The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells - PubMed (original) (raw)

. 2014 Mar 15;28(6):576-93.

doi: 10.1101/gad.227496.113.

Kostandin V Pajcini, Teresa D'altri, Lili Tu, Rajan Jain, Lanwei Xu, Michael J Chen, Stacey Rentschler, Olga Shestova, Gerald B Wertheim, John W Tobias, Michael Kluk, Antony W Wood, Jon C Aster, Phyllis A Gimotty, Jonathan A Epstein, Nancy Speck, Anna Bigas, Warren S Pear

Affiliations

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

Dawson M Gerhardt et al. Genes Dev. 2014.

Abstract

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

Keywords: Notch; hematopoietic stem cell; transcriptional activation domain.

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Figures

Figure 1.

Figure 1.

The Notch1 TAD deletion is a hypomorphic mutation. (A) The Notch1 TAD was deleted by homologous recombination using a targeting vector designed to delete the genomic region of mouse Notch1 corresponding to the TAD (extending roughly from a Xho site to a SacI site). (B) Deletion of the TAD (609 bp) was verified by PCR using primers flanking the TAD. (C) Loss of the Notch1 TAD does not affect Notch1 mRNA expression. mRNA was prepared from +/+ and ΔTAD/ΔTAD MEFs and subsequently used for qPCR. Transcripts from +/+ and ΔTAD/ΔTAD cells were amplified with unique primers. PCR products specific for ΔTAD/ΔTAD transcripts yielded product below the limit of detection in +/+ cells. Primers specific for +/+ transcripts yielded product below the limit of detection in ΔTAD/ΔTAD cells. “F” indicates forward primer, and “R” indicates reverse primer. (D) Notch1 expression. Nuclear extracts were prepared from splenic CD4+ T cells derived from transplanted +/+, +/ΔTAD, and ΔTAD/ΔTAD FL cells and were used for Western blot. Blots were probed with antibody specific for Notch1 cleaved at Val1744. β-Actin was the loading control. (E) Cross-sections of wild-type and ΔTAD/ΔTAD hearts at E18.5. The ΔTAD/ΔTAD heart shows a ventricular septal defect (VSD). (RV) right ventricle; (LV) left ventricle. (F) Deletion of the Notch1 TAD is a hypomorphic mutation. Notch1+/in32 mice were bred with +/ΔTAD to generate Notch1in32/ΔTAD embryos. Notchin32/ΔTAD embryos were harvested at E9.5. Control embryos from +/ΔTAD × +/ΔTAD matings were harvested at E10.5. Normal gross development was observed in ΔTAD/ΔTAD E10.5 mutant embryos. Retarded development and an enlarged pericardial sac were observed in Notch1in32/ΔTAD embryos. See also Supplemental Figure S1.

Figure 2.

Figure 2.

Notch1 signaling in the E14.5 FL. (A) Notch1 is expressed on the surface of FL hematopoietic progenitors and HSCs. E14.5 FLs from wild-type B6 embryos were stained for SLAM-LSK markers and Notch1 or isotype control antibody. Flow cytometry plots are representative of three independent experiments. (B) Average fold mean fluorescent intensity (MFI) of Notch1-expressing E14.5 FL hematopoietic cells (CD45+) and SLAM-LSKs over isotype control. (C) Notch1 is cleaved in E14.5 FL HSCs (CD45+ SLAM-LSKs). Wild-type DN3 thymocytes, DP thymocytes, and BM SLAM-LSKs were used as additional controls for detection of cleaved Notch1. Following surface staining, cells were fixed, permeabilized, and stained for ICN1 (Val1744). (D) Graph represents average fold MFI of ICN1 over background staining. (E) Expression of Hes1 mRNA in sorted hematopoietic cells. All values were normalized to Ef1α. Populations are similar to C.

Figure 3.

Figure 3.

