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First published online 28 August 2008
doi: 10.1242/dev.024778

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Temporal regulation of ephrin/Eph signalling is required for the spatial patterning of the mammalian striatum

¹ Université Libre de Bruxelles (U.L.B.), IRIBHM (Institute for Interdisciplinary Research), 808 Route de Lennik, B-1070 Brussels, Belgium.
² Karolinska Institute, Department of Cell and Molecular Biology, SE-171 77 Stockholm, Sweden.
³ Uppsala University, Department of Neuroscience, 75123 Uppsala, Sweden.

View larger version (65K): [in this window] [in a new window]   Fig. 1. In vitro recapitulation of the striatal sorting. (A) Schematic model of development of matrix and striosome compartments in the mouse striatum. Striosomal neurons (in red) are generated first and migrate from the lateral ganglionic eminence (LGE) to the striatal mantle, followed by matrix neurons (in blue). The two populations transiently mix within the striatal mantle, then segregate from each other to form a mosaic of matrix/striosome compartments. (B) A novel organotypic assay to recapitulate the sorting of matrix versus striosome neurons. GFP+ cells are dissociated from the LGE at distinct embryonic ages (E12/E15), and plated onto postnatal striatal slices. The relative distribution of the GFP+ cells is assessed by an M/S value that represents the ratio of the densities of cells that settled in the DARPP32-negative matrix (M) and DARPP32-positive striosome (S) compartments. (C-H) Overlay with E15-LGE derived cells. GFP+ cells preferentially settled in the DARP32-negative matrix compartment (arrows in F-H), while avoiding the DARPP32-positive striosomes (arrowheads). (I-N) Overlay with E12-LGE derived cells. GFP+ cells preferentially settled in the DARP32-positive striosome compartments (arrowheads). (O,P) Quantification of the relative distribution of the GFP+ cells between the striatal compartments. (O) Schematics of the quantification of matrix/striosome cell sorting. The number of GFP+ pixels counted in each striosome (S) and within corresponding areas in adjacent matrix compartment (M) enable to determine a M/S ratio, reflecting the relative distribution of the GFP+ cells in each compartment. (P) E15-LGE and E12-LGE derived cells display very different M/S values (*P<0.0001), respectively greater than and less than 1, reflecting their preference for complementary compartments, contrary to a control thalamic cell population. Scale bar: 200 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Temporal and spatial pattern of expression of ephrin/Eph genes. (A-I) In situ hybridization for EphA4 (A,D,G), ephrin A5 (B,E,H) and ephrin B2 (C,F,I) in mouse embryos. (A-C) At E12.5, ephrin A5 and ephrin B2 are expressed in the ventricular zone (VZ), as well as in the presumptive striatum (Str, arrows in B,C), whereas EphA4 is expressed only weakly in the VZ (arrows in A). (D-F) At E14.5, ephrin A5 and ephrin B2 are expressed throughout the striatal mantle. EphA4 expression also starts to be detected, although mostly remains in the VZ. (G-I) At E16.5, the striatum is diffusely stained for both ephrin ligands and for EphA4. (J-O) In situ hybridization for EphA4 (J,M) and ephrin A5 (K,N) and ephrin B2 (L,O), at peri-natal stages. EphA4 receptor displays a matrix-like pattern of expression surrounding patches of negative expression (arrows in M), ephrin A5 is expressed in a progressively more restricted pattern, resulting in a distribution in patches (arrows in N), whereas ephrin B2 is expressed in a diffuse and progressively weaker pattern.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Matrix/striosome pattern of expression of ephrin A5 and EphA4. (A-C,G-I) Comparison between EphA4 and DARPP32 distribution on alternate sections of P2 (A-C) and P4 (G-I) mouse striatum. EphA4 expression (determined by in situ hybridization) was converted into false green (A,G) and DARPP32 immunostaining into false red (B,H). Overlays (C,I) reveal that the majority of the EphA4-negative patches appear positive for DARPP32 (arrowheads in C,I), indicating that the EphA4 receptor strictly belongs to the matrix compartment. (D-F,J-L) Comparison between EphA4 and ephrin A5 staining on alternate sections. EphA4 expression was converted into false green (D and J) and ephrin A5 expression into false red (E,K). Overlays (F,L) reveal a partially complementary pattern, with ephrin A5 being preferentially expressed in a subset of EphA4-negative patches, particularly in the centre of the striatum (arrowheads in F and L), but also in the EphA4+ compartment, and progressively downregulated between P2 and P4.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Ephrin/Eph inhibitors disrupt the sorting of the matrix and striosomal neurons in vitro. (A-F) When mouse E16 LGE-derived cells, enriched for the matrix neurons, are overlaid on postnatal striatal slices preincubated with control Fc reagents (A-C) they display a preference for the DARPP32-negative matrix compartment (arrows). This preference is partially lost when slices are preincubated with soluble ephrin inhibitors EphA3-Fc/EphB2-Fc (D-F), revealing more GFP+ cells in DARPP32-positive compartments (arrowheads). (G-L) When E12-LGE derived cells, enriched for the striosome neurons are overlaid on postnatal striatal slice preincubated with control Fc reagents (G-I), they display a preference for the DARPP32-positive striosome compartments (arrowheads). This preference is partially lost when slices are preincubated with soluble ephrin inhibitors (J-L), revealing more GFP+ cells in DARPP32-negative compartments (arrows). (M) Quantification of the relative densities of GFP+ cells in matrix (M) and striosome (S) compartments. Treatment with ephrin soluble inhibitors results in a decrease of matrix preference for E16 cells, and conversely a decreased striosome preference for E12 cells (*P=0.001, **P=0.0047). Scale bar: 100 µm.

