Growth factor independent-1 Maintains Notch1-Dependent Transcriptional Programming of Lymphoid Precursors (original) (raw)
Figure 3
Gfi1 is required to enforce ICN1 activation of lymphoid genes.
(A) Schematic for ICN colony forming unit (CFU) assay +/− Gfi1. CFU from FACS sorted ICN+eGFP expressing Lin− cells from either Gfi1f/f or RosaCreERT2+ Gfi1f/f were plated at 3000 cells/plate in 1 µM 4-OHT or EtOH control and cultured for 7 days (left). CFU/plate were then enumerated, disrupted and replated at 3000 cells/plate for additional two platings (middle). Representative pictures of RosaCreERT2+Gfi1f/f in EtOH or 4-OHT are shown (right). (B) Schematic of FACS-sorted ICN+eGFP expressing Lin− cells from either Gfi1f/f or RosaCreERT2+Gfi1f/f plated at 3000 cells/plate in EtOH and cultured for 7 days, then replated in 1 µM 4-OHT for an additional two platings (left). Enumeration of CFU (right). (C) Schematic of CFU conditions: similar to (B), but after 7 days in 1 µM 4-OHT, CFU were disrupted and RNA was isolated for microarray analysis (left). Principal component analysis of CFU with FACS-sorted HSC and lymphoid progenitor populations is shown. Note that Gfi1f/f CFU, but not Gfi1Δ/Δ CFU, cluster with LMPP. (D) Heatmap of 125 statistically different T-lineage commitment genes between Gfi1f/f CFU and Gfi1Δ/Δ CFU in indicated HSC and progenitor populations. Note that although Gfi1Δ/Δ CFU cluster with DN2 & DN3 cells, many of the 125 genes show differential expression suggesting Gfi1 Δ/Δ CFU are not characteristic of normal DN2 or DN3 cells. (E) GSEA enrichment plots and leading edge heatmaps demonstrate Gfi1f/f CFU transcriptionally mimic the expression of the indicated genesets, specific for lymphoid progenitors, while Gfi1Δ/Δ CFU fail to induce these genes. (F) Flow cytometric plots of Flt3+ LSK (LMPP) in BM progenitors from Gfi1+/+ and Gfi1−/− mice (N = 6/genotype); averages with SEM are shown. (G) Relative gene expression of indicated genes from FACS sorted Flt3− and Flt3+ LSK from Gfi1+/+ and Gfi1−/− mice. Averages with SD are displayed from triplicates (N = 2).