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doi: 10.1242/10.1242/dev.00162

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Domain-specific olivocerebellar projection regulated by the EphA-ephrin-A interaction

¹ Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA
² Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA

View larger version (42K): [in a new window]   Fig. 1. Expression and activity of EphA receptors in the E10 chick IO. (A-C) Whole-mount RNA in situ hybridization of the brainstem. Ventral views are shown. Rostral is at the top. Arrowheads indicate expression in the IO. (D-O) Coronal sections through the IO subjected to RNA in situ hybridization (D-F, H-J, L-N) or affinity probe in situ (G,K,O). Dorsal is at the top. EphA3 is expressed in a narrow area of the medial IO, whereas EphA5 and EphA6 show broader distribution. White and black arrowheads in I indicate areas Mm and Ir, respectively, which show strong EphA5 expression. Total receptor activity detected with ephrin-A2-AP corresponds to the RNA expression patterns. Scale bars: 500 µm. (P-Q) Areas defined by EphA receptor expression are schematized in a ventral view of the brainstem (P) and serial coronal sections (Q, left side). Lamellar organization of the IO is shown on the right side of Q.

View larger version (44K): [in a new window]   Fig. 2. Expression and activity of ephrin-A ligands in the E10 cerebellum. (A-F) RNA in situ hybridization (A,B,D,E) and affinity probe in situ (C,F) of whole mount cerebellum. Capital letters indicate domain names. (A-C) Dorsal is at the top. (D-F) Anterior is at the top. Expression of ephrin-A2 is strongest in domain C (lobules I-IXab), followed by domain A (lobules I-VIII) and E (lobules II-VII). ephrin-A5 is moderately expressed in domains A (lobules I-V), C (lobules I-V), D (lobules I-VIII) and F (lobules III-VIII). Total ligand activity detected with an EphA3-AP probe corresponds to the RNA expression patterns. (G) ephrin-A2 expression in a coronal section of the cerebellum (lobulus VII). Parasagittal pattern is seen in the Purkinje cell layer (pl). Asterisk, deep cerebellar nuclei. (H) A diagram of parasagittal domains in the cerebellum. Please note that expression patterns of ephrin-A2 and ephrin-A5 are shown only in domains A-C. Scale bars: 500 µm.

View larger version (32K): [in a new window]   Fig. 3. Organotypic hindbrain culture. (A) The whole hindbrain containing the cerebellum and brainstem was isolated from E10 embryo. The cerebellum was cut along the dorsal midline (red line), and the both halves of the cerebellum were separated and turned over. (B) Comparison of ephrin-A expression in domains A-C between the native cerebellum and explant. Dark purple: ephrin-A2(++), ephrin-A5(+) (rostral domain C). Light purple: ephrin-A2 (+), ephrin-A5(+) (rostral domain A). Light blue: ephrin-A2 (+ or ++), ephrin-A5(-) (middle parts of domains A and C).

View larger version (64K): [in a new window]   Fig. 4. Anterograde tracing of IO axons. (A,D,G,J,M) DiI insertion sites in the IO (red stars). Ventral views. IO areas are shown as in Fig. 1P. (B,E,H,K,N) Projection patterns of DiI-labeled axons (white arrowheads) in the contralateral side of the cerebellum. (C,F,I,L,O) Domain patterns of ligand activity detected with EphA3-AP. Black arrowheads indicate projection areas of DiI-labeled axons shown by white arrowheads in B,E,H,K,N. (P) Scheme of cerebellar domains in explant (see Fig. 3B). In each panel, rostral is at the top. (A-C) Axons from the main part of Area L project to the rostral parts of domain C. (D-F) The most caudal part of Area L maps to the rostral domain A. (G-I) Area Ic maps to the rostral oval region of domain B. (J-L) Area Ir maps to the middle region of domain C. (M-O) Area Ml maps to the caudal domain B. Scale bar, 500 µm.

View larger version (40K): [in a new window]   Fig. 5. Domain patterns of retrogradely labeled IO axons. (A,E,I,M,Q) A diagram of DiI (red star) and DiA (green star) insertion sites in one half of the cerebellum. (B-D,F-H,J-L,N-P,R-T) Retrogradely labeled neurons in the IO. Ventral views are shown. Rostral is at the top. Dashed line indicates the ventral midline. Scale bar, 500 µm. (U) A diagram of IO areas (see Fig. 1P).

