First published online August 18, 2003
doi: 10.1242/10.1242/dev.00654
Reelin signaling directly affects radial glia morphology and biochemical maturation
Eva Hartfuss1,
Eckart Förster2,
Hans H. Bock3,
Michael A. Hack1,
Pierre Leprince4,
Juan M. Luque5,
Joachim Herz3,
Michael Frotscher2 and
Magdalena Götz1,*
1 Max-Planck-Institute of Neurobiology, Neuronal Specification, Am Klopferspitz
18a, D-82152 Martinsried, Germany
2 Institute of Anatomy, University of Freiburg, Albertstr.17, D-79104 Freiburg,
Germany
3 Department of Molecular Genetics, UT Southwestern, 5323 Harry Hines Blvd,
Dallas, TX 75390-9046, USA
4 University of Liège, Center for Cellular and Molecular Neurobiology, 17
Place Delcour, B-4020 Liège, Belgium
5 Instituto de Neurociencias UMH/CSIC, Campus de San Juan s/n, E-03550 San Juan
de Alicante, Spain
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Fig. 5. Reelin signaling affects Blbp content and morphology of radial glial cells
in vitro. (A) Fluorescent micrographs of Rc2- (red) and Blbp- (blue)
immunoreactive cells dissociated from E14 cortex and cultured for 24 hours in
control (top) or reelin-conditioned (bottom) medium. Note the increase in the
number of Blbp-immunoreactive cells cultured in reelin-conditioned medium
(middle bottom panel). Arrows indicate double-labelled cells. (B-D) Histograms
of the percent of Rc2-only (red bars) or Rc2- and Blbp-immunoreactive cells
(yellow bars) dissociated from different brain regions of E14 embryos cultured
for 2 or 24 hours in control or reelin-conditioned medium. Note that the
proportion of Blbp-immunoreactive cells significantly increases (Student's
t-test, ***P<0.01) after a 24 hour exposure to
reelin-conditioned medium. This increase also occurred in cells from reeler
cortex to the same number of Blbp-positive cells as induced in wild-type
cortex, suggesting that the decrease in Blbp-positive cells of the reeler
cortex can be fully rescued by the addition of reelin. [Number of cells
analysed: (B) 2 hours, n(Ctrl-med)=1652, n(Rln-med)=1523;
(B) 24 hours n(Ctrl-med)=3478, n(Rln-med)=3457; (C)
n(Ctrl-med)=1263; n(Rln-med)=1274; (D) n(GE)=973,
n(SC)=886.] (E) Western blot of chemically defined culture medium
conditioned for 48 hours by cells stably transfected with a control (CTRL) or
reelin-expression plasmid (see D'Arcangelo
et al., 1997 ; Beffert et al.,
2002; Förster et al.,
2002) showing a high amount of reelin in the reelin-conditioned,
but not the control medium. (F,G) Morphological analysis of radial glial cells
after exposure to reelin in vitro. (F) Two corresponding micrographs depicting
examples of an Rc2-only positive precursor without processes (arrowheads) and
two Rc2/Blbp-doubleimmunoreactive bipolar cells with processes longer than
their cell soma after culturing in reelin-conditioned medium. The histograms
in G depict the proportion of Rc2-positive cells from E14 wild-type (light
gray) or reeler (dark gray) cortex with a bipolar morphology. Note that cells
with bipolar morphology increase significantly (Student's t-test,
***P<0.01) after exposure to reelin-conditioned medium
for 24 hours. [Number of cells analysed: Ctrl-med, n(wild type)=429,
n(Rln)=204; Rln-med, n(wild type)=396,
n(Rln)=232.]
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Fig. 3. Morphologically identified subpopulations of ventricular zone cells in
wild-type and reeler CNS. (A-A') Single frames of a 3D-reconstruction of
precursors traced from the ventricular surface by DiI in E14 mouse cortex.
Frame A' represents a rotation of 20° (y-axis) with regard
to frame A. Note the two cells indicated by the curved arrows previously
hidden behind cells with long radial processes. For the quantification
depicted in D, cells labeled from the ventricular surface were classified as:
L, long precursor (the radial process reaching the pial surface, red in D); S,
short precursor (with the process ending below the cortical plate, light-gray
in D); CS, club-shaped precursors without a basally oriented process
(dark-gray in D); GC, precursors with a radial process terminating with a
growth cone-like structure (orange in D) as exemplified in A,A',B.
(C,C') Maximum intensity of entire stacks used for 3D reconstructions of
DiI-labeled VZ cells in E14 cortices of wild-type (C) and reeler (C')
littermates. Note that the majority of DiI-labeled ventricular zone cells has
long radial processes in all regions and at all stages analysed in the
wild-type CNS (left side), while their number is significantly reduced in the
reeler cortex, but not the GE. Student's t-test was used for
significance analysis and three asterisks in D indicate P<0.01.
