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First published online September 12, 2006
doi: 10.1242/10.1242/dev.02564

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Ephrin A/EphA controls the rostral turning polarity of a lateral commissural tract in chick hindbrain

¹ SORST, Japan Science and Technology, Japan.
² Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan.
³ MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London SE1 1UL, UK.

View larger version (88K): [in a new window]   Fig. 1. Development of a lateral commissural tract in chick hindbrains. (A) Schematic showing an open-book hindbrain preparation, with DiI anterograde labelling. DiI crystals were inserted into the lateral margin of the caudal hindbrain, 700-1200 µm caudal to the gVIII root. (B-D) DiI anterograde labelling on fixed hindbrains at stage 24 (n=3), stage 26 (n=3) and stage 27 (n=4), respectively, showing the progression of a laterally located commissural tract. (D) The labelled commissural tract grew to the base of the cerebellar plate. High magnification of the blue-boxed area in D shows that some axons from the tract turn to invade the cerebellar plate (white arrow in the inset in D). (E-H) DiI anterograde labelling of organotypically cultured hindbrains at stage 21 (n=3), stage 23 (n=8), stage 25 (n=10) and stage 27 (n=3), cultured for 2 days in vitro (div). The lateral commissural tract labelled by DiI anterograde labelling in the organotypic cultures closely resemble that in the fixed samples both in its appearance and developmental course. At stage 27+2 div (H), axons could be seen to leave the longitudinal tract and invade the developing cerebellar plate. White arrowheads in C,D,G,H indicate retrogradely labelled neurons contralateral to the DiI injection sites. (I) An organotypically cultured stage 26 hindbrain, with the lateral commissural tract labelled by DiI (white arrow) and the central projection of the right gVIII labelled by DiD (white arrowhead). The lateral commissural tract was located lateral to the central projections of gVIII (n=6). (J,K) Immunohistochemistry using an antibody against a neurofilament-associated protein (3A10) on a stage 24 flat-mounted hindbrain. (K) A higher magnification view of the boxed area in J. Blue arrowheads in J,K indicate the position of the lateralmost longitudinal tract. This lateralmost tract appears to converge from a turning point 900-1000 µm caudal to gVIII (indicated by a red arrow in J). Three longitudinal tracts located in the lateral extreme of the hindbrain can be seen in K: the central projection of gV (purple arrow) and of gVIII (root of gVIII indicated by red asterisk), and the lateral commissural tract (blue arrowhead). White vertical line in B indicates the midline. The asterisks in B-I indicate the injection sites of DiI. Scale bar: 400 µm in B-J; 100 µm in the inset in D and in K.

View larger version (68K): [in a new window]   Fig. 2. Caudal hindbrain possesses a graded nonpermissive/repulsive activity for the cC-VC axons. (A,B) Two types of grafting experiments. The positions of the grafts were approximately 1.2 mm (A) and 1.8 mm (B) caudal to gVIII, respectively. Almost all cC-VC axons stalled or turned away before entering the graft in B (n=12), whereas a small proportion of the cC-VC axons still grew through the graft in A. (C) A piece of rostral hindbrain along the cC-VC path was cut and put back as control. Majority of cC-VC axons could grow through the graft (n=10). All grafts were soaked in DiO before transplanting into the hosts, thus the borders of the grafts were discerned by DiO signal combined with bright-field illumination. DiO signal was displayed only in C, but removed for better illustration of axons in the grafts in other images. White dots outline the border of grafts in A-C. (D) Quantification of the percentage of axons entering the graft (*P<0.0001, Mann-Whitney U test) (error bar indicates s.d.). Scale bar: 400 µm.

View larger version (75K): [in a new window]   Fig. 3. PI-PLC and EphA3-Fc could alleviate the inhibitory activity of the caudal hindbrain. (A,B) The experimental procedures followed illustrated using the same grafting scheme as in Fig. 2B. With normal culture medium (A1) or with only hFc added to culture medium (B1), almost all cC-VC axons were prevented from entering the caudal graft. However, adding PI-PLC (A2) or unclustered EphA3-Fc (B2) to the culture medium led to many axons entering the graft, reducing the inhibition in the graft. White dots outline the grafts. Scale bar: 400 µm.

View larger version (57K): [in a new window]   Fig. 4. Distribution of EphA and ephrin A in stage 25 hindbrains. (A) EphA3-AP in situ binding on a stage 25 hindbrain revealed ephrin A activity. The blue bracket indicates the rostrocaudal extent of the lateral ephrin A gradient in the caudal hindbrain. (B) Ephrin A2-AP in situ binding on a stage 25 hindbrain revealed EphA activity. The blue bracket indicates EphA3-positive domain correlating with the position of presumptive cC-VC neurons. A ventral column of cells adjacent to the floor plate is also EphA positive (see also lower panels in C). (C) Top panel shows a transverse section of a hindbrain with its cC-VC neurons retrogradely labelled (retrogradely labelled cC-VC neurons indicated by white arrowhead, and cC-VC commissures indicated by white arrow). The same section was subjected to ephrin A2-AP in situ binding, shown in the middle panel in C; black arrow shows the EphA-positive commissure. The top and middle panels were superimposed and DiI signals were traced onto the AP signals by red dots (bottom panel). cC-VC neurons and their axons appear to be EphA positive. Scale bar: 400 µm in A,B; 100 µm in C.