Increased apoptosis in FL ΔTAD/ΔTAD LT-HSCs. (A) Flow cytometry analysis of E11.5 AGM regions and E11.5 FLs from +/+ and ΔTAD/ΔTAD embryos. Cells were gated on 7-AAD−TER−119− populations. Endothelial cells were identified as CD144+, hematopoietic progenitors are CD45+, and HSCs are CD45+CD144+. (B) Absolute numbers of cells gated as CD45+CD144+ (as depicted in A) in E11.5 AGM regions and E11.5 FLs from three independent experiments. (C) Cell cycle analysis of +/+ and ΔTAD/ΔTAD E14.5 FL SLAM-LSKs. Representative flow cytometry plot of the cell cycle by DAPI and Ki-67. (D) The bar graph represents percentages of SLAM-LSKs in each cell cycle stage from three independent experiments. (E) Increased apoptosis in ΔTAD/ΔTAD E14.5 FL SLAM-LSKs. Representative flow cytometry plots of Annexin V+ cells from +/+ (dotted line) and ΔTAD/ΔTAD (bold gray line) E14.5 FL SLAM-LSKs. Annexin V expression on internal control Lin+ cells of +/+ (solid black line) and ΔTAD/ΔTAD (light-gray shading) was used to determine the positive gate for Annexin V staining. (F) The bar graph represents the normalized percentage of Annexin V+ 7-AAD− cells from E14.5 +/+ and ΔTAD/ΔTAD FL SLAM-LSKs (n = 4). Values were determined by subtracting the mean percentage of +/+ Annexin V+ Lin+ cells (calculated as percent Annexin V+ cells ± SEM, which was 1.600 ± 0.147; n = 4) from the mean percentage of Annexin V+ +/+ SLAM-LSKs and by subtracting the mean percentage of ΔTAD/ΔTAD Annexin V+ Lin+ cells (4.025 ± 0.728; n = 4) from the mean percentage of Annexin V+ ΔTAD/ΔTAD SLAM-LSKs.

Figure 4.

Figure 4.

Competitive defects and reduced LT-HSC frequency in ΔTAD FL HSCs. (A) Schematic for noncompetitive E14.5 FL transplants. (B) Multilineage reconstitution of primary recipients by ΔTAD/ΔTAD E14.5 FL cells. Representative flow cytometry plots from the thymus and spleen of +/+ or ΔTAD/ΔTAD reconstituted recipients at 16 wk. (C) E14.5 FL cells (2 × 106) from B6 (CD45.2+) +/+, +/ΔTAD, or ΔTAD/ΔTAD embryos were transplanted into lethally irradiated SJL (CD45.1+) recipients. Bar graph represents mean reconstitution at 16 wk in BM, measured by the percentage of CD45.2+ cells. (D) E14.5 FL cells (1 × 106) from B6 (CD45.2+) +/ΔTAD or ΔTAD/ΔTAD embryos were transplanted in competition with 1 × 106 wild-type CD45.1+ E14.5 FL cells into lethally irradiated SJL CD45.1 recipients. The bar graph represents mean reconstitution at 16 wk in peripheral blood, measured by the percentage of CD45.2 cells. (E) SLAM-LSK gating strategy to identify LT-HSCs. (F) Percentage of LSK from +/+, +/ΔTAD, or ΔTAD/ΔTAD E14.5 FL cells. All cells were first gated on DAPI−CD45.2+. (G) Number of LSKs (left panel) and SLAM-LSKs (right panel) per 106 cells from +/+, +/ΔTAD, or ΔTAD/ΔTAD E14.5 FLs.

Figure 5.

Figure 5.

