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Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons

¹ Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
² Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

View larger version (38K): [in a new window]   Fig. 1. Chick commissural axons follow a complex pathway on the contralateral side of the floor plate. (A) Small crystals of DiI were iontophoretically applied to dorsal spinal cord regions containing commissural neuron cell bodies in fixed open-book spinal cord preparations obtained from E4-E5.5 chick embryos. The microelectrode used to deliver the dye is shown in the lower left corner of the micrograph. (B) At E4, commissural axons anterogradely labeled with DiI extend across the floor plate (indicated by the white lines) in the transverse plane. At the contralateral floor plate margin, axons turn rostrally and extend for approximately 100 µm alongside the floor plate in the longitudinal plane. (C,D) Labeled axons extending on only the contralateral side of the spinal cord (indicated by the white bracket in A; dorsal is upwards and rostral is towards the right). (C) At E5, axons turn away from the floor plate (after extending for 100 µm adjacent to this structure) and project obliquely into more dorsal regions of the white matter. (D) At E5.5, commissural axons execute a final rostral turn and grow longitudinally within the dorsal marginal zone (the white arrowheads indicate decussated axons labeled by an adjacent DiI application). A small number of commissural axons are shown at the bottom of this panel traveling adjacent to the floor plate. The dorsal limit of the explant shown in this panel extends beyond the top edge of this micrograph. Scale bars: in A, 500 µm for A; in D, 50 µm for B-D. contra, contralateral; fp, floor plate; ipsi, ipsilateral.

View larger version (60K): [in a new window]   Fig. 2. Chick commissural axons follow three distinct contralateral pathways. (A) Combined phase and fluorescent micrograph showing three distinct trajectories followed by DiI labeled commissural axons on the contralateral side of an E5.5 chick open-book spinal cord explant (dorsal is upwards and rostral is towards the right). (a) A small population of axons crosses the floor plate and extends directly into more dorsal regions of the white matter before initiating a rostral turn into the longitudinal axis. (b) The major class of axons exhibits an arcuate trajectory that consists of a rostral turn at the contralateral floor plate boundary, followed by growth alongside the floor plate for 100 µm, diagonal growth away from the floor plate, and then a rostral turn into the longitudinal axis within the dorsal marginal zone. (c) A third, small population of axons turns orthogonally at the contralateral floor plate boundary and projects rostrally adjacent to this structure for distances greater than 100 µm. (B) Schematic representation of an open-book spinal cord preparation (marginal surface facing upwards) showing each contralateral pathway depicted in A. For each application of DiI into the dorsal spinal cord (see Materials and Methods for details), the relative proportion of axons within a cohort of DiI labeled axons (n=50) that followed each pathway was calculated. Scale bar: 100 µm in A. d, dorsal; fp, floor plate; rp, roof plate; v, ventral.

View larger version (37K): [in a new window]   Fig. 3. Chick commissural growth cones decrease in complexity as they execute their final turn. (A) Schematic representation of the most predominant contralateral commissural pathway. The numbers indicate the positions along the pathway at which growth cone morphology was examined. In B, rows 1-5 show the morphology of growth cones at the corresponding regions of the contralateral pathway depicted in A. Commissural growth cones are large and highly elaborated with numerous filopodial extensions and well-spread lamellipodia within more proximal regions of the contralateral pathway (rows 1 and 2). By contrast, upon reaching the dorsal limit of their extension (row 3), axons terminate in growth cones that are simple and elongated. This simple morphology is maintained in more distal segments of the pathway (rows 4 and 5), as axons project rostrally in the longitudinal plane. Scale bar: 100 µm.

View larger version (46K): [in a new window]   Fig. 4. Mouse commissural axons extend along the major contralateral pathway followed by their chick counterparts. (A-C) Commissural axons were anterogradely labeled with DiI in open-book spinal cord explants obtained from E11-E13 mouse embryos as described in Material and Methods. (A) At E11, leading commissural axons extend ventrally and are found within, or just on the contralateral side of, the floor plate (region between the white lines). This preparation was visualized under both fluorescence and phase-contrast optics and shows a single growth cone emerging from the floor plate and executing a rostral turn (as well as other axons that have just entered the floor plate). (B,C) Decussated commissural axons projecting on only the contralateral side of the floor plate between E12 and E13 (dorsal is up and rostral is to the right). (B) As observed in the chick spinal cord, most crossed commissural axons extend alongside the floor plate for approximately 100 µm before turning diagonally into more dorsal regions of the white matter. (C) By E13, most commissural axons have executed a final rostral turn into the longitudinal axis within an intermediate region of the marginal zone. Scale bars: in A, 100 µm; in C, 100 µm for B,C.

