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First published online August 25, 2008
doi: 10.1242/10.1242/dev.022616

Development 135, 3113-3124 (2008)
Published by The Company of Biologists 2008
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Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis

Ayano Kawaguchi1,*, Tomoko Ikawa1, Takeya Kasukawa2, Hiroki R. Ueda2,3, Kazuki Kurimoto4, Mitinori Saitou4 and Fumio Matsuzaki1,5,*

1 Laboratory for Cell Asymmetry, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
2 Functional Genomics Unit, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
3 Laboratory for Systems Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
4 Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
5 CREST, Japan Science and Technology Agency.


Figure 1
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  		Fig. 1. Hierarchical clustering of single-cell cDNAs and definition of clusters. Cluster dendrograms showing the results from (A) SigABC genes (probe sets significantly different across Groups A, B and C; n=114 probe sets; see Table S1 in the supplementary material) and (B) `Over-20 copies' probe sets (probe sets for which at least one of the 70 cDNA samples showed an expression level of >20 copies per cell; n=10493 probe sets). The dendrogram in A defines four clusters. In both dendrograms, each sample name represents one cell, and its color indicates the cluster to which it belongs. The first letter of the name indicates the cell group; for example, A-11L is a Group A cell. The values in red at the branches are AU (approximately unbiased) P-values (%) that indicate how strongly the cluster is supported by the data. For example, for a cluster with an AU P-value >95%, the hypothesis that `the cluster does not exist' is rejected with a significance level of 5%. The horizontal branch length represents the degree of dissimilarity in gene expression among the samples.

 

Figure 2
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  		Fig. 2. Expression levels of Cluster II/III genes in single-cell cDNAs. Data from the probe sets that were expressed in Cluster II/III, but not in Cluster I, were used for clustering (n=117 probe sets; Welch's t-test, FDR<0.1, log fold-change cut-off >2.5). Each column indicates one cell, and each row indicates one probe set on the microarray. The expression levels are color-coded from red (high) to black (low). These probe sets were categorized into several groups based on their expression pattern along the clusters: probe sets that showed a typical expression pattern are grouped by color (blue, green or yellow). The in situ hybridization patterns of these genes are shown in Fig. 3. For the I- II+ III+ IV- genes (yellow), the log fold-changes from Cluster II to Cluster III cells are also indicated.

 

Figure 3
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  		Fig. 3. mRNA expression of Cluster II/III genes in the E14 mouse brain. In situ hybridization of all of the (A) yellow (I- II+ III+ IV-) and (B) green (I- II- III+ IV-) genes, and some of the (C) blue (I- II+/- III+ IV+) genes from Fig. 2. Magnified views of Ac,b,f are shown in Am,n,o, respectively; Bu,v show magnified views of Bh,k, respectively; Ci shows a magnified view of Cg. Signals for the I- II+ III+ IV- genes typically exhibited a two-band pattern in the SVZ and part of the VZ (arrows, Ac,d,h,i,j,k,m; see also Bj,r). For some genes, the band in the VZ was dominant (arrowhead, Aa,b,f,g,n,o). In both cases, the VZ signals were 20-40 µm from the apical surface (dotted line). GenePaint set IDs are indicated at the bottom right of some panels. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Expression of Cluster I genes in single-cell cDNAs and in the E14 brain. (A) Expression levels of Cluster I genes. Data from the probe sets that were expressed in Cluster I, but not in Cluster II/III, were used for clustering (n=175 probe sets; Welch's t-test, two-tailed, FDR<0.1 log fold-change cut-off <-2.5). Each column indicates one cell, and each row indicates one probe set on the microarray. The expression levels are color-coded from red (high) to black (low). Unlike the Cluster II/III genes (see Fig. 2), no particular pattern was seen in the Cluster I genes, except for the I+ II- III- IV+ genes (5730410E15Rik, Dab1). (B) Examples of in situ hybridization of the E14 mouse brain for Cluster I genes. In all cases, signals were seen in the VZ. The 5730410E15Rik and Dab1 genes were expressed in both the VZ and CP (n,o), consistent with the single-cell gene expression profiles (A). GenePaint set IDs are indicated at the bottom right of some panels.

 

Figure 5
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  		Fig. 5. Expression of marker and signaling pathway genes in single-cell cDNAs, and cell-to-cell variation in the expression of determinants. (A) Expression of marker and signaling pathway genes. Each column indicates one cell, and each row indicates one probe set on the microarray. The expression levels are color-coded from red (high) to black (low). All Cluster I cells expressed some neuronal differentiation-related genes at various degrees. Neurog2 and Dll1 are marked with asterisks. Cells strongly expressing Dll1 were mostly Cluster II cells. Hes3, Dll4 and Jag2 showed low signal levels and are not included in this figure. (B-E) Cell-to-cell variations in gene expression in the Cluster I population. Scatter diagram of the Hes1 and Fgfr3 (B), Neurog2 (C), Dll1 (D) or Neurod2 (E) expression levels from cDNAs of 70 single cells. Each symbol indicates one cell, and axes are on a natural logarithmic scale.

 

Figure 6
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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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© The Company of Biologists Ltd 2008