Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate - PubMed (original) (raw)

. 2010 Sep;137(18):3001-11.

doi: 10.1242/dev.051565. Epub 2010 Aug 11.

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Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate

Mary Redmond Hutson et al. Development. 2010 Sep.

Abstract

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

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Figures

Fig. 1.

Fig. 1.

Clonal analysis of the secondary heart field. (A-C) Secondary heart field progenitors differentiate into beating myocardium (red), smooth muscle (elongated green) and endothelial cells (rounded green) after 96 hours in culture. B and C are enlarged regions from A. (D-K) Clonal analysis shows the that SHF contains a multipotent progenitor cell that is capable of differentiating into myocardium (D,G,J,K), smooth muscle (E,H-K) and endothelial cells (F,I,K). Tripotency is demonstrated in K with a clone expressing multiple cardiac cell lineage markers. Blue labels the nuclei in all the cells and the nuclei plus the cytoplasm in the MF20-positive cell (blue arrowhead). Arrows indicate nuclei expressing no differentiation markers.

Fig. 2.

Fig. 2.

Identification of stem cells in the SHF. (A) Cells doubly positive for Isl1 and Nkx2.5 (white arrowhead) represent a myocardial and smooth muscle lineage. (B) Cells doubly positive for Isl1 and Flk1 (white arrowhead) represent a smooth muscle and endothelial cell lineage. Cultures are a mixed population, with some cells expressing only one marker (green arrowhead) or no marker (blue asterisk). (C-E) Asymmetrical localization of Numb (green, white arrows) in SHF cells suggests existence of a stem cell population [C,D are also stained for transitin, a type IV intermediate filament protein that has been shown to co-localize with Numb (Wakamatsu et al., 2007)]. (D) Cell with condensed chromatin and asymmetric localization of Numb. (E) Asymmetric localization of Numb in Isl1-expressing cells.

Fig. 3.

Fig. 3.

SHF explants cultured for 24 hours with FGF8 or BMP2, alone or in combination, and analyzed for proliferation (pHH3) or myocardial differentiation (MF20). (A) Low concentrations of FGF8 (2.5-5.0 ng/ml) significantly increased SHF proliferation (*_P_≤0.003 compared with control). SU5402 treatment significantly decreased proliferation when compared with controls (*_P_=0.01) and 2.5 ng and 5.0 ng FGF8-treated groups (**_P_≤0.008). (B) FGF8 treatment decreased myocardial differentiation with the 5 ng/ml dose (*_P_=0.004). SU5402 significantly increased differentiation when compared with controls (*_P_=0.014) and all FGF8 treatment groups (**_P_≤0.03). (C) Increasing concentrations of BMP decreased proliferation compared with controls (*_P_≤0.0001). This effect was negated by Noggin exposure (_n_=8 for each treatment group). (D) Increasing concentrations of BMP increased myocardial differentiation compared with control (*_P_≤0.004). This effect was inhibited by Noggin (**P<0.0001). Each increase in BMP2 dose significantly increased differentiation (*_P_≤0.02) (_n_=8 for each treatment group). (E) FGF8 (2.5 ng/ml) plus 25 ng/ml BMP2 treatment increased proliferation compared with controls (*_P_=0.003). Treatment with low dose BMP2 (25 ng/ml) and high FGF8 (5 ng/ml) dose significantly increased proliferation compared with control (*_P_=0.0001). Proliferation levels were significantly reduced when explants were cultured with 5 ng/ml FGF8 and 300 ng/ml BMP2 (*_P_=0.0001) (_n_=12 for each treatment group). (F) 25 ng/ml BMP2 plus 2.5 ng/ml FGF8 treatment did not increase differentiation compared with controls and FGF8. Myocardial differentiation was significantly decreased in explants grown with 5 ng/ml FGF8 and 25 ng/ml BMP2 (*_P_=0.0001) Myocardial differentiation was significantly increased in explants grown in 300 ng/ml BMP and 5 ng/ml FGF8 (*_P_=0.003) compared with controls (_n_=12 for each treatment group).

Fig. 4.

Fig. 4.

Dissecting the role of the FGF intracellular signaling pathways in SHF explants grown for 24 hours. (A,B) Inhibiting of the Ras/Erk pathway using the MEK inhibitor had no effect on proliferation (A) but significantly increased myocardial differentiation (B) compared with controls (B,*_P_=0.03). Inhibition of the AKT pathway with the PI3K inhibitor decreased proliferation (A,*_P_=0.001) but had no effect on differentiation. Inhibition of the PKC pathway with the PLCγ inhibitor decreased proliferation (A, *_P_=0.001) but had no effect on differentiation. Blocking the FGF signaling with SU5402 decreased proliferation (A, *_P_=0.003) in the same way as the PI3K and PLC-γ inhibitors, and increased differentiation (B; *_P_=0.02) in the same way as the MEK inhibitor. (C,D) Treatment with low dose BMP2 (25 ng/ml) plus the MEK inhibitor did not affect proliferation. Combining BMP2 and the Mek inhibitor significantly increased myocardial differentiation compared with controls (*_P_=0.001) but was not significantly increased compared with BMP alone.