Impaired competitive reconstitution by Notch1 TAD-deficient FL HSCs and Rbpj-deficient FL cells. (A) Schematic representation of competitive transplants from B6 (CD45.2+) FL E14.5 +/+, +/ΔTAD, and ΔTAD/ΔTAD FL and BM cells from adult B6/SJL F1 (CD45.1+/CD45.2+) mice were FACS-sorted for SLAM-LSK. Three-hundred-fifty FL SLAM-LSKs and 350 BM SLAM-LSKs were transplanted in competition (1:1) into lethally irradiated CD45.1+ recipients. (B) Representative flow cytometry plots from peripheral blood (16 wk post-transplant) of recipients showing reconstitution by CD45.2+ cells. (C) Donor cell reconstitution at week 16 was measured by CD45.2+ percentage in peripheral blood of recipients. The plot of data points is from five independent experiments. (D) Competitive reconstitution of Rbpj-deficient FL hematopoietic cells. E14.5 FL cells from _Rbpj_f/f; Vav-Cre Rosa26YFP and Rbpj+/+; Vav-Cre Rosa26YFP control embryos were transplanted in competition with CD45.1+/CD45.2+ adult BM cells at a ratio of 10,000 FL cells:200,000 BM cells. (E,F) Donor cell reconstitution was measured by the percentage of YFP+ cells in the blood (E) and BM (F) at 16 wk post-transplant.

Figure 6.

Figure 6.

Decreased binding of the Notch1 transcriptional complex to the Hes1 promoter element in extracts prepared from ΔTAD MEFs. (A) B6 E14.5 FL cells were cultured on OP9-DL1 cells for the times indicated (4 h or 10 h) in the presence of vehicle control (DSMO) in the “Notch on” state or GSI in the “Notch off” state. SLAM-LSKs were sorted from the coculture, and RNA obtained from the SLAM-LSKs was used for microarray analysis. Selected genes with decreased expression in the presence of GSI are shown. (*) Genes with decreased expression after both 4 h and 10 h of treatment in the presence of GSI; _Q_-value (percent) is used to express the false discovery rate. (B) qPCR measurements from E14.5 FL SLAM-LSKs for Hes1, Jag1, and Itgal (Integrin-α/LFA-1) mRNA transcripts. All mRNA values were normalized to Ef1α. (C) +/+ MEFs were treated for 24 h with GSI or DMSO vehicle control. GSI was washed out at 18 h, and cells were cultured in vehicle control for an additional 6 h. Hes1 mRNA was normalized to Ef1α values. Graph is representative of four independent experiments. (D,E) Graph of absolute values of Hes1 mRNA (D) and Jag1 mRNA (E) from ΔTAD/ΔTAD and +/+ MEFs. Values were normalized to Ef1α from four independent experiments for Hes1 and two independent experiments for Jag1. (F) Schematic for oligonucleotide immunoprecipitation. (G) +/ΔTAD nuclear lysates were incubated with the Hes1 promoter oligonucleotide. (Left panel) Western blot for cleaved Notch1 (Val1744) in +/ΔTAD nuclear lysates (input) shows increased ΔTAD protein relative to wild-type (+) Notch1 protein. At three concentrations (1 μg, 0.75 μg, and 0.5 μg) of oligonucleotide, wild-type protein binding to Hes1 promoter is enriched relative to ΔTAD protein binding. (H) Association of Maml1 with Hes1 oligonucleotide in the presence of +/+, +/ΔTAD, or ΔTAD/ΔTAD MEF nuclear lysates. Western blot for total Maml1 (left panel) and oligonucleotide-bound Maml1 (right panel) from +/+ or ΔTAD/ΔTAD nuclear lysates incubated with 1 μg of Hes1 oligonucleotide. (I) Model of impaired formation and/or stabilization of Notch ternary complex with loss of the Notch1 TAD.

Figure 7.

Figure 7.

Model of Notch1 +/+ and ΔTAD/ΔTAD FL development and function. (Top panel) Formation of the Notch1 transcriptional complex promotes optimal transcription of Notch target genes, allowing for generation of HSCs from the AGM, expansion of HSCs in the FL, and robust function of FL HSCs in competitive transplants. Lack of the Notch1 TAD (bottom panel) impairs formation of the Notch1 transcriptional complex (top panel), resulting in reduced transcription of Notch target genes. Lack of the TAD allows for generation of HSCs from the AGM but leads to a decreased number of HSCs in the FL as well as impaired function of FL HSCs in competitive transplants.

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