E2A proteins promote development of lymphoid-primed multipotent progenitors - PubMed (original) (raw)

E2A proteins promote development of lymphoid-primed multipotent progenitors

Sheila Dias et al. Immunity. 2008.

Abstract

The first lymphoid-restricted progeny of hematopoietic stem cells (HSCs) are lymphoid-primed multipotent progenitors (LMPPs), which have little erythromyeloid potential but retain lymphoid, granulocyte, and macrophage differentiation capacity. Despite recent advances in the identification of LMPPs, the transcription factors essential for their generation remain to be identified. Here, we demonstrated that the E2A transcription factors were required for proper development of LMPPs. Within HSCs and LMPPs, E2A proteins primed expression of a subset of lymphoid-associated genes and prevented expression of genes that are not normally prevalent in these cells, including HSC-associated and nonlymphoid genes. E2A proteins also restricted proliferation of HSCs, MPPs, and LMPPs and antagonized differentiation of LMPPs toward the myeloid fate. Our results reveal that E2A proteins play a critical role in supporting lymphoid specification from HSCs and that the reduced generation of LMPPs underlies the severe lymphocyte deficiencies observed in E2A-deficient mice.

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Figures

Figure 1

Figure 1. A dose dependent requirement for E2A in the generation of CLPs, ETPs and DN2 cells

(A) Lin− BM cells from E2A+/+, E2A+/− and _E2A_−/− mice were analyzed for surface expression of c-kit and IL-7Rα (upper panels) and the _c-kit_lowIL-7Rα+ subpopulation was analyzed for expression of Sca-1 and Flt3 (lower panels) by flow cytometry. CLPs are Lin−c-_kit_lowIL-7Rα+Sca-1+Flt3+. (B) Surface expression of c-kit and CD25 on Lin− thymocytes; numbers represent the percentage of cells in each gate. (C) Relative number of CLPs in BM and ETPs, DN2 and DN3 cells in the thymus of adult E2A+/− (grey) and _E2A_−/− (white) mice, as compared to wild type mice (black; set to 1). A minimum of 5 mice were analyzed in each group; bars represent the mean ± SD; ** p<0.01.

Figure 2

Figure 2. A dose dependent requirement for E2A in the generation of LMPPs

(A) Lin− BM cells from E2A+/+, E2A+/− and _E2A_−/− mice were analyzed for surface expression of c-kit and Sca-1 (upper panels). The LSK subset was analyzed for expression of c-kit and Flt3 (middle panels) or Flt3 and VCAM-1 (lower panels). Numbers represent the percent of cells in the indicated gate. LMPPs are LSK Flt3high and LSK VCAM-1−. (B) Number of HSCs, MPPs, LMPPs and CLPs in E2A+/− (grey) and _E2A_−/− (white) mice relative to WT (black; set to 1). A minimum of 6 mice were analyzed in each group; bars represent the mean ± SD; ** p<0.01. (C) The total number of HSCs (black), MPPs (grey) and LMPPs (white) cells in E2A+/+, E2A+/− and _E2A_−/− mice is shown and the sum of these populations is the total number of LSK cells.

Figure 3

Figure 3. E2A proteins restrict HSC, MPP and LMPP proliferation

(A) BrdU-incorporation in BM LSKs as determined 24 hours of in vivo exposure to BrdU. LSKs were analyzed for surface expression of Flt3 and intracellular BrdU; numbers indicate the percent of BrdU+ LSKs. The graph summarizes results of 5 independent experiments. (B) Percent of HSCs, MPPs, and LMPPs that were BrdU+ in E2A+/+ and _E2A_−/− mice, respectively. (C) Representative FACS analysis for Annexin-V and PI staining on cultured HSC, MPP or LMPPs cells.

Figure 4

Figure 4. Analysis of the E2A+/+ and _E2A_−/− LMPP transcriptome

(A) Clustering of genes that are differentially expressed in replicate samples (one per column) of E2A+/+ and _E2A_−/− LMPPs (Affymetrix MOE430 2.0 arrays). Expression of these genes in E2A+/+ HSCs is shown for comparison. The clustering includes all genes with expression levels >50 in at least one of the four LMPP arrays and differing by > 2- fold (using a lower 90% confidence bound of fold change). Each row corresponds to one unique identifier and a subset of CLP-associated genes is indicated (see Figure S3 for complete list of genes). The right-most clustering shows the lineage association of these differentially expressed genes in LT-HSC, preGM, CLP, MkP and preCFU-E (as defined in (Pronk et al., 2007b). Red indicates high, blue low, and white intermediate expression levels. (B) Relative expression of Notch1 and Ccr9 mRNA in purified E2A+/− and _E2A_−/− LMPPs, as compared to E2A+/+ progenitors (normalized to Hprt and set to 1), determined by QPCR. (C) QPCR analysis of Ccr9 and Flt3 mRNA in _E2A_−/− fetal liver multipotent progenitors, 36 hours after transduction with control or E47 producing retrovirus. (D) E2A+/+, E2A+/− and _E2A_−/− LSKs analyzed for surface expression of Flt3 and CCR9. CCR9 expression in E2A+/+ LK−S cells is shown for comparison. (E) Absolute number of CCR9+ LMPPs in the BM (femurs and tibias) of E2A+/+, E2A+/− and _E2A_−/− mice. Bars represent the mean ± SD; * p<0.05.

Figure 5

Figure 5. Expression of key lymphoid transcription factors in the absence of E2A

(A) QPCR for Ikzf1, Sfpi1, and Gfi1 mRNA in purified E2A+/+, E2A+/− and _E2A_−/− LMPPs. (B) Distribution of conserved potential E2A, Ikaros and PU.1 binding sites in the promoters (−7.5 kb to +2.5 kb) of E2A-dependent lymphoid-associated genes or a set of randomly selected LMPP genes whose expression is not E2A-dependent. Binding sites were identified using rVISTA through the ECR Browser.

Figure 6

Figure 6. _E2A_−/− LMPPs restrict the MEP fate but show increased myeloid clonogenic potential

(A) HSCs, MPPs, and LMPPs were isolated from the BM LSK population by cell sorting and single cells were seeded in wells of Terasaki plates in conditions supporting multilineage myeloid differentiation. (B) Frequency of E2A+/+ and _E2A_−/− clones giving Mk in vitro (black). One of 4 representative experiments is shown. (C) Representative examples of colonies positive (left) or negative (right) for the presence of megakaryocytes (Mk). Wright-Giemsa staining of the Mk-containing clone (bottom); G = granulocyte; M = macrophage. (D) Number of clones generated from 180 single cell cultures of E2A+/+ or _E2A_−/− HSCs, MPPs, or LMPPs. Color-coding from blue to red represents the size of colonies from smallest (3–9 cells) to largest (90–100% of well). One of 4 representative experiments is shown. (E) Frequency of clonable E2A+/+ (black) and _E2A_−/− (white) LSKFlt3+ progenitors determined at day 7 of culture in methylcellulose (mean of 3 replicate wells ± SD; * p<0.05; freq., frequency). One of 3 representative experiments is shown.

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