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doi: 10.1242/10.1242/dev.00467

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Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping

Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (61K): [in a new window]   Fig. 1. Mechanisms that establish the retinotectal topographic map and lack of an effect of electroporation of a control RCAS vector on its development. (A) Normal development of the retinotopic map in the chick retinotectal projection. Initially, RGC axons extend posteriorly past the AP (anterior-posterior) location of their future TZ (circle). In addition, RGC axons originating from the same DV (dorsal-ventral) retinal location enter and extend across the tectum with a broad distribution along its LM (lateral-medial) axis. RGC axons form interstitial branches along their shafts at the AP level of their TZ; the branches are extended along the LM axis toward the TZ where they arborize. Later, overshooting segments of the primary axons are eliminated. Graded expression of Eph receptors in the retina and their ephrin ligands in the tectum are indicated. (B) Schematic of midbrain electroporation procedure on an E1.5 chick embryo. Cathode (+) and anode (–) electrodes were positioned on the opposite sides of the midbrain. (C) Dorsal view of an E12 chick brain. Between E6, when RGC axons first enter the tectum anteriorly, and E12, the tectal lobes rotate such that anterior tectum moves ventrally and away from the midline. This rotation results in the developmental AP (anterior-posterior) axis of the tectum (dashed line) being roughly perpendicular to the AP axis of the brain. For analysis, the optic tectum (ot) was removed, cut along the AP tectal axis, and the medial and lateral halves were mounted whole as shown in the drawing at right. The asterisk is in the same location in the photo and drawing. (C) In situ hybridization using an S35-labeled ephrin-B1 probe on a coronal section through an E13 tectum transfected on E1.5 with an RCAS-ephrin-B1-IRES-eGFP. The transfection results in a columnar pattern of ectopic ephrin-B1 expression from the neuroepithelium (ne) to the stratum opticum (so). (D) Medial (M) half of an E13.5 tectum transfected with RCAS-eGFP at E1.5. Transfections domains express the green eGFP reporter. DiI was focally injected into NV (nasal-ventral) retina (red dot, left inset). DiI-labeled RGC axons (red) are visible in posterior tectum and arborize at the correct location for their TZ in mid-tectum. Branches are unaffected in areas of eGFP expression (right inset). RGC axons are also unaffected by the eGFP. cb, cerebellum; fb, forebrain. Scale bar in D: 300 µm and 100 µm in right inset.

View larger version (29K): [in a new window]   Fig. 3. The normal preferential extension of interstitial branches toward their TZ is altered by ectopic domains of ephrin-B1 consistent with a repellent action. (A) Quantification scheme. The tectum was divided into three domains: medial of the TZ, within the LM extent of the TZ, and lateral of the TZ. Branches in each of these bins were scored as either directed laterally or medially, and as within or outside a transfection domain. The blue and green arrowheads represent the relative strength and direction of the branching preference. A Directional Coefficient (DC) was calculated by subtracting the percentage of branches directed laterally from the percentage of branches directed medially. A positive DC indicates a preference to branch medially, whereas a negative DC indicates a preference to branch laterally. (B,C) In control E11-E14 chicks most branches formed along temporal RGC axons, outside the LM extent of the TZ, extend towards the TZ. At the LM position of the TZ branches show no preference in orientation. The temporal control cases (B) included 14 normal, non-transfected tecta (n=499 branches). The nasal-ventral control cases (C) included 3 RCAS-eGFP-transfected tecta, 1 tectum electroporated with RCAS-ephrin-B1-IRES-eGFP, but in which no eGFP reporter labeling was detected, and 7 normal non-transfected tecta (n=399 branches). (D) In chicks transfected with RCAS-ephrin-B1-IRES-eGFP (n=11 tecta, 700 branches), quantitation of branch directionality, irrespective of their relationship to ectopic domains of ephrin-B1, shows a disruption in normal directionality and a bias to extend laterally. (E) Branches from RCAS-ephrin-B1-IRES-eGFP-transfected cases that were located within an ectopic domain of ephrin-B1 expression were directed laterally, regardless of position (n=11 tecta, 386 branches). (F) Branches in RCAS-ephrin-B1-IRES-eGFP transfected cases that were located outside an ectopic domain of ephrin-B1 exhibited normal branching preferences toward their TZ (n=11 tecta, 314 branches). Statistical tests of significance of quantitation of directional extension of interstitial branches: Nasal-ventral controls (C): in the lateral bin, more branches were directed medially than laterally, Student's paired t-test, P<0.01; at the LM location of the nascent TZ, there is no difference in branch directionality, P=0.87; in the medial bin, more branches are directed laterally than medially, P<0.04. Temporal controls (B) show the same branch directionality as the nasal-ventral controls (C): lateral bins, P=0.92; TZ bins, P=0.80; medial bins, P=0.84 (2 test). Test of significance for all interstitial branches in ephrin-B1 transfected tecta (D) compared to nasal-ventral controls (C): lateral bins, P<<0.001 (2 test); TZ and medial bins are not significantly different. Test of significance in ephrin-B1-transfected tecta for branch directionality within ectopic domains of ephrin-B1 (E) compared to nasal-ventral controls (C): lateral bins, P<<0.001; TZ bins, P<0.04; medial bins, P=0.367 (2 test). Test of significance in ephrin-B1-transfected tecta for branch directionality within ectopic domains of ephrin-B1 (E) versus outside of the domains (F): lateral bins, P<<0.001; TZ bins, P<0.04; medial bins, P<0.1 (2 test). Test of significance in ephrin-B1 transfected tecta for branch directionality outside of ectopic domains of ephrin-B1 (F) compared to nasal-ventral controls (C): lateral bins, P=0.863; TZ bins, P=0.85; medial bins, P=0.52 (2 test).

