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.
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.
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).
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.
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.
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.
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.
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.
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).