Fig. 5.

Fig. 5.

FGF8 promotes smooth muscle differentiation after 48 hours in culture. (A-D) SHF explants grown for 48 hours and stained for MF20 (red) and SMLK (green). (A) Control culture grown in 2% FBS. (B) SHF cultures grown with 10 μg/ml of FGF8 showing increased smooth muscle differentiation (green) compared with control. (C) SHF grown for 48 hours with Mek inhibitor. (D) Forty-eight-hour culture grown with BMP2. Arrows indicate differentiating smooth muscle cells. (E) FGF8-treated SHF explants had significantly more smooth muscle (green) compared with control or BMP2-treated cultures (*_P_≤0.05). FGF8-treated explants (5 ng/ml) had significantly less myocardium than did controls (**_P_=0.04) or BMP2-treated cultures (**_P_=0.001). After 48 hours, nearly all of the BMP2-treated culture differentiated into myocardium (#).

Fig. 6.

Fig. 6.

Inhibition of Ras/Erk and PLCγ pathways disrupts arterial pole alignment_._ (A-C) Inhibitor treatment disrupted heart looping. Left side view of HH16 embryos after 24 hour exposure to either vehicle control (A), 10 μM of MEK inhibitor (B) or 10 μM of the PLCγ inhibitor (C). Abnormal heart looping was observed in both inhibitor treatment groups. Normally, the outflow limb (white arrows) is partially obscured by the inflow limb (black arrows). (D-F) Arterial pole alignment in heart from day 9 embryos was disrupted after inhibitor treatment. (D) Control heart showed normal alignment (arrows) of the aorta (ao) and pulmonary trunk (p). Hearts from MEK inhibitor-treated (E) and PLCγ inhibitor-treated (F) embryos had ‘side-by-side’ vessels (arrows), indicating overriding aorta or DORV. (G-I) Cross-sections through hearts in D-F and stained with Hematoxylin and Eosin. The aorta was in a side-by-side orientation with the pulmonary trunk in the MEK inhibitor-treated (H) and the PLCγ inhibitor-treated (I) animals. (J-L) Hearts from day 9 embryos after neural crest ablation alone (J) or neural crest ablation (NCA) followed by treatment with the MEK inhibitor (K) or PLCγ inhibitor (L). All of the hearts from the NCA embryos had a common outlet (PTA).

Fig. 7.

Fig. 7.

MEK and PLCγ inhibitors disrupt development of the SHF. (A) Inhibition of Ras/Erk signaling with the Mek inhibitor caused premature myocardial differentiation in the SHF. A HH16 embryo, sagittally sectioned through outflow tract and SHF, was immunostained for MF20 (red) to mark myocardium and Hoechst to label the nuclei. The MF20 was ectopically expressed in the SHF mesoderm (arrows) that is continuous with the myocardial rim (asterisk) of the outflow tract. (B) Graph showing the number of proliferating cells in the SHF of embryos treated with the PLCγ and MEK inhibitors compared with control embryos. Only inhibition of the PLCγ pathway significantly decreased SHF proliferation (*_P_=0.03). (C,D) Mek inhibitor treatment disrupted development of the coronary stems. Cross-section through the base of aorta at the left coronary stem insertion from a control embryo (C) and an embryo treated with the MEK inhibitor (D) and stained with SM22. Arrow in C shows normal single coronary stem. Arrows in D show the abnormal and multiple small vessels traversing the aortic wall.

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References

    1. Abu-Issa R., Kirby M. L. (2007). Heart field: from mesoderm to heart tube. Annu. Rev. Cell Dev. Biol. 23, 45-68 - PubMed
    1. Abu-Issa R., Smyth G., Smoak I., Yamamura K.-I., Meyers E. (2002). Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129, 4613-4625 - PubMed
    1. Barron M., Gao M., Lough J. (2000). Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev. Dyn. 218, 383-393 - PubMed
    1. Brown C. B., Wenning J. M., Lu M. M., Epstein D. J., Meyers E. N., Epstein J. A. (2004). Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse. Dev. Biol. 267, 190-202 - PubMed
    1. Cai C. L., Liang X., Shi Y., Chu P. H., Pfaff S. L., Chen J., Evans S. (2003). Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877-889 - PMC - PubMed

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