/Ϫ samples (lanes 1, 6, and 12). However, a 2.0 kb aberrant transcript was detected in ϩ/Ϫ and Ϫ/Ϫ brains. Equal load- ing was indicated by reprobing with a GAPDH probe (lower panel). (B) Cell surface biotinlyation. To verify the synthesis and proper subcellular distribution of the ephrin-B3lacZ encoded ephrin-B3-␤-gal fusion protein, primary cell cultures from ϩ/ϩ (lane 1) and Ϫ/Ϫ (lanes 2 and 3) animals col- lected at embryonic day E11.5 were sub- jected to cell surface biotinlyation to label all that are exposed on the extracellular surface. Following biotinlyation, total cell pro- tein lysates were immunoprecipitated with anti-␤-gal (lanes 1 and 2) or anti-Nck (lane 3) antibodies. Washed immunoprecipitates were then immunoblotted with strepavidin- conjugated HRP. This resulted in the detec- tion of a specific 145 kDa biotinylated protein only in cultures of ephrin-B3lacZ brains. No bio- tinlyation of the cytoplasmic Nck protein was observed, even though plenty of this protein was immunoprecipitated as evident by the reprobe of the immunoblot with the anti-Nck antibodies (lower panel). The position of pro- tein molecular weight standards is provided on the left. (C) Expression of the ephrin-B3-␤-gal fusion protein in whole embryos. ephrin-B3neo/ϩ and ephrin-B3lacZ/ϩ embryos were collected at the indicated stages of development and reacted with X-gal for the detection of ␤-gal activity. Only the ephrin-B3lacZ embryos showed a dark blue reaction product, which was mainly restricted to the developing nervous system and heart. No staining was apparent in the ephrin-B3neo embryos. specimens (Figure 2A, lanes 1, 6, and 12). The blot was To verify that this chimeric protein is synthesized and reprobed for lacZ and no detectable transcript was ob- properly localized to the plasma membrane, primary served even in the homozygote samples (data not cell cultures derived from ϩ/ϩ and Ϫ/Ϫ embryos were shown). This indicates that splicing around the frt- reacted with NHS-biotin. This treatment covalently at- flanked neo cassette inserted between the ephrin-B3 taches biotin to all proteins that are exposed to the extra- exons 4 and 5 does not occur (see below). As exon 5 cellular surface of the cell (Henkemeyer et al., 1996). contains the coding information for the last 20 amino Following the biotinlyation of cell surface proteins, total acids of the extracellular domain and the entire trans- cell protein lysates were prepared and subjected to im- membrane and cytoplasmic domains, any protein syn- munoprecipitation experiments with anti-␤-gal antibod- thesized from the ephrin-B3neo mutation, if stable, should ies. The washed immunoprecipitates were then resolved be secreted from the cell as it would be missing impor- on a SDS–polyacrylamide gel and immunoblotted with tant membrane-attachment sequences. Unfortunately, strepavidin-conjugated horseradish peroxidase (HRP) specific antibodies that recognize the ephrin-B3 extra- to detect biotinylated proteins. This resulted in identifi- cellular domain are unavailable to perform biochemical cation of the expected 145 kDa ephrin-B3-␤-gal fusion analysis to determine whether a soluble monomeric form protein only in the ephrin-B3lacZ mutant cells (Figure 2B, of the protein is produced. Nevertheless, it is well estab- lane 2). No signal was observed in the lysates obtained lished that Ephs and ephrins are normally localized to from the ϩ/ϩ cells (lane 1). As an important control, no the membrane at specific sites of cell–cell contact (e.g., signal was detected when an antibody directed against Henkemeyer et al., 1994) and that clustering is required the cytoplasmic protein Nck was utilized in the immuno- for active signaling (e.g., Davis et al., 1994). Therefore, precipitations (lane 3, upper panel), even though this we interpret the ephrin-B3neo mutation to provide either protein was abundant inside the cell (lane 3, lower a loss-of-function (null) or, alternatively, a hypomorphic panel). This biochemical analysis demonstrates that the phenotype. ephrin-B3lacZ mutation results in production of the ex- The ephrin-B3lacZ mutation was designed to express pected ephrin-B3-␤-gal fusion protein and that it is pro- a membrane-bound ephrin-B3-␤-gal fusion protein that cessed and localized to the plasma membrane. replaces the ephrin-B3 cytoplasmic domain with ␤-gal. As a further test of the properties of the engineered Mirror Movement Disorder in ephrin-B3 Mutant Mice 89

mutations, ephrin-B3neo/ϩ and ephrin-B3lacZ/ϩ embryos were collected at E9.0 and E11.5 days development and stained as whole-mounts for ␤-gal activity (Figure 2C). This led to a dark blue reaction product only in the ephrin-B3lacZ embryos, indicating that the ephrin-B3-␤- gal fusion protein is specifically expressed from this mutation and not from the ephrin-B3neo allele. Moreover, the expression observed for the ephrin-B3-␤-gal fusion protein precisely recapitulated the known pattern (Gale et al., 1996; Bergemann et al., 1998), with highest levels in the developing nervous system and heart. Most nota- ble at E9.0 is the high expression in the hindbrain, and at E11.5 in the floor plate down the ventral midline of the neural tube. ephrin-B3neo Homozygotes Exhibit a Hopping Locomotion and Other Mirror Movements that Require Forward Signaling All mice homozygous for the ephrin-B3neo