Morpholinos for splice modificatio

Morpholinos for splice modification

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Reelin is a positional signal for the lamination of dentate granule cells
Shanting Zhao, Xuejun Chai, Eckart Förster, Michael Frotscher
  1. Fig. 3.

    Co-cultivation of reeler (rl–/–) dentate gyrus with wild type rescues radial fiber orientation and neuronal lamination. (A) Schematic diagram illustrating the experimental design. The rat dentate outer molecular layer was co-cultured next to the reeler dentate gyrus to provide the reeler dentate gyrus with a reelin-containing zone in normotopic position. (B) NeuN staining of a rat hippocampal culture co-cultured with the reeler hippocampus, as indicated in A. Note the formation of a compact cell layer in the reeler dentate gyrus (arrow) adjacent to the rat outer molecular layer. Dashed line indicates border between the two cultures. The boxed area is shown in C and D at a higher magnification. (C) Boxed area shown in B, immunostained for GFAP. Note the long, vertically oriented radial fibers (arrowheads) in the reeler dentate gyrus adjacent to the rat outer molecular layer. The dotted line indicates the border between the cultures. (D) Same detail as is shown in C, counterstained for NeuN. Arrows indicate the compact cell layer formed in the vicinity of the rat outer molecular layer. Scale bars: 150 μm in B; 40μ m in C,D.

  2. Fig. 1.

    The reeler phenotype is preserved in slice cultures of hippocampus. (A) Slice culture of wild-type hippocampus, prepared on P0 and incubated for 7 days in vitro (DIV). Staining for NeuN reveals dense packing of pyramidal neurons in CA1 and CA3, and of granule cells in the granular layer (g) of the dentate gyrus (DG). (B) Slice culture of reeler hippocampus, prepared on P0 and incubated for 7 DIV. NeuN-stained pyramidal neurons and granule cells show the migration defect characteristic of the reeler hippocampus. Pyramidal neurons in CA1 form a double layer (asterisks), and the granule cells are scattered all over the dentate gyrus. (C) Double-labeling for NeuN (red) and GFAP (green) in a slice culture of wild-type dentate gyrus. Long GFAP-positive radial glial fibers run perpendicular to the granular layer. g, granular layer; h, hilus; m, molecular layer. (D) Detail of reeler dentate gyrus double-labeled for NeuN and GFAP. Granule cells do not form a circumscribed layer, and GFAP-positive cells have short processes, thus resembling typical astrocytes. Scale bars: 100μ m in A,B; 20 μm in C,D.

  3. Fig. 2.

    Treatment of reeler slice cultures with recombinant reelin increases the length and density of GFAP-positive fibers. (A) Portion of the hilus of the dentate gyrus in an untreated control culture of reeler hippocampus. GFAP-positive cells have relatively thick, short processes, reminiscent of astrocytes. Two characteristic cells are labeled by arrows. (B) Portion of reeler dentate gyrus after incubation with recombinant reelin for 7 days. The length of GFAP-positive fibers has dramatically increased. The fibers run in all directions, often crossing each other at right angles (arrowheads). (C) Same control culture as that shown in A, counterstained for NeuN. As is characteristic for the migration defect in the reeler mutant, dentate granule cells are scattered all over the dentate gyrus (DG). (D) Same culture as that shown in B, counterstained for NeuN. Treatment with recombinant reelin, while increasing the length of GFAP-positive fibers, did not rescue the granule cell migration defect characteristic of the reeler dentate gyrus. CA3, hippocampal region CA3. Scale bars: 20 μm in A,B; 75 μm in C,D. (E,F) Incubation of the cultures with recombinant reelin significantly (**) increased both the length and the density of GFAP-positive fibers in the hilar region of reeler cultures (n=10; P<0.01).

  4. Fig. 4.

    A specific position of the rat hippocampal slice culture is required to induce a compact cell layer in the reeler dentate gyrus. Two reeler cultures (rl–/–1 and rl–/–2) are co-cultured with a rat hippocampal slice. A compact cell layer (arrow) has only formed in rl–/–1, which was co-cultured next to the outer molecular layer of the rat dentate gyrus. In rl–/–2, which was cultured next to the stratum oriens of CA1, the reeler-specific loose distribution of neurons in the dentate gyrus is retained (arrowhead). Dashed lines represent borders between cultures. Scale bar: 200 μm.

