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Fig. 6. Delta expression and effect of a Notch signaling inhibitor.
(A,A') Dll1 mRNA expression in the E14 mouse
brain. (A') Magnified view of boxed region from A. Signals were most
intense in the part of the VZ 20-40 µm away from the apical surface
(arrow). Scale bar: 50 µm. (B) Dll3 mRNA expression in the
E14 mouse brain. (C) Anti-Dll1 (green) and anti-Ki67 (red)
immunostaining of the E14 cerebral wall. (D-K) Effect of Notch
inhibitor in E14 cerebral slice culture. DMSO vehicle (D,F,H) or 10 µM DAPT
(E,G,I) was present in the medium for 20 hours. (D,E) Anti-Tbr2 (red),
anti-Ki67 (green) and DAPI (blue) staining. (F,G) Anti-PH3 (green) and DAPI
(blue) staining. (H,I) Anti-Vcam1 (red) staining. Vcam1 immunoreactivity was
diminished by the DAPT treatment (n=7 slices for DMSO, n=8
slices for DAPT). (J) The percentage of Tbr2+ cells amongst
Ki67+ cells was significantly increased by DAPT treatment
(Mann-Whitney test, two-tailed, **P=0.0022, n=8
slices for DAPT and n=7 slices for DMSO). (K) The frequency of
non-surface PH3+ cells among total PH3+ cells was
significantly increased by DAPT treatment (Mann-Whitney test, two-tailed,
***P=0.0002, n=8 slices for DAPT and
n=11 slices for DMSO). Error bars indicate s.d. The number of
apoptotic cells in the VZ was not increased by DAPT treatment compared with
the DMSO control, as determined by anti-cleaved caspase 3 immunoreactivity
(not shown). (L-O) Time-course changes in gene expression in slice
culture treated with DMSO (L,N) or DAPT (M,O). In situ hybridization for
Dll1, Gadd45g, Svet1 or Sstr2 was performed on samples
treated for 7 (L,M) or 20 (N,O) hours. (P) Delta-Notch signaling and
differentiation of progenitor cells. Cluster I cells are apical progenitor
cells, Cluster II cells are nascent basal progenitor cells in the VZ, and
Cluster III cells are basal progenitor cells in the SVZ. Cluster II cells (and
probably young neurons) express Delta only transiently in the apical half of
the VZ, and maintain neighboring Cluster I cells in the undifferentiated
state. The choice of the Cluster II cell fate by a daughter cell occurs before
and/or during early G1 phase, and the attenuation of Notch signaling
presumably triggers this step. During migration to the SVZ, Cluster II cells
lose their apical process and become unable to receive a strong Delta
signal.
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