allele display abnormal locomotion as characterized by a kangaroo- like hopping gait similar to that described for EphA4 receptor mutant mice (Dottori et al., 1998). The hopping locomotion observed in ephrin-B3neo mutants involves both forelimbs and hindlimbs, as evident by a foot-track study (Figure 3). Remarkably, while hopping was 100% penetrant in the ephrin-B3neo homozygotes in all genetic backgrounds tested, not a single ephrin-B3lacZ homozy- gote has shown this locomotion defect. This result indicates that the presence of the membrane-bound ephrin-B3 extracellular and transmembrane domains is important for normal motor control and that the cyto- plasmic domain is not. Figure 3. Foot Track Analysis of ephrin-B3neo and ephrin-B3lacZ Mu- The hopping gait associated with ephrin-B3neo homo- tant Mice zygotes seemed to be a result of a general inability to Representative foot tracks of ϩ/ϩ, ephrin-B3neo/ephrin-B3neo, and perform asymmetric movements as other synchronous ephrin-B3lacZ/ephrin-B3lacZ adult mice are shown. To visualize the mirror movements were also evident, such as during gait abnormalities, the forepaws and hindpaws of the mice tested were labeled with different colored inks. running or swimming. When placed on a narrow beam or on top of a wire grid, ephrin-B3neo mutants exhibited a hopping motion (see movie [http://www.neuron.org/ peared a few days later in the ephrin-B3neo mutants when cgi/content/full/29/1/85/DC1]). In addition to these types the pups started spontaneous crawling (around P7– of locomotion “challenge” tests that require motor cor- P10). tex input, we observed other types of symmetrical volun- The postnatal timing of the appearance of symmetri- tary movements in the ephrin-B3neo mutants. For in- cal limb movements is consistent with a defect affecting stance, during grooming, when the mutants scratched voluntary movements that are controlled by the motor their torso or head with one hindlimb, the other hindlimb cortex. Another formal possibility is that there may be always moved simultaneously with the working limb and a defect in the local wiring within the spinal cord, which is scratched the air. Moreover, as shown in Figure 3 and known to contain an ill-defined central pattern generator in the digital movie that accompanies this manuscript, that functions to help provide a rhythmic movement to the overall behavior of the ephrin-B3lacZ homozygotes the limbs during locomotion-type activities (Grillner and was indistinguishable from wild-type mice. Wallen, 1985; Armstrong, 1986; Kiehn and Kjaerulff, Although the hopping gait of ephrin-B3neo mutant ani- 1998). This spinal pattern generator involves a complex mals becomes apparent as soon as they start walking, interplay of proprioceptive impulses from muscular we performed tests to determine if mirror movements spindles and tendon organs that travel in feedback cir- can be observed at earlier neonatal stages. Mild pain cuits that bring about the reflexes basic to voluntary stimuli to newborn pups were administered by tail pinch- and other movements, and of static reflexes, which ing to induce limb movements. From the day of birth counteract earth’s gravity. To determine whether the (P0) until around the third day after birth (P3), all control ephrin-B3neo homozygotes have a defect in reflexive re- and mutant pups showed random or alternate limb sponses, mutant and control mice were subjected to a movements in response to the pain stimuli. However, mild pinch of one of the feet and the mouse was ob- from around P4, the ephrin-B3neo mutants began to show served as to whether the manipulated limb twitched clear symmetric movements of the forelimbs in response singularly as the normal reflex, or whether both limbs to the stimuli, while the ϩ/ϩ and ϩ/Ϫ littermates showed twitched simultaneously. All animals tested this way, random or alternate limb movements when their tails including the hopping ephrin-B3neo homozygotes, dis- were pinched. Mirror movements in the hindlimbs ap- played unilateral pain reflexes (see movie). This indi- Neuron 90

Figure 4. Anterograde Tracing of the Corticospinal Tract in ephrin-B3neo Mutant Mice The axonal tracer BDA was stereotaxically injected into the left motor cortex and allowed to anterogradely fill the CST in vivo. Shown are coronal sections of a ϩ/Ϫ mouse (A, C, E, G, and I) and a Ϫ/Ϫ mouse (B, D, F, H, and J) at the level of the cortex/injection point (A and B), the pons (C and D), the decussation (E and F), and the cervical spinal cord (G and H). CST axon pathfinding from the cortex to the decussation level was normal in both the ϩ/Ϫ and Ϫ/Ϫ brains. Note that at the level of the pons, labeled CST axons are ipsilateral (arrows in [C] and [D]), while at the level of the caudal medulla, the axons have decussated and are now positioned contralaterally where they extend toward the dorsal region of the caudal hindbrain (arrows in [E] and [F]). In the cervical spinal cord (G and H), labeled CST axons are found as a tight bundle of fibers that project down the dorsal column of the spinal cord where they are restricted to the contralateral side of the midline (arrows mark the midline and asterisks mark the central canal). In the control (G), the CST axons respect the midline boundary and send collateral fibers exclusively into the contralateral (right) spinal gray matter. In the Ϫ/Ϫ specimen (H), the main bundle of the CST resides in the contralateral dorsal column; however, in addition to projecting axon collaterals into the correct contralateral side, labeled CST fibers are observed to have projected across the midline and into the ipsilateral (left) side of the spinal cord (arrowheads in [H]). (I and J) Longitudinal sections of CST-traced spinal cords at the level just dorsal to the central canal. The thin dotted lines indicate the midline in both panels. In the ϩ/Ϫ control (I), collateral branches of the CST are observed to have exclusively entered the contralateral spinal cord. In the Ϫ/Ϫ mutant (J), numerous collateral fibers are observed to have crossed the midline and project into the ipsilateral (left) side of the spinal cord. cates that the neuronal circuitry underlying the reflex ipsilateral internal capsule and the cerebral peduncle arc is apparently normal in these mice and, therefore, (Figure 4A) to reach the ventral brain stem where they supports the idea that abnormalities in motor cortex pass through the medullary pyramids and then, at cau- control are responsible for the mirror movements. dal end of medulla, cross the midline and turn dorsally to enter the contralateral dorsal column of the spinal cord (Figures 4C and 4E). At this point, the decussated Defective Pathfinding of Corticospinal Tract CST axons then project down into the spinal cord in neo Axons in ephrin-B3 Mutant Spinal Cords the contralateral dorsal column where they branch off The appearance of mirror-image type movements devel- collateral fibers into appropriate levels of the cord gray neo oping in the ephrin-B3 homozygotes days shortly after matter (Figures 4G and 4I). In wild-type mice, these col- birth suggested a close temporal relationship between laterals enter only the contralateral side of the spinal this phenotype and the CST, which innervates the spinal cord, where they target to the selected interneuron and cord postnatally (Gribnau et al., 1986; Stanfield, 1992; motor neuron populations. The course of CST axons in Kuang and Kalil, 1994; Terashima, 1995; Joosten, 1997; ephrin-B3neo homozygotes appeared normal down to the Gianino et al., 1999). To determine whether the CST is pyramidal decussation (Figures 4B, 4D, and 4F). How- defective in the ephrin-B3neo homozygotes, we per- ever, CST axons in the mutants showed a consistent formed both anterograde and retrograde axon tracing abnormal projection pattern in the spinal cord (Figures studies in wild-type, heterozygous, and mutant adult 4H and 4J). Although the thick longitudinal bundle of mice. For anterograde tracing, unilateral stereotaxic in- fasciculated CST axons was found at its normal position jections of biotinylated dextran amine (BDA) was used traversing down the contralateral dorsal column, we ob- to deposit this axon tracer into one side of the motor served that collateral fibers were able to branch into cortex to label layer V neurons (Gianino et al., 1999). either the contralateral or ipsilateral side of the spinal After allowing the BDA to fill the CST axons in vivo, mice cord gray matter. This abnormal branching of collateral were sacrificed and sections of the brain and spinal fibers across the midline was observed in all ephrin- cord were reacted with strepavidin-conjugated HRP to B3neo homozygotes examined by this anterograde method identify the labeled fibers. (n ϭ 4) and was not detected in either the ϩ/ϩ (n ϭ 3) In normal animals, the CST axons course through the or ϩ/Ϫ (n ϭ 4) animals tested. Mirror Movement Disorder in ephrin-B3 Mutant Mice 91

Figure 5. Retrograde Tracing of the Corticospinal Tract in ephrin-B3neo Mutant Mice Supraspinal descending pathways terminating in the lumbar spinal cord were labeled by unilateral injection of HRP into the right side of the spinal cord. Examples of a ϩ/ϩ adult mouse (A, B, E, G, I, and K) and a Ϫ/Ϫ littermate (C, D, F, H, J, and L) are shown. Sections through the spinal cord (K and L) verified that the tracer injections were unilateral. (A to D) Retrograde tracing of the CST labels layer V neurons in the motor cortex on both the left and right sides of the brain. In the ϩ/ϩ brain, the vast majority of the labeled neurons are in the expected left side of the cortex, contralateral to the injection site (A). The few labeled neurons found in the right ipsilateral side of the cortex in the ϩ/ϩ brain (B) represents a less abundant CST pathway that courses uncrossed down the ventral aspect of the spinal cord. In the Ϫ/Ϫ mutant brain, the ipsilateral motor cortex showed a significant increase in the number of retrogradely labeled CST neurons. The intensity and number of labeled ipsilateral neurons (D) was almost equal to the labeling observed in the contralateral cortex (C). (E–J) Unilateral injection of HRP tracer also retrogradely labeled other CNS nuclei, including the paraventricular nucleus in the hypothalamus (E and F), the red nucleus in the midbrain (G and H), and the reticular nuclei along the brainstem (I and J). The retrograde tracing of these CNS nuclei appeared no different between the control and mutant mice analyzed. Note that the paraventricular nucleus is mainly ipsilateral, the red nucleus is mainly contralateral, and the reticular nuclei are bilaterally distributed. Scale bar, 100 ␮m.