  5. Fig. 5.

    Rescue of neuronal lamination is induced by reelin. (A,B) Reeler hippocampus co-cultured next to rat hippocampus; the dotted line represents the border between the two cultures. Neuronal somata stained for Neurotrace (green); counterstained for reelin (anti-reelin G10; red). A compact neuronal layer (arrows) has only formed in those portions of the reeler culture that are juxtapositioned to reelin-synthesizing cells in the outer molecular layer of the rat culture. (C) Reeler hippocampal culture co-cultured next to a rat olfactory bulb (OB) culture. The border between the two cultures is marked by a dashed line. A dense neuronal layer (arrow) in the reeler culture has formed near the border of the rat olfactory bulb culture, containing numerous reelin-synthesizing mitral cells (red). (D) A compact neuronal layer in the reeler dentate gyrus next to reelin-synthesizing neurons in a rat co-culture fails to form when the two cultures were incubated in the presence of the reelin-blocking CR-50 antibody. Scale bars: 175 μm in A,B,C; 150 μm in D.

  6. Fig. 6.

    Western blots for reelin under the different experimental conditions. Lane 1, supernatant of reelin-transfected 293 cells. Arrowheads indicate the full-length protein and its characteristic fragments. Lane 2, supernatant of control 293 cells. Lane 3, freshly prepared culture incubation medium. Lane 4, freshly prepared supernatant (200 μl) from reelin-transfected 293 cells, added to 800 μl incubation medium (as was used in the experiments with reeler cultures). Lane 5, lysate of rat hippocampus (P5). Lane 6, lysate of slice cultures of P5 rat hippocampus incubated in vitro for 7 days. Lane 7, lysate of slice cultures of P0 reeler hippocampus incubated in the presence of reelin (DIV 7). Lane 8, same incubation medium as in lane 4, after 2 days of incubation. Lane 9, incubation medium from wild-type slice cultures after 2 days of incubation.

  7. Fig. 7.

    Rescued neurons in the compact cell layer are calbindin-positive granule cells. (A,C) Low-power micrographs of reeler hippocampal cultures co-cultured next to the rat hippocampus to promote the formation of a dense neuronal layer (arrows) in the reeler cultures (staining for Neurotrace). (B) Boxed area in A counterstained for calbindin (red). Note that the neurons in the compact cell layer of the reeler culture are calbindin-positive, like the granule cells in the rat dentate gyrus. The dotted line represents the border between cultures. (D) Boxed area in C immunostained for calretinin (red). Only hilar neurons in the reeler mouse dentate gyrus are immunoreactive, indicating that these neurons do not participate in the formation of the densely packed cell layer (as a species difference, hilar mossy cells in the rat are not calretinin-positive). (E) Calretinin-immunopositive mossy cells (red) intermingle with granule cells in a slice culture of reeler hippocampus (culture from P0 mouse, DIV7; counterstained with Neurotrace, green). (F) High-power magnification of a calretinin-immunoreactive mossy cell (red) in the dentate area of a reeler culture (P0; DIV7). The cell was double-labeled for ApoER2 (green puncta). Scale bars: 200 μm in A,C; 85 μm in B; 55 μm in D; 80μ m in E; 4 μm in F.

  8. Fig. 8.

    Rescue of granule cell orientation and layer-specific commissural input. (A,B) Scattered distribution of biocytin-labeled granule cells in the dentate gyrus of a reeler hippocampal slice culture. Granule cell dendrites and axons (red in B) extend in all directions. In A, neuronal somata were counterstained for Cresyl Violet to show the loose distribution of granule cells. (C,D) Many biocytin-labeled granule cells in the rescued granular layer of a reeler slice culture show normal dendritic and axonal orientation. Neuronal somata are counterstained for Cresyl Violet (C) to illustrate the formation of a granular layer in the reeler culture. Dashed line represents border between cultures; the boxed area is depicted in D. (E,F) The laminated projection of commissural fibers to the reeler dentate gyrus is only rescued when a compact granule cell layer has formed. (E) Co-culture of rat dentate gyrus and P4 reeler dentate gyrus. `Commissural' fibers from the rat dentate gyrus are scattered all over the reeler dentate gyrus, like their target granule cells, counterstained for Cresyl Violet. (F) By contrast, when a rat dentate gyrus is co-cultured with P0 reeler dentate gyrus, a granular layer and a compact `commissural' projection (arrowheads) have formed. Dashed lines represent borders between cultures. Scale bars: 45 μm in A,B; 50 μm in C; 30 μm in D; 70 μm in E; 50 μm in F.

  9. Fig. 9.

    Schematic diagram hypothesizing a dual function of reelin in the dentate gyrus. (A) Reelin (blue), synthesized by Cajal-Retzius cells (dark blue) in the marginal zone (m) provides a positional signal for radial glial fibers (green, arrow). The glial cell bodies are located in the secondary proliferation zone, the future hilus (h). (B) Glial cell processes have reached the pial surface, providing a scaffold for the migration of neurons (red). (C) Reelin provides a stop signal for migrating granule cells. Following migration, granule cells accumulate in a layer directly underneath the marginal zone. Alternatively, some radial glial cells may retract their long hilar process (soma translocation), thereby accumulating directly underneath the granular layer (right radial glial cell). Here they may divide and become neurons, inheriting the radial glial apical processes (Miyata et al., 2001).