View larger version (127K): [in a new window]   Fig. 2. Ectopic domains of ephrin-B1 inhibit/repel the arborization of RGC axons, but do not affect their trajectories. Ectopic domains of ephrin-B1 are marked by the green eGFP reporter and RGC axons and arborizations are labeled red by anterogradely transported DiI. (A'',B'',C'',D'' and E'' are merged images of A and A' etc.) (A-B'') Lateral (L) half of an E14 tectum transfected with RCAS-ephrin-B1-IRES-eGFP. DiI was focally injected into nasal (N) dorsal retina. (A-A'') RGC axons extend without deviation through ectopic domains of ephrin-B1. (B-B'') Close up views reveal that ectopic domains of ephrin-B1 ring the TZ (arrowheads). (C-D'') Ventral RGC axons in tecta transfected with RCAS-ephrin-B1-eGFP. In both cases, a dense TZ is present in the appropriate location. However, the TZs appear to be shaped by the domains of ectopic ephrin-B1-eGFP. (C-C'') Ectopic domains of ephrin-B1 hem in the TZ; dense arborizations of the TZ fill areas with little or no ectopic expression of ephrin-B1 (arrowheads). (D-D'') The TZ is split into a larger and smaller component of dense arborization by an area of sparse arborization coincident with an ectopic domain of ephrin-B1 (arrowheads). (E-E'') A TZ in medial tectum at E13 interspersed with small patches of ectopic domains of ephrin-B1. The tectum was transfected with RCAS-ephrin-B1-IRES-eGFP. The usually uniformly dense TZ is perforated with areas of sparse arborization (arrowheads in E) that in most instances are coincident with ectopic domains of ephrin-B1 (arrowheads in E'). V, ventral. Scale bar in E'': 900 µm (A), 300 µm (B,C,D) and 200 µm (E).

View larger version (95K): [in a new window]   Fig. 4. Interstitial branches extended within ectopic domains of ephrin-B1 are directed away from their correct TZ. (A) Medial half of an E13 tectum transfected with RCAS-ephrin-B1-IRES-eGFP. A focal injection of DiI was made in NV (nasal-ventral) retina (arrowhead, inset). RGC axons form a TZ in MP (medial-posterior) tectum. Ectopic domains of ephrin-B1 are marked by the green eGFP reporter and RGC axons, interstitial branches and arborizations are labeled red by anterogradely transported DiI. The boxed area lateral to the TZ is enlarged in B-D. (B-D) Most branches that extend within an ectopic domain of ephrin-B1 are directed laterally, away from their TZ (arrows). Branches that extend outside ectopic domains of ephrin-B1 are appropriately directed toward their TZ, as most branches are in control cases. The disruption in the guidance of interstitial branches is confined to ectopic domains of ephrin-B1. Along a single axon, a branch outside an ectopic domain of ephrin-B1 extends toward the TZ (lower arrowhead) whereas a branch inside a domain of ectopic ephrin-B1 is directed aberrantly away from the TZ (upper arrowhead). Scale bar in D: 800 µm (A) and 250 µm (B-D).

