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. Author manuscript; available in PMC: 2021 Jun 9.
Published in final edited form as: Nature. 2020 Dec 9;588(7839):705–711. doi: 10.1038/s41586-020-2998-x

Figure 2. Lymphatics are required for CM proliferation and survival.

Figure 2.

a, H&E staining shows no obvious defects in cardiac valves (arrows) or ventricular wall compaction in E17.5 Prox1ΔLEC/ΔLEC hearts (TAM injected at E13.5 and E14.5). N = 4 embryos/genotype. b, α-Laminin staining shows no differences in Prox1+ CM size between E17.5 controls and Prox1ΔLEC/ΔLEC hearts. Right panel shows quantification of Prox1+ CM size (α-Laminin+ area). Average cell size was measured from five fields/ventricle, 8–10 Prox1+ CMs/field, 3 embryos per genotype; N = 152 (control) and 155 (Prox1ΔLEC/ΔLEC). c-f, Immunostaining with proliferation markers (EdU, pH3, Ki67 and AuroraB) together with CM markers (cardiac Troponin C [cTnC], Prox1, αActinin and/or Mef2c). In all images, arrows indicate the double positive CMs selected for counting. N = 4 embryos/genotype from 3 separate litters. g, Quantification of the immunostaining in c-f shows reduced number of EdU+, Ki67+, AuroraB+ and pH3+ CMs in E17.5 Prox1ΔLEC/ΔLEC hearts. N = 4 embryos/genotype from 3 separate litters. **p=0.003 (EdU, Ki67 and AuroraB), **p=0.002 (pH3). h, Active Caspase-3 immunostaining shows increased CM apoptosis in Prox1+ CMs in E17.5 Prox1ΔLEC/ΔLEC hearts. Arrows indicate apoptotic CMs. N = 4 embryos/genotype from 3 separate litters. ***p=0.0003. Control embryos are TAM treated Cre− and Cre+;Prox1+/+ littermates. Data are presented as mean ± S.E.M. p values were calculated by unpaired two-tailed Student’s t test. n.s, not significant. Scale bars, 1 mm (a), 25 μm (b, c-f, h). Lower magnification of panels c-e and h are included in Supplementary Fig 1.