In order to confirm the anterograde tracing results contralateral cortex in the mutant brains analyzed, while and to examine other supraspinal tracts that innervate in wild-type animals the value was 27.6% Ϯ 6.1% (p Յ the spinal cord, a retrograde tracing study was also 0.002). As some cortical neurons do project ipsilaterally employed (Li and Waters, 1991; Akintunde and Buxton, to form the ventral CST, this lower value observed in 1992; Gianino et al., 1999). A mixed HRP compound the control mice represents the normal uncrossed frac- (Romero et al., 1999) was unilaterally injected into the tion. All other major axon pathways appeared normal ventral right half side of the lumbar spinal cord and in the mutants and, indeed, these tracings provide an allowed to retrograde label CNS neurons that form the excellent internal control for our observations of the supraspinal tracts, including the corticospinal, vestibu- motor cortex. For instance, labeling in the paraventricu- lospinal, and rubrospinal tracts. Serial sections of the lar nucleus within the hypothalamus was primarily ipsi- brain and spinal cord were then reacted to detect HRP- lateral (E and F), while the labeling in the red nucleus traced axons and cell bodies. Presented in Figure 5 are (G and H) was primarily contralateral, and the labeling representative results of such unilateral injections into of neurons in the reticular nuclei (I and J) was bilateral. wild-type (n ϭ 5) and ephrin-B3neo homozygous (n ϭ 5) adults, and shown are sections of the motor cortex Electrostimulation of the Motor Cortex Confirms (A–D), the paraventricular nucleus (E and F), the red Bilateral Communication of the CST nucleus (G and H), and the reticular nuclei (I and J). As In order to confirm the functional significance of the we wanted to study both ipsilateral and contralateral abnormal collateral branching of the CST back into the projections, great care was taken to ensure that only ipsilateral side of the nervous system in the ephrin-B3neo one side of the spinal cord was labeled in the injections homozygous adult, we performed an electrostimulation (K and L). Of all the tracts that were labeled, only the study by placing an electrode unilaterally into the motor CST appeared abnormal in the mutants. In the wild-type cortex of anesthetized mice (Li and Waters, 1991). Al- animal (A and B), most of the retrograde-labeled CST though ϩ/ϩ and ϩ/Ϫ animals showed normal twitching neurons are located in layer V of the contralateral motor of forelimbs and hindlimbs contralateral to the stimula- cortex. However, in the mutants, there are many more tion side, the Ϫ/Ϫ animals showed bilateral limb labeled neurons in the ipsilateral motor cortex (D) and twitching in response to the stimulation (data not the density appears very similar to the number of neu- shown). This revealed a functional bilateral innervation rons in the contralateral cortex (C). Cell count analysis of the spinal cord by the motor cortex and is consistent of the motor cortex revealed that the ipsilateral cortex with the aberrant branching of CST axons observed in contained 66.1% Ϯ 8.7% as many labeled cells as the the mutants. Neuron 92

Figure 6. Expression of Ephrin-B3 and EphA4 in the Postnatal Brain and Spinal Cord Coronal sections of the motor cortex (A–C and F–H) and spinal cord (D, E, I, and J) were collected and stained for ␤-gal activity to de- tect the expression of lacZ at birth (A–E) and at postnatal day 5 (F–J). Representative im- ages obtained for ephrin-B3lacZ/ϩ heterozy- gotes (A, D, F, and I) and EphA4lacZ/ϩ hetero- zygotes (B, C, E, G, H, and J) are shown. At these two stages, ephrin-B3-␤-gal staining is restricted to the midline of the spinal cord, including in the middle of the dorsal column (outlined in [D]). Very little expression is de- tected elsewhere, and there appears to be lower levels at the midline at P5 compared to P0. Low (B) and high (C) magnifications of the motor cortex revealed a low but detectable level of ␤-gal staining for EphA4 at birth. At P5, EphA4 is upregulated to very high levels in layers II–V of the (G). In the high magnification of lamina V and VI (H), intense ␤-gal staining is observed in the large pyramidal neurons of the motor cortex. EphA4 is also expressed, albeit at lower lev- els, in the spinal cord at P0 and at P5. Scale bars for (A)–(C) and (F)–(H), 50 ␮m; (D), (E), (I), and (J), 100 ␮m.