View larger version (133K): [in this window] [in a new window]   Fig. 5. Altered vivo striatal patterning in ephrin A5/EphA4 mutants. (A-C) When injected with BrdU at E16/E17 and sacrificed at P2, control animals (double heterozygotes for ephrin A5 and EphA4, n=9) display a matrix-like distribution of BrdU. (D-F) Ephrin A5 KO animals (n=5) display a similar distribution of BrdU+ neurons. (G-I) EphA4 KO animals (n=4) display a much more uniform distribution of the BrdU+ matrix neurons across the striatum and more diffuse DARPP32 staining. In addition, many more BrdU+ neurons are found at ectopic locations within DARPP32-positive striosomes. (J-L) Ephrin-A5/EphA4 DKO animals (n=6) show an almost completely uniform distribution of the BrdU+ matrix neurons across the striatal mantle (J), a more diffuse DARPP32 staining (K) and numerous ectopic BrdU+ matrix neurons within the striosomes (inset in L). Arrowheads show DARPP32+ positive striosomes. Scale bar: 100 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 6. EphA4 signalling is required for striatal neuron sorting in vivo. The mean M/S values determined for control (n=9), ephrin A5 KO (n=5), EphA4 KO (n=4) and ephrin-A5/EphA4 DKO (n=6) littermates reveal an abnormal, more even distribution of presumptive matrix neurons in EphA4 KO and DKO genotypes, whereas their distribution in ephrin A5 KO remains similar to controls.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Schematic comparison of cell sorting processes occuring during hindbrain segmentation and striatal compartmentalization. (A) In the developing hindbrain, neural cells generated from adjacent rhombomeric segments express repulsive ephrin ligands (in red) or Eph receptors (in green) in a mutually exclusive pattern. This allows a strict restriction of cell intermingling that results in the characteristic segmented pattern of the rhombomeres. (B) In the developing striatum, striosome neurons (S) are generated first (t1) and express high levels of ephrin ligands (in red) and low levels of Eph receptors (in green). Matrix neurons are generated later (t2) and express high levels of receptors and low levels of ligands. Matrix and striosome cells first intermix and then partially segregate, through the effects of bidirectional interactions, to form the mature mosaic pattern of the striatum (t3). See text for further discussion.

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