View larger version (54K): [in a new window]   Fig. 5. Mouse commissural axons exhibit distinct contralateral trajectories. (A) Schematic diagram of an open-book spinal cord explant in which the placement of DiI was varied within the dorsal spinal cord (the dorsal and ventral regions of the spinal cord are indicated by brackets). The numbers in A correspond to the positions of three separate DiI applications that labeled ventrally directed axons whose trajectories are shown in panels 1-3 in B. (B) Cell bodies immediately adjacent to the roof plate (1) extend axons that project ventrally and subsequently turn rostrally on the ipsilateral side of the spinal cord for distances up to 450-500 µm. In this micrograph, the floor plate (indicated by the white lines) is shown at the top. Panels 2 and 3 show axons projecting on only the contralateral side of the spinal cord (dorsal is upwards and rostral is towards the right). Cell bodies located within successively more ventral regions (2 and 3 in (A)) give rise to axons that cross through the floor plate. A minor population of axons (2) maintained extended (>100 µm) contact with the floor plate after executing a rostral turn at the contralateral margin of this structure. An axon that is projecting diagonally away from the floor plate and along the pathway depicted in C is shown outside of the plane of focus. No axons were ever observed to recross the floor plate in these studies. A much larger population of axons (3) maintains only transient (100 µm) contact with floor plate before assuming a more complex pathway that culminates in a rostral turn in an intermediate region of the white matter. For these analyses, 30 axon cohorts labeled by the application of DiI into each spinal cord region depicted schematically in A (see Materials and Methods for additional details) were examined. Scale bar: 100 µm. contra, contralateral; d, dorsal; ipsi, ipsilateral; v, ventral.

View larger version (70K): [in a new window]   Fig. 6. Postcommissural axons do not re-enter the spinal cord gray matter. Transverse vibratome sections taken from E5.5 chick (A) and E13 mouse (B) embryos were labeled with anti-L1 antibodies to reveal the position of postcommissural axons within the M/L axis of the spinal cord. In both chick and mouse embryos, decussated commissural axons are excluded from the gray matter and are confined to fiber tracts within the marginal zone of the spinal cord (indicated by red arrowheads in A,B). (C) The contralateral trajectory of commissural axons in a schematic representation of a spinal cord cross-section. After exiting the floor plate, most commissural axons navigate along a complex pathway within the spinal cord white matter and assemble into an ascending tract within the dorsal (chick) or intermediate (mouse) marginal zone. In C, an axon is shown exiting the marginal zone (indicated by red arrow) to emphasize the observation that crossed commissural axons do not re-enter the gray matter. c, caudal; d, dorsal; fp, floor plate; r, rostral; v, ventral.

View larger version (50K): [in a new window]   Fig. 7. Commissural axons execute their final turn and project alongside a B-class ephrin boundary in both the chick and mouse spinal cord. EphB3-Fc detects B-class ephrin protein in the floor plate and in dorsal regions of E5.5 chick and E13 mouse transverse (A,E) and open-book (B,F) spinal cord preparations. In the dorsal spinal cord, ephrinB protein is expressed within a broad domain that extends from the ventricular zone to the marginal zone (A,E). (C,G) At high magnification, the top halves of the explants shown in B,F, respectively (the dorsal edge of each explant is indicated by the white arrowheads). (D,H) Combined fluorescence and phase-contrast micrographs of separate preparations (taken at the same AP level and shown at the same magnification as the explants in C and G) reveal the position of DiI-labeled commissural axons relative to B-class ephrin expression domains. Decussated commissural axons in both chick and mouse spinal cord explants initiate a final turn and project longitudinally adjacent to a B-class ephrin boundary in the dorsal (chick) or intermediate (mouse) marginal zone. Scale bars: in E, 100 µm for A,E; in F, 250 µm for B,F; in H, 125 µm for C,D,G,H.

View larger version (37K): [in a new window]   Fig. 8. In vitro blockade of EphB-ephrinB interactions results in the misprojection of commissural axons into dorsal regions of the spinal cord that express B-class ephrins. (A) A combined fluorescence and phase-contrast micrograph of an E11 mouse open-book spinal cord explant in which commissural axons were anterogradely labeled with DiI at 0 hours in culture. In this preparation, a single axon is shown within the floor plate (bounded by white lines). (B) EphB3-Fc appropriately detects B-class ephrin expression in the floor plate and in a dorsal region of cultured E11.5 open-book spinal cord explants. (C-G) Commissural axons were anterogradely labeled with DiI in open-book spinal cord preparations obtained from E11-E11.5 mouse embryos and cultured for 48 hours in the presence of 50 µg/ml Fc alone (C-E), EphB3-Fc (F) or ephrinB1-Fc (G). Each panel depicts only the trajectory followed by commissural axons on the contralateral side of the spinal cord. (C) In explants cultured in the presence of Fc alone, commissural axons grew a considerable distance away from the floor plate and turned at an appropriate position within an intermediate region of the spinal cord white matter. (D,E) At high magnification, the turns executed by decussated commissural axons in C. In explants cultured in the presence of EphB3-Fc (F) or ephrinB1-Fc (G), commissural axons aberrantly projected into more dorsal regions of the spinal cord where B-class ephrins are expressed.

View larger version (33K): [in a new window]   Fig. 9. Repulsive guidance cues that are likely to influence the contralateral commissural pathway. Several repellent guidance cues expressed in the spinal cord are ideally positioned to shape the contralateral trajectory of commissural axons. Slit proteins expressed by both floor plate cells and motoneurons may force newly crossed commissural axons to turn into the longitudinal axis (Li et al., 1999). A graded distribution of soluble repellent cues (e.g. one or more slits or semaphorins) secreted by floor plate cells or other ventral cell types may also act to deflect decussated commissural axons (shown in red) away from the ventral midline and into more dorsal spinal cord regions. In complementary fashion, secreted cues emanating from cells situated at or near the dorsal midline, including one or more slits, semaphorins and BMPs, may act coordinately with the local effects of B-class ephrins to specify the dorsoventral position of the longitudinal commissural tract. d, dorsal; fp, floor plate; rp, roof plate; v, ventral.