Expression of Ephrin-B3 and EphA4 inserts lacZ sequences at the ATG start of translation in the CST Pathway (deleting EphA4 translation) and thus produces an un- As CST axons make the pathfinding choice to defascicu- conjugated ␤-gal, which aggregates to one or a few late from the main longitudinal bundle and enter the cytoplasmic compartments within the interior of the cell. deeper layers of the spinal cord during postnatal devel- Although not detected in the motor cortex at birth (P0; opment, it seemed appropriate to investigate for the Figure 6A), ephrin-B3 is highly concentrated in the spinal expression of both ephrin-B3 and EphA4 at a number cord, where it localizes to the midline (Figure 6D). The of different time points after birth. For this analysis, we staining observed is intense and labels the two midline took advantage of the ephrin-B3lacZ allele and the pre- cells, where it appears to precisely mark their apical viously described EphA4lacZ mutation (Helmbacher et al., membranes, those which are juxtaposed. The basolat- 2000). Both mutations incorporate lacZ insertions, which eral membranes of these midline cells show little, if any, allow a very high resolution view of the expression of staining for the ephrin-B3-␤-gal fusion protein. EphA4 these two by an easy stain for ␤-gal activity. In on the other hand showed a fairly low level of expression addition, as the ephrin allele generates an ephrin-B3-␤- at P0 in the motor cortex and spinal motor neurons gal fusion protein, we anticipated that this might pro- (Figures 6B, 6C, and 6E). Shortly after birth, most of the duce a protein that shows normal subcellular localiza- pioneer CST axons have decussated the caudal hind- tion, as previously observed for a similarly placed brain and are actively pathfinding down the contralateral EphB2-␤-gal fusion (Henkemeyer et al., 1996). Indeed, dorsal column, with the remaining axons close behind this appears to be the case as the staining for ephrin- (Gianino et al., 1999). While the expression of ephrin-B3 B3-␤-gal appears to be highly localized to specific re- continues in a line down the middle of the spinal cord gions of the plasma membrane in vivo (see below) and during postnatal stages (Figure 6I), EphA4 is greatly in cultured cells (data not shown). The EphA4lacZ mutation upregulated in the cortex in a layer-specific manner (Fig- Mirror Movement Disorder in ephrin-B3 Mutant Mice 93

Figure 7. Summary of Expression and Corticospinal Tract Pathfinding Defect Observed in ephrin-B3neo Mutants (A) Normal appearance of the CST with the left side cortical neurons colored in green and the right side colored in black. Coronal sections at the level of the forebrain (top), the hindbrain rostral of the decussation (middle), and in the spinal cord (bottom). In the spinal cord, the CST fibers are located as a fasciculated longitudinal bundle in the contralateral dorsal column. Collateral fibers branch off the midline fascicle and project into the ventral horn to innervate the appropriate contralateral motor neurons, which are colored according to their cortical input. (B) Expression of EphA4 (red) and ephrin-B3 (blue) in the CST during postnatal development. Ephrin-B3 is concentrated at the midline of the nervous system, while EphA4 is expressed in the neurons of the motor cortex that send CST fibers down into the spinal cord. Only the EphA4- positive neurons in the left motor cortex and their associated contralateral fibers are shown. In wild-type mice, cortical neurons project into the contralateral side of the spinal cord to innervate their appropriate targets in the (right) ventral horn. The midline expression of ephrin-B3 is notably absent at the CST decussation level (middle image). (C) In animals homozygous for the ephrin-B3neo allele, no functional ephrin-B3 is produced. In this situation, EphA4-expressing cortical neurons are still able to decussate in the caudal hindbrain and project down the contralateral dorsal column; however, collateral fibers of the CST are able to project across the midline and enter the ipsilateral spinal cord. This leads to aberrant bilateral innervation of the cortical neurons, as they are now able to synapse with populations of motor neurons in both the right and left sides of the spinal cord. ures 6G and 6H). There is obvious strong expression symmetrical movements of these mutant mice. Our abil- of EphA4 in layer V, which contains the cortical motor ity to trace these CST axons histochemically and to test neurons that form the CST. In addition, EphA4 staining their circuits by electrostimulation clearly demonstrates in layer IV is even more intense than in layer V, while the bilateral nature of these cortical connections with expression in layers II and III is comparable to layer V. the spinal cord. The postnatal upregulation of EphA4 in layer V cortico- The finding that the cytoplasmic domain of ephrin-B3 spinal neurons is consistent with a role for forward sig- is dispensable for this function provides strong evidence naling in the CST growth cones only after they have that this molecule specifically acts as the guidance cue crossed the midline. (the ligand) to stimulate the forward signal into Eph re- ceptor–expressing axons. However, previous studies of Discussion a targeted mutation in the gene encoding the EphA4 receptor, which