View larger version (78K): [in a new window]   Fig. 5. Overall ephrin-B1 protein in transfection domains exhibits a graded distribution that parallels the endogenous ephrin-B1 gradient. (A) Schematic of a coronal section through an E10 tectum. Dividing cells are present in the neuroepithelium (ne) and stratum griseum et fibrosum superficiale (SGFS). RGC axons extend through the stratum opticum (SO) at the pial surface of the tectum. Boxes indicate areas shown in B and C. (B,C) E10 tectum incubated with EphB2-Fc reveals the distribution of ephrin-B1 along radially aligned processes (arrows) and in the SO (brackets). (B) Lateral tectum has low levels of ephrin-B in the SO. (C) Medial tectum has high levels of ephrin-B in the SO. The images in B and C are of the same section, taken sequentially using the same confocal settings and processed identically. (D-F) Coronal section through E7 lateral tectum after electroporation at E1.5 with RCAS-ephrin-B1-IRES-eGFP. Tectum was stained with EphB2-Fc to reveal the distribution of ephrin-B1. Infected cells and processes are in green and EphB2-Fc staining is red. Many infected cells are present in the ne as well as the SGFS. Lateral is to the left and medial is to the right of each panel. (E) EphB2-Fc reveals the presence of ephrin-B1 in the SO (bracket) and along radially aligned processes (arrow). Within the transfection domain (between arrowheads) a gradient of ectopic ephrin-B1 that parallels the endogenous ephrin-B1 gradient is apparent. (F) The eGFP reveals the extent of the transfection (between arrowheads). The level of eGFP is relatively consistent across most of the transfection domain. At these ages (E7-E10), only ephrin-B1 is expressed within the tectum; therefore the EphB2-Fc staining reveals the distribution of ephrin-B1 protein selectively (Braisted et al., 1997). Scale bar: 40 µm (B,C) and 50 µm (D-F).

View larger version (26K): [in a new window]   Fig. 6. Bifunctional action of ephrin-B1 as a repellent and attractant to direct interstitial axon branches and limit arborization to develop the DV retinotectal map. A and B summarize our findings and C and D summarize a model of the bifunctional action of ephrin-B1 during normal map development in chick and mouse, and the graded expression of EphB and EphA receptors by RGCs and ephrin-B1 and ephrin-As in the tectum (or SC). (A) Interstitial branches of primary RGC axons are normally directed towards their future termination zone (TZ; open circle) either medially or laterally, dependent upon primary axon location along the LM tectal axis. Within ectopic domains of ephrin-B1 (green ovals, and indicated as peaks on the endogenous LM gradient of ephrin-B1), normal bidirectional branch extension is disrupted and branches preferentially extend laterally, regardless of axon position. (B) Ectopic domains of ephrin-B1 shape and inhibit the arborization of interstitial branches at the TZ (red shape). These data suggest that a high level of ephrin-B1 is a repellent for interstitial branches and their arbors. (C) Proposed bifunctional action of ephrin-B1 during normal development of the DV retinotopic map. RGC axons initially extend posteriorly past their future TZ and preferentially form branches along their shafts at the level of the AP location of their TZ (Yates et al., 2001). Both the initial axon overshoot and the formation of interstitial branches are controlled, in part, by a repellent action of ephrin-As on EphA-expressing RGC axons (Yates et al., 2001). Branches extended by RGC axons positioned lateral to their TZ are attracted medially by ephrin-B1 up its gradient toward the TZ (Hindges et al., 2002). Branches extended by RGC axons positioned medial to their TZ are repelled laterally by ephrin-B1 down its gradient toward the TZ. Together these findings suggest that the response to ephrin-B1 of interstitial branches extended by the same DV population of RGC axons, and therefore expressing the same subtypes and levels of EphB receptors, is context-dependent. If an interstitial branch forms along an axon positioned lateral to its TZ, the branch initially extends in a domain of lower ephrin-B1 than found at its TZ. At this level of ephrin-B1, for a given axon, it acts as an attractant and guides branches medially up the gradient of ephrin-B1. Conversely, an interstitial branch that forms along an axon positioned medial to its TZ, encounters a level of ephrin-B1 higher than that at its TZ. At this level, ephrin-B1 acts as a repellent and directs branches laterally down the gradient of ephrin-B1. Therefore, ephrin-B1 may act as a bifunctional guidance molecule to control the position-dependent bidirectional extension of interstitial branches of RGC axons originating from the same DV retinal site. Alternatively, EphB receptor signaling may act as a `ligand-density sensor' and titrate signaling pathways that promote branch extension toward the optimal ephrin-B1 concentration found at the TZ; branches located either medial or lateral to the TZ would encounter a gradient of increasingly favored attachment in the direction of the TZ. (D) Arbors are formed at the TZ exclusively by interstitial branches (Yates et al., 2001). Overshooting segments of the primary RGC axons are eliminated during this process. Based on our findings, ephrin-B1 may also function to help restrict the extent of an arbor along the LM tectal axis. Ephrin-As may help restrict the posterior extent of the arbor (Nakamoto et al., 1996; Yates et al., 2001). Retinal axes: D, dorsal; V, ventral; T, temporal; N, nasal. Tectal axes: L, lateral; M, medial; A, anterior; P, posterior.