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. 2008 Jun 15;22(12):1617-35.
doi: 10.1101/gad.1649908.

Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice

Kazuki Kurimoto  1 , Yukihiro Yabuta, Yasuhide Ohinata, Mayo Shigeta, Kaori Yamanaka, Mitinori Saitou
Affiliations

Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice

Kazuki Kurimoto et al. Genes Dev. .

Abstract

Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction but specifically avoiding developmental programs such as the epithelial-mesenchymal transition, Hox cluster activation, cell cycle progression, and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors. In contrast, it is dispensable for the activation of approximately half of the genes up-regulated in PGCs, uncovering the Blimp1-independent events for PGC specification. Notably, however, highly PGC-specific genes exhibited distinct correlations to Blimp1 in wild-type embryos, and these correlations faithfully predicted their expression impairments in Blimp1 mutants. Moreover, their expression overlaps within single cells were severely damaged without Blimp1, demonstrating that Blimp1 exerts positive influence on their concerted activation. Thus, Blimp1 is not a single initiator but a dominant coordinator of the transcriptional program for the establishment of the germ cell fate in mice.

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Figures

Figure 1.
Figure 1.
Single-cell analysis of the expression of key genes during germ cell specification, both in the wild-type and Blimp1 KO embryos. (A) Schematic representation of embryos used for analysis. Dotted squares indicate the regions from which single-cell cDNAs were prepared. Arrows indicate embryonic stages covered for the analysis. (ICM) Inner cell mass; (PS) prestreak; (E/MS) early-/mid-streak; (LS/0B) late-streak/no-bud; (E/MB) early-/mid-bud; (EHF) early head fold. (B) Transition of the ratio of Hoxb1-positive (black), Sox2-positive (red), and stella-positive (blue) cells within wild-type Blimp1 or T (PS stage)-positive, Oct4-positive cells (left), Blimp1 KO Blimp1Δ-positive, Oct4-positive cells (middle), and wild-type Blimp1-negative, Oct4-positive somatic neighbors (right). The thin lines in the middle panel represent data from the wild-type embryos shown on the left. (C,D) Immunofluorescence analysis of Oct4 (panels i,ii) and Sox2 (panels iii,iv) in Blimp1-mVenus embryos at the LS (C) and MB (D) stages. (Panels ii,iv) Merged images with DAPI are shown. Blimp1-positive cells initially repressed Sox2 protein (C, panel iii) but subsequently specifically regained it (D, panel iii). This is consistent with the result obtained by the single-cell cDNA analysis (shown in B), despite the different technical sensitivities between the two methods or the differences between mRNA and protein detection. Bar, 100 μm. (E) Immunofluorescence analysis of Sox2 in Blimp1-mVenus wild-type (panels i,ii) and Blimp1 KO (panels iii,iv) embryos at the MB stage. (Panel ii,iv) Merged images with DAPI are shown. Bar, 100 μm.
Figure 2.
Figure 2.
Global profile of the germ cell specification pathway. (A) Number of cells subjected to the microarray analysis. Developmental stages and marker gene expressions are indicated. (*) Data from the previous study (Kurimoto et al. 2006) are used for the E3.5 ICM cells. (L) <10 copies per cell. (B) UHC analysis of the global expression profiles of the wild-type Blimp1-positive single cells. Gene expression levels of Blimp1, Hoxb1, Sox2, and stella determined by Q-PCR are indicated with symbols: (+) >10 copies per cell; (L) <10 copies per cell; (−) not detected. (C) PCA. Each circle represents the averaged expression profile of each indicated cell group. (D,E) Gene expression differences between the indicated cell groups are shown by scatter plots. Among the 5694 genes selected by ANOVA, those expressed in either of the indicated cell groups (the number indicated by green) are ranked in the order of their expression level differences between the two groups. Genes and their numbers significantly up-regulated (red) or down-regulated (blue) in the left-hand cells are indicated.
Figure 3.
Figure 3.
Complex expression dynamics of the specification and somatic genes. (A–C) Expression dynamics of the specification genes (A), somatic genes (B), and genes commonly regulated in the E/MB stage PGCs and their somatic neighbors (C). The specification and somatic genes are classified based on their dynamics: group I (A, panel i), group II (A, panel ii), group IV (B, panel i), and group V (B, panel ii) (see text for details). Each group is subclassified based on the developmental intervals where the genes’ greatest expression level changes are observed (red dashed line): Intervals between the PS-stage and LS/0B-stage Hoxb1-negative cells, between the LS/0B-stage Hoxb1-positive and Hoxb1-negative cells, and between the LS/0B-stage Hoxb1-positive and E/MB-stage PGCs. Normalized log2 expression levels are plotted. Numbers of the genes that belong to each subclass and representative genes are shown. (D) Functional annotation of the genes over-represented in the indicated groups. EASE scores (see the Materials and Methods) are represented with bars. (E) Immunofluorescence analysis of Kit (panel i), AP2γ (panel ii), and Sox3 (panel iii) in Blimp1-mVenus wild-type embryos at the MB stage. Merged images with DAPI are shown in the right panel. Bar, 50 μm.
Figure 4.
Figure 4.
