Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.
Bottom Line: Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear.With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch.Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2.
Affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, United States The Knight Cardiovascular Institute, Oregon Health & Science Universtiy, Portland, United States Institute for Cell Engineering, Johns Hopkins University, Baltimore, United States.Show MeSH
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Mentions: To directly test the potential of RFP+ cells in the PA2, we labeled proliferating cells with EdU for 2 hr at E9.0, dissected out PA2s from embryos (asterisks, Figure 3D), and cultured the arches in 3D Matrigel culture. RFP+ cells were monitored in real-time with fluorescent time-lapse movies. After 2 days, the cluster of RFP+ cells expanded and exhibited multidirectional migration (Figure 3—figure supplement 2). The migrated RFP+ cells became contractile by day 4 (Figure 3—figure supplement 2; Videos 1 and 2). Although the range of their migration was somewhat limited, likely due to the extracellular environment of Matrigel, RFP+ cells continued to expand in the PA2 and differentiated into cardiomyocytes. When cultured in an uncoated dish, RFP+ cells expanded in the PA2 and progressively migrated out of the arch in a unidirectional fashion soon after being attached to the surface. However, no beating activity was observed on day 2 (Figure 3F). They migrated out of the arch progressively soon after being attached to the surface (Figure 3F). The migrated RFP+ cells started to spontaneously contract by day 4 (Figure 3F´). The expansion and migration of RFP+ cells appear to continue over 2 weeks and the beating area was correspondingly expanded (Figure 3F″; Video 3). The beating cells were positive for EdU, Nkx2.5, and cTnT/α-Actinin and exhibited a periodic Ca2+ oscillation pattern similar to that of adult cardiomyocytes (Figure 3G–I). To ascertain that the resulting cardiac cells were not derived from contamination from the adjacent OT, we isolated PAs from Nkx2.5GFP embryos (Biben et al., 2000) that express GFP in cardiac cells including the OT, but not in the PAs. Freshly dissected PA2s did not contain GFP+ cells, but cells expressed GFP 2–3 days after migration from the arch (Figure 3—figure supplement 3). We concluded that RFP+ Isl1+ cells expand in the PA2 and differentiate into cardiac cells as they migrate out of the arch. It is worth noting that PA1 cells also migrated out from the arch, but they remained GFP–and eventually formed myotube-like structures (Figure 3—figure supplement 3, Figure 3J). This is consistent with the previous finding that pharyngeal mesoderm is also the source of head skeletal muscles (Nathan et al., 2008).Video 1.3D-cultured PA2 in Matrigel.
Affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, United States The Knight Cardiovascular Institute, Oregon Health & Science Universtiy, Portland, United States Institute for Cell Engineering, Johns Hopkins University, Baltimore, United States.