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. 2008 Oct 7;105(40):15529-34.
doi: 10.1073/pnas.0808357105. Epub 2008 Oct 1.

Notch1 regulates the fate of cardiac progenitor cells

Alessandro Boni  1 , Konrad Urbanek, Angelo Nascimbene, Toru Hosoda, Hanqiao Zheng, Francesca Delucchi, Katsuya Amano, Arantxa Gonzalez, Serena Vitale, Caroline Ojaimi, Roberto Rizzi, Roberto Bolli, Katherine E Yutzey, Marcello Rota, Jan Kajstura, Piero Anversa, Annarosa Leri
Affiliations

Notch1 regulates the fate of cardiac progenitor cells

Alessandro Boni et al. Proc Natl Acad Sci U S A. .

Retraction in

Abstract

The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notch1 and raised the possibility that Notch1 regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPCs) in the niches express Notch1 receptor, and the supporting cells exhibit the Notch ligand Jagged1. The nuclear translocation of Notch1 intracellular domain (N1ICD) up-regulates Nkx2.5 in CPCs and promotes the formation of cycling myocytes in vitro. N1ICD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jagged1 activation of the Notch1 pathway. Importantly, inhibition of Notch1 in infarcted mice impairs the commitment of resident CPCs to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notch1 favors the early specification of CPCs to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notch1 may have critical implications in the control of heart homeostasis and its adaptation to pathologic states.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CPCs, c-kit, and Notch1 receptor. (A–D) Cardiac niche in which CPCs express c-kit (A, C, and D, green) and Notch1 (B and D, magenta). Connexin 43 (C and D, white dots, arrowheads) is present between CPCs and between CPCs and myocytes (C and D, α-sarcomeric actin, α-SA, red). The interstitium contains fibronectin (D, yellow) and collagen (D, light blue). (E) Jagged1 (white, arrows) is present between CPCs (c-kit, green) and myocytes. (F) Delta-like4 (yellow) is detected in the wall of a small coronary vessel (α-smooth muscle actin, α-SMA, magenta). (G) Dot plots showing the distribution of c-kit and Notch1 in CPCs. (H) Cytospin of FACS-sorted CPCs illustrating the coexpression of c-kit (green) and Notch1 (red). (I) CPCs (c-kit, green) that are positive for Notch1 (red) do not express GATA4. Two CPCs that are negative for Notch1 express GATA4 (white dots, arrows). (J) Distribution of Notch1–4 isoforms in CPCs.
Fig. 2.
Fig. 2.
Notch1 regulates CPC commitment. (A) Hes1 transcript was measured in CPCs at 2, 5, and 8 days (d) under control conditions (Ctrl) and in the presence of Jagged1 (Jag1) and Jag1 and γ-secretase inhibitor (Jag1+γ). Hes1 quantity is shown in fold changes versus the corresponding Ctrl. *, P < 0.05 vs. Ctrl; **, P < 0.05 vs. Jag1. (B) After Jagged1 stimulation, CPCs become c-kit-negative and display nuclear localization of N1ICD (yellow). One CPC continues to express c-kit (green) together with the extracellular domain of the Notch1 receptor (red, arrow). (C and D) Jagged1-stimulated CPCs express N1ICD (green) together with Nkx2.5 (red). The area included in the square is shown at higher magnification in the Inset. (E and F) After γ-secretase inhibition, Jagged1-stimulated CPCs are negative for N1ICD and Nkx2.5. (G) Percentage of CPCs positive for N1ICD and Nkx2.5 at 2, 5, and 8 days. For symbols and P values, see A. (H and I) The expression of GATA4 (yellow) is shown in CPCs stimulated by Jagged1 in the absence (H) and presence (I) of γ-secretase inhibitor. (J) Percentage of CPCs positive for GATA4 at 5 days. For symbols and P values, see A. (K) Nkx2.5 and GATA4 transcripts were analyzed in CPCs at 5 days. For symbols and P values, see A.
Fig. 3.
Fig. 3.
