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. 2015 Sep 18;10(9):e0138525.
doi: 10.1371/journal.pone.0138525. eCollection 2015.

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells

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Free PMC article

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells

Gabriella Lania et al. PLoS One. .
Free PMC article

Abstract

The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Generation of the ES cell line inducible system mES-Tbx1TetOFF.
A: top, docking site at the Rosa-Tet locus of the EBRTc cell line [17]; middle: exchange vector (PTHC-Tbx1-myc) carrying a Tbx1 cDNA; bottom: recombinant allele after Cre-induced recombination. B: qRT-PCR analysis of Tbx1 expression in undifferentiated mES-Tbx1TetOFF cells. Values are relative to that at tetracycline removal time (0h) and are the average of three different experiments. Error bar: S.E.M.
Fig 2
Fig 2. Differentiation assay of the mES-Tbx1TetOFF cell line.
A: Scheme of experimental protocol. B: qRT-PCR assay of Tbx1 expression in uninduced (+Tet) mES-Tbx1Tet OFF cells. A peak of expression is evident at day 6. C: Schematic representation of the experimental strategy used for pulse Tbx1 expression. D: qRT-PCR analysis of Tbx1 expression at day 6 after 24hrs without tetracycline. E: Immunofluorescence with an anti-P-H3 antibody on two colonies of mES-Tbx1Tet OFF cells with and without tetracycline at day 6. F: graphic representation of quantitative evaluation of mitotic activity using flow cytometry.
Fig 3
Fig 3. Tbx1 expression pulse at day 5 produces persistent changes of expression of cardiac markers.
A: qRT-PCR assay at Day 6, Day 8 and Day 11 of mES-Tbx1Tet OFF cells with (red) and without (blue) pulse at Day 5. The data are represented as mean of fold change relative to Day 5 value (not shown) of four different experiments. Error bars indicate SEM. (* = p-value<0.05; ** = p-value <0.005; *** = p-value <0.0005). B: Flow cytometric evaluation of the effects of Tbx1 pulse on VEGFR2, NKX2.5 and GATA4 cell population at Day 8. Flow cytometry at Day 8 shows a decrease of the numbers of VEGFR2+ and GATA4+ cells (B, B”) and an increase of the number of NKX2.5+ cells (B’) after Tbx1 pulse. C: The histograms show the mean percentage of positive cells in three different experiments. Error bar indicate SEM. D: Immunofluorescence detection of VEGFR2 with (Tet-) or without (Tet+) Tbx1 pulse. FSC: Forward Scatter.
Fig 4
Fig 4. Expression profile of VEGFR2+ cells isolated at Day 4.75 of differentiation from mES-Tbx1TetOff cells and subjected to additional 48hrs in culture, with or without tetracycline.
A: qRT-PCR assay of Vegfr2 expression of cells bound to magnetic beads (VEGFR2+ cells) and unbound (UN). B: RT-PCR analysis of VEGFR2+ and unbound cells. C: qRT-PCR of Tbx1 expression in VEGFR2+ cells. D,E: qRT-PCR evaluation of Vegfr2 and Smarcd3 expression in VEGFR2+ cells with and without Tet.
Fig 5
Fig 5. Clonal analysis of VEGFR2+ cells.
A: schematic representation of experimental strategy. B: Examples of RT-PCR analysis on VEGFR2+ cells in absence and in presence of Tbx1 pulse. C: Color map panels of marker positivity of individual clones with or without Tbx1 pulse. D: Graphic representation of clone multiplicity, based on the number of positive markers with and without tetracycline. Blue represents the percentage of clones that were positive for all three markers; Red represents the percentage of clones positive for two markers; Green represents the percentage of clones that are positive for one marker.
Fig 6
Fig 6. Vegfr2 expression in wild type and Tbx1 -/- mouse embryos E9.5 and E9.0.
A, A’: In situ hybridization of wild type and Tbx1 -/- embryos. B-C': Immunohistochemisty with an anti-VEGFR2 antibody on sagittal and transverse sections. The arrows indicate the expression domain in the pSHF. The arrowheads indicate the DMP region (n = number of embryos examined). Scale bars: 200 μm in A, A’, B, B’. 50 μm in C, C’. s: number of somites.
Fig 7
Fig 7. Vegfr2 and Tbx1 expression in E8.5 mouse embryos.
A-D': In situ hybridization of Tbx1 (A-D) or Vegfr2 (A'-D') shown in a rostro-caudal series of transverse sections of wild type embryos. Arrows indicate Vegfr2 expression in the pharyngeal mesenchyme. E-F: VEGFR2 immunohistochemistry in similar sections as in A and B. G-H: VEGFR2 immunohistochemistry in corresponding sections of a Tbx1 -/- embryo. Arrows indicate VEGFR2+ cells in the pharyngeal mesenchyme. OFT: cardiac outflow tract. IFT: cardiac inflow tract. Scale bars: 200 μm in A-D, A’-D’. 50 μm in E-H. s: number of somites.

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