can bind ephrin-B3, led to suggestions We provide a genetic analysis of the function of ephrin- that EphA4 functioned as the cue and ephrin-B3 as the B3 in the mouse. Our studies indicate that loss of ephrin- receptor to transduce a reverse signal into CST axons B3 results in a striking motor defect as manifested by (Dottori et al., 1998). In that study, the a hopping locomotion and other symmetrical move- data used to draw conclusions was restricted to late ments of the limbs. By generating a second mutation embryonic stages and birth, a window of time that pre- that selectively disrupts only the cytoplasmic domain of cedes when CST axons are pathfinding down the spinal ephrin-B3, we demonstrate that these mice do not hop cord. As CST axons make the choice to branch off collat- or show other mirror-type movements, indicating that eral fibers from the main longitudinal bundle to innervate reverse signaling mediated by ephrin-B3 is dispensable the spinal cord gray matter during postnatal develop- for normal corticospinal function. We show that, in the ment, we investigated for the expression of ephrin-B3 absence of ephrin-B3-mediated forward signaling, the and EphA4 at a number of more relevant time points contralateral CST axons can aberrantly traverse the mid- after birth. Our data indicate that, at the time after which line in the spinal cord and reenter the ipsilateral side of CST axons have crossed the midline at the pyramidal the nervous system. This leads to bilateral innervation decussation and are actively pathfinding down the dor- of the CST with the spinal motor column (Figure 7) and sal column of the contralateral spinal cord, the EphA4 such a wiring defect is consistent with the observed receptor is upregulated in CST neurons and ephrin-B3 Neuron 94

is concentrated solely at the midline. The midline is pro- in the situation described here for EphA4 and ephrin- posed to form a repulsive barrier that is highly selective B3. In the previous two examples, bidirectional signaling for certain axons, including the CST fibers. Taken to- is required, on the ipsilateral side, to guide axons toward gether, our results provide convincing evidence that and across the midline. In the case of the CST axons, ephrin-B3 is functioning as an important molecular com- Eph-ephrin signaling becomes important only after the ponent of this midline barrier. We therefore propose that has crossed the midline, and this signal ephrin-B3 functions in a non-cell-autonomous fashion apparently ensures that they do not recross back into to send a repulsive cue to the contralateral EphA4- the ipsilateral side. Perhaps, in some instances, the expressing CST axons, which transduces a forward sig- function of Eph receptors (i.e., EphB2) is to coax/drive nal into the growth cones to keep them from crossing the growth cone toward and across the midline, while the spinal cord midline. that of EphA4 is to then take over the driving seat and The situation observed here for EphA4 and ephrin- ensure that the growth cone, from this point forward, B3 is quite similar to another group of important axon avoids crossing the midline again. Indeed, this theme guidance molecules, the Robo receptors and their mid- of being able to guide axons at the midline can even be line-expressed Slit ligands (Kidd et al., 1998; Brose et recognized within the middle of the retina, as EphB2- al., 1999; Kidd et al., 1999). In both cases, the receptors mediated reverse signaling is important for the funneling EphA4 and Robo appear to be upregulated in their corre- of dorsal axons toward and into the sponding commissural axons after the growth cone has optic disk/optic nerve (Birgbauer et al., 2000). In addition crossed the midline. Such upregulation of a receptor to our studies of mutant mice, it is important to note that functions in axon repulsion would ensure that the that roles for Eph receptors and ephrins in midline axon growth cone will not reenter a region that it now needs to guidance have also been recognized in C. elegans (Zal- avoid. It will be important to fully document the postnatal len et al., 1999) and Xenopus (Nakagawa et al., 2000). expression of EphA4 in the CST to verify whether recep- Although we focused most of our attention on the tor expression is induced only after the axon has passed behavioral analysis and CST pathfinding defects in the the midline in the caudal medulla. It is worth pointing ephrin-B3neo mutants, there are other phenotypes asso- out that ephrin-B3 also shows dynamic expression pat- ciated with these mice. The morphology of the dorsal terns at the midline. For instance, there is little if any columns appeared slightly abnormal in that they had a expression of ephrin-B3 specifically at the midline re- more shallow dip/groove into which it penetrated the gion of the postnatal caudal medulla where the CST dorsal aspect of the spinal cord (see Figures 5K and 5L). fibers decussate to the contralateral side (depicted in This was also observed in the EphA4 mutants (Dottori et Figure 7B). This absence of ephrin-B3 specifically in the al., 1998). Furthermore, consistent with expression of window of the caudal medulla where CST axons need EphA4 in cortical layers II/III and V, histological examina- to cross the midline may have functional significance tion of adult brains revealed agenesis of