Notch1 and Nkx2.5 function. (A) GATA6 and Vezf1 transcripts were measured in CPCs at 5 days. For symbols and P values, see Fig. 2A. (B–F) Jagged1-stimulated CPCs express Ki67 (B and D, yellow), Nkx2.5 (C, D, and F, magenta), N1ICD (E and F, green), and α-SA (B–D and F, white). (G) Expression of Hes1 in control (C) and Jagged1-treated (Jag1) CPCs. Protein complex between N1ICD and RBP-Jk in control (Ctrl) and Jag1 CPCs. The RBP-Jk band detected in the supernatant (SN) of Ctrl CPCs corresponds to RBP-Jk not bound to N1ICD. IP, immunoprecipitation; K, kidney (positive control). (H) Band shift assay in nuclear extracts of P19 cells nontransfected (NT) and transfected (T) with an RBP-Jk expression vector. Shifted bands correspond to the RBP-Jk/oligonucleotide complex (arrow); the band is supershifted (arrowhead) in the presence of RBP-Jk antibody (Ab). Nkx2.5, oligonucleotide probe in the absence of nuclear extracts; Co, specific competitor; NS-Co, nonspecific competitor. (I) ChIP assay in P19 cells was performed as described in Materials and Methods with primers for sequences associated with the genes for Nkx2.5, Hes1 (positive control), and MEF2C (negative control). The amount of DNA in each sample (input) is shown. Immunoprecipitation was performed without primary antibody (no Ab) and with anti-RBP-Jk antibody (RBP-Jk Ab). Arrows indicate the amplified bands obtained with primers that recognize Nkx2.5 (Nkx2.5) and Hes1 (Hes1) promoters. IgG, isotype control. (J) ChIP in CPCs was performed with the same protocol used for P19 cells. For detail, see I. (K) Nkx2.5 promoter activity was measured by luciferase assay in CPCs after transfection with reporter plasmids carrying wild-type Nkx2.5 promoter or Nkx2.5 promoter containing a mutated RBP-Jk binding site.
Fig. 4.
Fig. 4.
Notch1 function in vivo. Border zone of untreated (A–C and E) and γ-secretase inhibitor-treated (F) infarcts in which the areas included in rectangles are shown at higher magnification in the Insets. (A) CPCs (c-kit, green, arrows) express Notch1 (yellow). Red, tropomyosin. (B) Jagged1 (white, arrows) is expressed in CPCs and between CPCs and myocytes (α-SA, red). (C) CPCs express N1ICD (magenta, arrows) and Nkx2.5 (yellow). (D) Number of CPCs and percentage of CPCs positive for N1ICD and Nkx2.5 in the border zone of untreated and treated infarcts. *, P < 0.05 vs. untreated. (E and F) Newly formed BrdU-positive myocytes (white, arrows) in the border zone of untreated (E) and treated (F) infarcts. (G) γ-Secretase inhibition decreases the fraction of BrdU-labeled myocytes. *, P < 0.05 vs. untreated.
Fig. 5.
Fig. 5.
Myocardial regeneration. (A–D) The area included in the square (A) is illustrated at higher magnification in B–D. Regenerated myocytes (B–D: α-SA, red, arrows) are BrdU-positive (B, white), N1ICD-positive (C, green), and Nkx2.5-positive (D, yellow). (E–H) Area of regeneration (new) located within the infarct (dead). New myocytes are BrdU-positive (E), N1ICD-positive (F), and Nkx2.5-positive (G). (H) Merge of E–G. (I) Cardiac function in untreated and treated mice.
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References

    1. Bray SJ. Notch signaling: A simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7:678–689. - PubMed
    1. Tanigaki K, Honjo T. Regulation of lymphocyte development by Notch signaling. Nat Immunol. 2007;8:451–456. - PubMed
    1. Iso T, Kades L, Hamamori Y. HES and HERP families: Multiple effector of the Notch signaling pathway. J Cell Physiol. 2003;194:237–255. - PubMed
    1. Estrach S, Ambler CA, Lo Celso C, Hozumi K, Watt FM. Jagged1 is a beta-catenin target gene required for ectopic hair follicle formation in adult epidermis. Development. 2006;133:4427–4438. - PubMed
    1. Calvi LM, et al. Osteoblastic cells regulate the hematopoietic stem cell niche. Nature. 2003;425:841–846. - PubMed

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