View larger version (58K): [in a new window]   Fig. 6. EphA receptor expression profiles of retrogradely-labeled areas in IO. (A,E,I,M,Q) DiI insertion sites (red star) in the cerebellum. (B,F,J,N,R) Retrogradely labeled neurons in the contralateral IO. Dashed lines indicate ventral outlines of the brainstem. Dorsal is at the top. Vertical lines indicate midline. (C,D,G,H,K,L,O,P,S,T) EphA3 and EphA5 expression detected by RNA in situ hybridization in the same and adjacent coronal sections through the IO (arrowheads). Scale bar, 500 µm. (U) A diagram indicating the positions of coronal planes of the sections. (A-D) The caudal domain B is innervated by axons from the medial IO, where both EphA3 and EphA5 are expressed (Area Ml). (E-H) The rostral domain B receives axons from the caudo-intermediate IO, where no EphA3, but strong EphA5 expression is detected (Area Ic). EphA6 is also expressed in this area (data not shown). (I-L) The middle region of domain C receives axons from the rostro-intermediate IO, where no EphA3 and moderate EphA5 expression is detected (Area Ir). EphA6 expression was not detected in this area (data not shown). (M-T) The rostral parts of domains C and A receive axons from the lateral IO, where both EphA3 and EphA5 are not expressed (Area L).

View larger version (44K): [in a new window]   Fig. 7. Summary of the olivocerebellar topography and the Eph/ephrin domains. (A) Overview of the olivocerebellar projection in the organotypic hindbrain culture. (B) Cerebellar domains defined by ephrin-A ligands (left) and areas in the inferior olive defined by EphA receptors. (C-G) Corresponding areas between the cerebellum (left) and inferior olive (right) revealed in the axon tracing experiments are shown in grey.

View larger version (46K): [in a new window]   Fig. 8. Retroviral modification of the EphA-ephrin-A interaction in the cerebellum disrupts the olivocerebellar mapping. (A-E) Ligand and receptor activity detected by affinity probe in situ, in virus-infected explants. Rostral is at the top. Cb, cerebellum. Asterisk, inferior olive. (A) In an uninfected control cerebellum, parasagittal patterns of endogenous ligand activity are seen. (B) The cerebellum of an RCAS-ephrin-A2-infected embryo shows massive expression of ephrin-A2. (C) Following infection with the RCAS-EphA3C virus, EphA3-AP fails to detect endogenous ligand activity. (D) Receptor activity in an uninfected control cerebellum. (E) Ectopic EphA3C expression induced by the RCAS-EphA3C virus. (F-O) Retrograde axon tracing in retrovirus-infected explants. (F) Dye insertion sites in the cerebellum of the organotypic hindbrain culture. The capital letters indicate domain names. The rostral domain C and the caudal domain B are labeled with DiA (green star) and DiI (red star), respectively. (G-I) Labeling pattern in the IO of an embryo infected with the RCAS-AP virus. The caudal domain B and the rostral domain C receive axons from Area Ml and Area L, respectively. (J-L) Representative labeling pattern of IO following infection with the RCAS-ephrin-A2 virus. The caudal domain B is invaded by axons from more lateral parts of the IO. The rostral domain C is innervated by axons from Area L. (M-O) In explants infected with the RCAS-EphA3C virus, the caudal domain B receives axons from Area Ml. In contrast, the rostral domain C receives axons from broad areas of the IO. (P-T) Anterograde tracing of Area Ml axons in virus-infected explants. (P) DiI insertion site (Area Ml) in the IO. (Q-R) In explants infected with RCAS-ephrin-A2 virus, labeled axons stop at the border of ectopic expression domains (arrowheads), and fail to project to their normal target domain. (S-T) Following the RCAS-EphA3C virus infection, Area Ml axons project to most parts of the cerebellum, including the domains with high endogenous ligand expression. Virus-derived expression was detected by affinity probe in situ (R,T). Scale bars, 500 µm.

View larger version (79K): [in a new window]   Fig. 9. Ephrin-A2 inhibits outgrowth of IO axons in vitro in a region-specific manner. Explants of the medial (A,C) and lateral (B,D) IO prepared from E8 embryos were grown on homogeneous carpets of 293T cell membrane, either mock-transfected (A,B), or transfected with pcDNA1-ephrin-A2 (C,D). Whereas axons from the lateral IO grow equally well on both membranes, axon outgrowth from the medial IO was significantly inhibited on the ephrin-A2 membrane. Scale bar, 500 µm.

View larger version (26K): [in a new window]   Fig. 10. Models for the mechanisms of the domain-specific olivocerebellar mapping. Schemes of the possible models discussed in the text are shown. R1-4, receptors; L1-4, ligands. Receptor-ligand pairs with the same numbers interact with high affinities. Large characters indicate high expression.