[Number of cells analysed: Ctx, n(E12)=90, n(E14)=250,
n(E16)=116, n(E18)=40; GE, n=267; SC
n=130; reeler Ctx, n=294; GE, n=61.]
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Fig. 1. Neurochemically identified subpopulations of radial glia in wild-type and
reeler telencephalon. Frontal vibratome sections of wild-type (A,C,E-G) and
reeler (B,D) telencephalon stained for Blbp (A-D,F,G), Rc2 (A-D) or reelin
(E-G). A-D show sections of embryonic day (E) 16 wild-type (A,C) and reeler
(B,D) cortex stained for Rc2 (red) and Blbp (green) as indicated in the
micrographs. A-D show maximum intensity pictures ( 50 µm),
A',A'' and B',B'' depict single optical
sections (5 µm). Note that Rc2-immunoreactivity is similar, but the
Blbp-immunoreactivity is strongly reduced in the reeler cortex, whereas no
difference is seen in the GE. E depicts a low power view of
reelin-immunoreactive cells in the telencephalon at E14. The broken line
indicates the outline of the ventricle and border between cerebral cortex
(Ctx) and ganglionic eminence (GE). (F,G) High power views of the cortex (F)
and ventral telencephalon (G), as indicated by the white boxes in E. Note the
close vicinity of reelin-immunoreactive neurons (red, examples indicated by
arrows) and the Blbp-immunopositive endfeet of radial glial cells (green) from
the cerebral cortex (F), but not from the GE (G).
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Fig. 2. Quantitative analysis of precursor subtypes in wild-type and reeler mice
throughout neurogenesis. The pie charts depict the quantitative
co-localization analysis of precursor cells immunoreactive for Rc2, Blbp and
Glast in acutely dissociated cells of wild-type and reeler cortex at E12, E14
and E16. The analysis was performed as described in detail in Hartfuss et al.
(Hartfuss et al., 2001).
Briefly, triple immunostainings were performed in different combinations of
Rc2, anti-Glast, anti-Blbp and anti-Ki67 to detect all dividing cells as
described in the above reference. Note that the number of Blbp-positive
precursors is severely reduced in the reeler compared with wild-type cortex.
[Number of cells analysed: wild-type, n(E12)=311,
n(E14)=1821, n(E16)=302; reeler, n(E12)=307,
n(E14)=1853, n(E16)=295.]
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Fig. 4. Neuronal differentiation in reeler cortex. A shows confocal pictures
(maximum intensity) of E16 frontal sections of wild-type and reeler cortex
stained for the neuron-specific antigen ß-tubulin-III. Note that the
thickness of the cortical plate is comparable in wild-type and reeler cortex,
while alterations in the organization of the cortical plate and fiber tracts
are already visible. To detect small quantitative changes in the number of
neurons, reeler mutant cells were crossed with Tau::EGFP mice that express GFP
in neurons (Tucker et al.,
2001). GFP-positive neurons were quantified at the
fluorescent-activated cell sorter (FACS) as depicted in B in examples of sort
profiles of a wild-type-Tau::GFP and a reeler-Tau::GFP cortex at E14.
Left histograms show the dot plot of cells in forward scatter (FSC;
x-axis) and side scatter (SSC; y-axis). The polygonal area
is indicating the gated, i.e. analysed, healthy cells. The histograms on the
right side show the number of events (x-axis) and the GFP-intensity
(y-axis) and cells with green fluorescence above background are
depicted in green. Note the identical number of green fluorescent neurons in
wild-type and reeler cortex.
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Fig. 6. Reelin signals directly to radial glial cells. (A,B) FACS profiles as
described in Fig. 4 of two
independent sorts selectively enriching for radial glial cells. In A,
GFP-negative (black fraction in the histogram) precursor cells were isolated
from the Tau::EGFP-mouse mouse line containing GFP in postmitotic neurons
(Tucker et al., 2001); in B,
GFP-positive cells (green fraction in the histogram) were isolated from the
hGFAP-GFP mouse line with GFP in radial glia
(Zhuo et al., 1997;
Malatesta et al., 2000).
Histograms in A' and B' depict the proportion of Rc2-only or Rc2-
and Blbp-immunopositive cells cultured after the sorting in control (red bars)
or reelin-conditioned (blue bars) medium for 24 hours. Note the significant
(Student's t-test,*P<0.04) increase in
Blbp-immunoreactive radial glia after exposure to reelin-conditioned medium,
indicating that the presence of neurons is not required to mediate this
effect. [Number of cell analysed: A, n(Ctrl)=237,
n(Rln-med.)=354; B, n(Ctrl)=215,
n(Rln-med.)=367.]
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© The Company of Biologists Ltd 2003