the corpus as it may provide the only nonrepulsive conduit through callosum in about 20% of the ephrin-B3neo mutants (8 which these growth cones can pass. Although we did out of 39 brains analyzed), which was not observed in not observe defects at the decussation level in the any ϩ/ϩ or ϩ/Ϫ littermates. Other commissural struc- ephrin-B3 mutants, further genetic analysis may reveal tures like the anterior, posterior, hippocampal, and ha- redundancies in the system. Clearly, there are many benular commissures did not appear to be affected. molecules expressed at the midline in the nervous sys- Skilled voluntary motor control is known to be initiated tem, and no single protein or signaling pathway will be in the primary motor cortex and to involve multiple cen- responsible for controlling all decisions on whether to ters both in the brain and in the spinal cord (Bures and cross or not to cross. However, our results presented Bracha, 1990; Holstege, 1995). The abnormal recrossing here provide yet another example of the importance of of the CST observed in the ephrin-B3neo mice and conse- Eph receptors and ephrins in the midline navigation of quent bilateral innervation of the spinal cord by each select populations of commissural growth cones in the cerebral hemisphere offers a simple explanation to the mammal. mirror movement disorder in these animals. Thus, in One of the most fascinating features of Ephs and contrast to the norm in which the motor cortex stimu- ephrins is their remarkable ability to utilize either the lates the contralateral side of the spinal cord to elicit forward or the reverse components of bidirectional sig- limb movement in the opposite side of the body, each naling to carry out their functions at the cerebral hemisphere in the ephrin-B3neo mice simultane- midline. For instance, guidance of the anterior commis- ously activates motor neurons in both sides of the spinal sure axons across the midline appears to require the cord. This was clearly indicated by our behavioral analy- reverse signal transduced into the B-ephrin-expressing sis of these animals in goal-directed (balance beam) axons with the EphB2 molecule acting as the cue (Hen- and precise locomotion (grid walking) environments, in kemeyer et al., 1996). However, in contrast, midline guid- which asymmetrical voluntary motor control by the cere- ance of the contralaterally projecting inner ear efferents bral cortex is essential for appropriate performance in rhombomere 4 requires the EphB2 cytoplasmic do- (Goldberger et al., 1990). This notion is further supported main and is, therefore, dependent on a forward signal by the apparent normal innervation of other descending mediated by EphB2 (Cowan et al., 2000). The studies pathways involved in motor activity, such as the rubro- presented here demonstrate a third example of how spinal and reticulospinal tracts. genetic analysis can be used to dissect out the roles of Although the aberrant innervation of the CST in the bidirectional signaling and how they are utilized to con- ephrin-B3neo mice appears sufficient to explain their ab- trol axon guidance decisions at the midline. However, normal behavior, the possibility remains that other neu- the role of Eph-ephrin signaling is apparently reversed ronal pathways in the brain and spinal cord may also Mirror Movement Disorder in ephrin-B3 Mutant Mice 95

be involved. This is suggested by the upregulation of (ephrin-B3T). Mice used in this analysis were of the 129 inbred back- EphA4 receptor expression in most neurons in the cere- ground or on a mixed 129/C57BL6 or 129/CD1 background, with bral cortex, including the corticocortical neurons in lam- no difference in phenotypes noted. ina II/III and V, as well as neurons in lamina IV. At the Corticospinal Tract Tracing spinal cord level, commissural interneurons normally For anterograde tracing, anesthetized adult mice received stereo- involved in generating rhythmic pattern motor activity, taxic unilateral injections of 10% BDA (Molecular Probes) directly namely the central pattern generator, are known to be into the sensorimotor cortex using a glass micropipette attached able to maintain reciprocal patterns of motor activity, to Nanoject injector (World Precision Instruments). After 4 weeks, even in absence of supraspinal innervation (Marder and mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (4% PF), brains and spinal cords were then removed, post- Calabrese, 1996; Feraboli-Lohnherr et al., 1999). Thus, fixed, cryoprotected, and sectioned at 50 um using a vibratome. it is possible that the abnormal wiring of these neurons Sections were washed in PBST (0.5% Triton X-100 in PBS), reacted may result in alterations in reciprocal motor activity such with 0.3% H2O2 in PBST for 30 min to quench endogenous peroxi- as simultaneous limb movement. However, this seems dases, incubated in PBST with strepavidin-conjugated HRP (Vector less likely since our anatomical and behavioral data on Labs), and then developed in 0.0015% diaminobenzidine, 0.003% the sensory motor pathway underlying spinal reflexes H2O2, and 0.4% NiSO4. For retrograde tracing, anesthetized mice were subjected to hemi- argues in favor of a normal local circuitry in the spinal laminactomy at the T13-L2 vertebral level and an HRP mixture (Ro- cord of these animals. mero et al., 1999) was