Figure 2
Figure 2. Global gene expression profiling of the primitive erythroid lineage. Labeled cRNA samples were hybridized to Illumina Mouse WG-6 v1.1 Expression BeadChip genome-wide arrays. Quality control of array data was performed using the Bioconductor lumi R package. The filtered genes were clustered into 7 major patterns using the maSigPro algorithm. We were able to survey a genome-wide probe set representing 46 630 murine transcripts, encompassing the emergence of EryP progenitors in the YS through successive stages of erythroblast differentiation in the circulation. Many of these probes target less well-annotated transcripts or transcript isoforms of known genes. There are 21 174 unique genes in the University of California-Santa Cruz mouse mm9 refseq protein coding gene database, and the Illumina mouse-6 v1.1 microarray used in this study contains probes for 18 970 or 89.6% of the well-annotated refseq mouse genes. Analyses were performed using all probes with Entrez ID annotations found with the lumiMouseAll.db version 1.6.1 annotation package. (A) Changes (increased or decreased) in transcript numbers during consecutive stages of EryP development. The graph represents the total numbers of transcripts showing a change of greater than 2-fold (P < .01). Dotted red line indicates increasing expression; dotted gray line indicates decreasing expression. Peaks in transcription variation were identified during the windows from E8.5-E9.5 (transition from the YS to the circulation stage, 273 transcripts) and from E11.5-E12.5 (fetal liver stage, 351 transcripts). (B) Plot representations of 7 specific clusters of transcripts with similar temporal expression patterns. Clusters were subclassified into 3 groups, representing genes that are progressively up-regulated (clusters 1, 2, and 3, red lines); down-regulated (clusters 4, 5, and 6, green lines); or up-regulated through E11.5 and then down-regulated rapidly over the next 24 hours of development (gray line, cluster 7). The peaks in transcription variation indicated in panel A are especially evident in clusters 1, 4, and 7 (relatively sharp increases or decreases in expression, E8.5-E9.5, corresponding to the transition from the YS to the circulation stage) and in clusters 3, 6, and 7 (abrupt increases or decreases in expression, E11.5-E12.5, corresponding to fetal liver stage, when EryP complete their maturation and enucleate). Each individual point (○) represents the mean gene expression of the cluster genes from one microarray experiment. Each line connects mean values for all replicates. (C) Overrepresented gene ontologies for the clusters shown in panel A. (D) Expression of a representative gene that is up-regulated (Gata1) and one that is down-regulated (Igf2) in the EryP microarray dataset, analyzed using qRT-PCR. Expression levels were normalized relative to ubiquitin b (Ubb).

Global gene expression profiling of the primitive erythroid lineage. Labeled cRNA samples were hybridized to Illumina Mouse WG-6 v1.1 Expression BeadChip genome-wide arrays. Quality control of array data was performed using the Bioconductor lumi R package. The filtered genes were clustered into 7 major patterns using the maSigPro algorithm. We were able to survey a genome-wide probe set representing 46 630 murine transcripts, encompassing the emergence of EryP progenitors in the YS through successive stages of erythroblast differentiation in the circulation. Many of these probes target less well-annotated transcripts or transcript isoforms of known genes. There are 21 174 unique genes in the University of California-Santa Cruz mouse mm9 refseq protein coding gene database, and the Illumina mouse-6 v1.1 microarray used in this study contains probes for 18 970 or 89.6% of the well-annotated refseq mouse genes. Analyses were performed using all probes with Entrez ID annotations found with the lumiMouseAll.db version 1.6.1 annotation package. (A) Changes (increased or decreased) in transcript numbers during consecutive stages of EryP development. The graph represents the total numbers of transcripts showing a change of greater than 2-fold (P < .01). Dotted red line indicates increasing expression; dotted gray line indicates decreasing expression. Peaks in transcription variation were identified during the windows from E8.5-E9.5 (transition from the YS to the circulation stage, 273 transcripts) and from E11.5-E12.5 (fetal liver stage, 351 transcripts). (B) Plot representations of 7 specific clusters of transcripts with similar temporal expression patterns. Clusters were subclassified into 3 groups, representing genes that are progressively up-regulated (clusters 1, 2, and 3, red lines); down-regulated (clusters 4, 5, and 6, green lines); or up-regulated through E11.5 and then down-regulated rapidly over the next 24 hours of development (gray line, cluster 7). The peaks in transcription variation indicated in panel A are especially evident in clusters 1, 4, and 7 (relatively sharp increases or decreases in expression, E8.5-E9.5, corresponding to the transition from the YS to the circulation stage) and in clusters 3, 6, and 7 (abrupt increases or decreases in expression, E11.5-E12.5, corresponding to fetal liver stage, when EryP complete their maturation and enucleate). Each individual point (○) represents the mean gene expression of the cluster genes from one microarray experiment. Each line connects mean values for all replicates. (C) Overrepresented gene ontologies for the clusters shown in panel A. (D) Expression of a representative gene that is up-regulated (Gata1) and one that is down-regulated (Igf2) in the EryP microarray dataset, analyzed using qRT-PCR. Expression levels were normalized relative to ubiquitin b (Ubb).

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