View larger version (70K): [in a new window]   Fig. 5. EphA5 and EphA3 are expressed in presumptive cC-VC neurons, and ephrin A2 protein is distributed in a gradient in caudal hindbrain. (A,B) EphA5 and EphA3 in situ hybridisation on stage 25 hindbrains, respectively. The blue brackets in both images indicate expression domains correlating with the location of cC-VC neurons. (C) Ephrin A2 immunohistochemistry on a lateral parasagittal section of a stage 26 brainstem. The graded distribution of ephrin A2 in the caudal hindbrain is indicated by arrows a, b and c. Inset shows ephrin A2 protein in tectum and cerebellum from the same brain. (D) Ephrin A2 immunostaining on three transverse sections from a stage 26 hindbrain. a-c correspond approximately to positions a-c in C. CB, cerebellum; gV, trigeminal ganglion; ov, otic vesicle. Scale bar: 400 µm in A,B; 200 µm in C, inset in C and D.

View larger version (86K): [in a new window]   Fig. 6. Ectopically expressed ephrin A2 induces stalling and turning of cC-VC axons. EGFP alone or together with full-length chick ephrin A2 were electroporated in ovo at around stage 20. Electroporated hindbrains were taken for culture at stage 27-28, and cC-VC axons were anterogradely labelled with DiI. (A1) EGFP alone has no effect on cC-VC axons (n=15/16). (B1,C1) At the interface of an ectopic ephrin A2 domain, most cC-VC axons either stalled (B1,C1) or turned away to extend caudally (C1) (n=15/17). (A2-C2) Same images as the A1-C1, respectively, with EGFP signals removed. A small amount of axons grew into the ephrin A2 expression domain in both B2 and C2, suggesting the possibility that cC-VC axons might represent a heterogeneous population with respect to their responsiveness to ephrin A. Scale bar: 200 µm.

View larger version (93K): [in a new window]   Fig. 7. Blocking ephrin A/EphA signalling in vitro led to some cC-VC axons turning caudally. cC-VC axons were anterogradely labelled on organotypically cultured stage 25-26 hindbrains. (A) Without PI-PLC, in 20/25 samples, all axons turned rostrally. (B) With PI-PLC, in 25/32 samples, some axons turned caudally (white arrow). (P<0.0001, Fisher's test). (C) With hFc, in 21/29 samples, all cC-VC axons turned rostrally. (D) With EphA3-Fc, in 17/27 samples, some cC-VC axons turned caudally (white arrow) (P<0.015, Fisher's test). Scale bar: 400 µm.

View larger version (67K): [in a new window]   Fig. 8. Interfering with ephrin A/EphA signalling in ovo led to inappropriate caudal turning of some cC-VC axons. RCASB vector or RCASB-EphA3C was electroporated in ovo into caudal hindbrain at stage 20-21. An EGFP expression vector was co-electroporated to reveal the domain of successful electroporation. At stage 27-28, electroporated hindbrains were taken for culture and cC-VC axons were anterogradely labelled. (A-D) RCASB vector was electroporated. (E-H) RCASB-EphA3C was electroporated. EGFP signals in A,E indicate the domain of electroporation. (B,F) The same images as in A,E, respectively, with EGFP signals removed to better reveal the axons at their turning point. (C,D,G,H) Higher magnifications of axon turning regions of A,B,E,F, respectively. (I) A different sample with a higher degree of caudal-turning error when RCASB-EphA3C was electroporated. (J) EphA3-AP in situ binding on a hindbrain co-electroporated with RCASB-EphA3C and EGFP. The electroporated domain of this sample indicated by EGFP is outlined with white dots. (K) Quantification of caudal turning error in hindbrains electroporated with RCASB-EphA3C versus RCASB mock vector (Control). The degree of caudal turning is represented as the ratio of caudal-over rostral-turning axons. Data are represented in a scatter chart, with the sample values sorted in an ascending order. The red and orange arrows indicate the data points of samples shown in E-H and in I, respectively. Arrows in F,H,I indicate axons turning caudally. Samples with RCASB-EphA3C showed significantly higher incidence and amount of inappropriate caudal turning (P<0.0001, Mann-Whitney U-test). Scale bar: 200 µm in A,B,E,F,I,J; 100 µm in C,D,G,H.