injected into 6–8 points in the right half side Persistent mirror movements are pathological in hu- of the exposed spinal cord at the dorsal root entry 1 mm in depth mans and can exist both as an isolated phenomenon, using glass pipette attached to a Nanoject injector. Four days post as well as in association with complex neurological syn- surgery, animals were perfused with 4% PF and the HPR tracer was dromes (Herzog and Durwen, 1992). The clinical mani- visualized as previously described (Romero et al., 1999). festation of the severity of this condition varies, in part ␤-Gal Staining due to the ability of some patients to partially compen- To detect the ␤-gal activity induced by the ephrin-B3lacZ and EphA4lacZ sate for the simultaneous motor activity. This is particu- mutations, mouse embryos and later-stage tissues were processed larly evident in individuals with congenital mirror move- as described (Henkemeyer et al., 1996). ments as their initial trouble in performing tasks, such as riding a bicycle, can be surmounted to some extent as Cell Surface Biotinlyation they mature. The observation of evoked bilateral motor Cell surface biotinlyation was performed essentially as described (Henkemeyer et al., 1996), except that the primary cell cultures were potentials with identical latency after transcranial mag- obtained from embryos collected at E11.5 and the biotinylation was netic stimulation of one side of the brain strongly sug- performed after 3 days in culture. gests that bilateral innervation of the spinal cord by the CST underlies this human disorder (Mayston et al., 1997; Acknowledgments Balbi et al., 2000). By definition, the ephrin-B3neo animals reported in this study have persistent mirror movement We acknowledge Derrick J. Rossi for purifying and mapping the ephrin-B3 genomic clones, Lily Li and Xinwei Cao for assistance in disorder and, in agreement with the human counterpart, the construction of the targeting vector, Drs. Erik Meyers and Gail bilateral connectivity of each cerebral motor cortex with Martin for the frt-flanked neo cassette, Dr. Susan Dymecki for the motor neurons in the spinal cord appears to be the Flp mouse, Yuri Yokoyama and Tammy Oliver for histological assis- underlying pathology. Taken together, our findings sug- tance, and Shawna Kennedy and Jancy Cunningham for genotyping. gest that the ephrin-B3neo mouse represents a valid ani- N. Y. was supported by a Human Frontier Science Program long- mal model for congenital mirror movement syndromes term fellowship. This work was funded by the Christopher Reeve National Paralysis Foundation (to M. I. R. and L. F. P.) and by the NIH and points toward both the EphA4 and ephrin-B3 genes (RO1-NS36671) and the Muscular Dystrophy Association (to M. H.). as possible candidate loci responsible for such disor- ders in humans. Received October 3, 2000; revised November 17, 2000.

Experimental Procedures References

Generation of ephrin-B3 Mutations Akintunde, A., and Buxton, D.F. (1992). Origins and collateralization To construct the targeting vector, a 0.6 kb KpnI–BamHI mouse geno- of corticospinal, corticopontine, corticorubral and corticostriatal mic fragment that contained a portion of the fourth intron and the tracts: a multiple retrograde fluorescent tracing study. Brain Res. entire fifth exon (last exon) of the ephrin-B3 gene was modified to 586, 208–218. create a SalI restriction enzyme site at the position of codon 262. Armstrong, D.M. (1986). Supraspinal contributions to the initiation A lacZ cassette (Calzonetti et al., 1995) was flanked with loxP se- and control of locomotion in the cat. Prog. Neurobiol. 26, 273–361. quences and then inserted in frame at the introduced SalI site in Balbi, P., Trojano, L., Ragno, M., Perretti, A., and Santoro, L. (2000). the modified ephrin-B3 exon to disrupt codons 264–340. An frt- Patterns of motor control reorganization in a patient with mirror flanked PGK-neo cassette (Meyers et al., 1998) was inserted at the movements. Clin. Neurophysiol. 111, 318–325. KpnI site in the fourth intron 126 bp upstream of the splice acceptor Bergemann, A.D., Zhang, L., Chiang, M.K., Brambilla, R., Klein, R., for the fifth exon, and 2.3 kb (5Ј arm) and 7.4 kb (3Ј arm) genomic and Flanagan, J.G. (1998). Ephrin-B3, a ligand for the receptor fragments were all subcloned into pPNT (Tybulewicz et al., 1991). EphB3, expressed at the midline of the developing neural tube. The targeting vector was electroporated into the ES cell line R1 Oncogene 16, 471–480. (Nagy et al., 1993) and 14 out of 430 total ES cell lines screened exhibited homologous recombination. Chimeric mice were made Birgbauer, E., Cowan, C.A., Sretavan, D.W., and Henkemeyer, M. from two lines and both transmitted the mutation through the germ- (2000). Kinase independent function of EphB receptors in retinal line. Animals were genotyped by PCR with three primers: GACGG axon pathfinding to the optic disc from dorsal but not ventral retina. CGGGCCAAGCCTTCGGAGAG, ATAGCCAGGAGGAGCCAAAGAG, Development 127, 1231–1241. and AGGCGATTAAGTTGGGTAACG, yielding products of 401 bp Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, (ephrin-B3ϩ), 142 bp (ephrin-B3neo and ephrin-B3lacZ), and 437 bp C.S., Tessier-Lavigne, M., and Kidd, T. (1999). Slit proteins bind Neuron 96

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