Single-cell microarray analysis of the Blimp1 KO PGC-like cells. (A) UHC analysis of the Blimp1 KO cells at the LS/0B (left) and E/MB (right) stages, using the 493 specification genes. (B) PCA. The wild-type cells are represented with open circles, with the color code as in Figure 2C. Blimp1 KO cells are represented with filled circles, with indications for the KO clusters and developmental stages. (C,D, top) Scatter plots of the specification gene activation (left) and somatic gene repression (right) in Blimp1 mutants at the LS/0B (C) and E/MB (D) stages. To estimate the impact of Blimp1 deficiency at the LS/0B stage, wild-type LS/0B Blimp1-positive, Hoxb1-negative/low cells were used for the comparison to the LS/0B Blimp1 KO cells; in wild-type LS/0B Blimp1-positive, Hoxb1-negative cells, 291 of 493 specification and 116 of 330 somatic genes were up-regulated and down-regulated in comparison to the E/MB somatic cells, respectively. The vertical axis indicates log2 expression level difference between the Blimp1- or Blimp1Δ-positive cells and the E/MB stage somatic neighbors. The horizontal axis represents the ranking of the expression level difference between the wild-type Blimp1-positive, Hoxb1-negative cells and the E/MB stage somatic neighbors. Black filled circles indicate genes in the wild-type Blimp1-positive, Hoxb1-negative/low cells. Red filled circles indicate specification and somatic genes in the Blimp1 KO Cluster 1 cells that are properly up- and down-regulated (expression level difference from the somatic neighbors was significant and expression level difference from the wild-type cells was within twofold), respectively. Red open circles indicate specification and somatic genes in the Blimp1 KO Cluster 1 cells that are up- and down-regulated in comparison to the somatic neighbors (expression level difference from somatic neighbors was significant), respectively, but not properly compared to the wild-type cells (expression level difference from the wild-type cells was more than twofold). Black open circles indicate genes in the Blimp1 KO Cluster 1 cells that are indistinguishable from the somatic neighbors (expression level difference was not significant). For the definition of a significant expression level difference from the somatic neighbors, see Materials and Methods. (Bottom) Histogram of the Blimp1 KO Cluster 1 genes with the color code as in the top panel. The proportion of the specification genes properly up-regulated in the Blimp1 KO embryos (red-filled bars) is negatively correlated with the ranking of expression-level differences between the wild-type cells and somatic neighbors (i.e., magnitude of specificity); this notion is supported by the χ2 test with the null hypothesis that the proportion of the properly up-regulated genes is the same irrespective of the magnitude of specificity (P < 0.05).
Figure 5.
Figure 5.
Correlations between the selected specification genes and Blimp1. (A) Expression frequency differences between the wild-type Blimp1-positive cells and Blimp1 KO Blimp1Δ-positive cells, plotted against the logistic regression coefficient between the Blimp1 expression level and the expression frequency of the selected genes in the wild-type Blimp1-positive cells. Red and blue dots represent genes that show statistically significant logistic regression with the Blimp1 expression level (P < 0.01) (see also Supplemental Figs. S7, S9) and that do not, respectively. (*) Statistically significant correlation (P < 0.05). (B) Expression level differences between the wild-type Blimp1-positive cells and Blimp1 KO Blimp1Δ-positive cells, plotted against the linear regression coefficient between the Blimp1 expression level and the expression levels of the selected genes in the wild-type Blimp1-positive cells. Averaged expression level differences are calculated using cells expressing individual genes. Red and blue dots represent genes that show statistically significant correlation coefficients (P < 0.01) (see also Supplemental Figs. S8, S9) and that do not, respectively. (**) Statistically significant correlation coefficient (P < 0.01).
Figure 6.
Figure 6.
Expression coverage analysis of the selected genes. The expression coverages of genes shown as Gene B by genes shown as Gene A in the wild-type Blimp1-positive cells (left), Blimp1 KO cells (middle), and Blimp1-negative somatic cells (right) at the E/MS (top), LS/0B (middle), and E/MB (bottom) stages are shown according to the color code at the bottom.
Figure 7.
Figure 7.
A model of the global transcriptional dynamics for the specification of germ cell fate in mice. (A) Schematic representation of germ cell specification in mice. The most proximal posterior epiblast cells are determined as Blimp1-positive, lineage-restricted PGC precursors at the PS stage (blue circle). These cells then exhibit a mesoderm-like state under the influence of the mesoderm induction (orange circle). In the presence of Blimp1, the PGC precursors start to activate PGC-specific properties and then repress somatic programs, hence becoming established PGCs (green circle). In the absence of Blimp1, the mesoderm-like-state cells are propelled toward a somatic mesodermal fate as the embryo develops. (B) Summary of genes associated with germ cell specification. We identified 493 “specification” genes and 330 “somatic” genes, representatives of which are shown with the GO functional classifications. Genes that represent common developmental programs between germ and somatic cells are also shown. Blimp1 functions as a dominant repressor of somatic genes and as a positive regulator of the specification genes with high specificity. Blimp1-independent mechanisms are involved in the induction of approximately half of the specification genes. (ExE) Extraembryonic ectoderm.
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