Building the cardiovascular system in early embryos

心血管系统在发育早期形成，以支持胚胎的生长和存活。本项目的目的是确定转录机制，使用心脏发育的鸟类模型的祖细胞规范的早期步骤。这些机制的进化保守性将在两栖动物非洲爪蟾（Xenopus laevis.CRE）中进行评估。与Nkx2.5和Cer1相关的顺式调节元件（CRE）是两个对正常心脏形成至关重要的基因，以前已被鉴定。使用Citrine荧光报告基因的初步实验表明，克雷斯在未来心血管细胞谱系的早期变得活跃，这将有助于心脏和血管。为了确定早期命运决定的上游调节因子，该项目将涉及在体内表征这些克雷斯。Citrine报告基因的电穿孔，随后进行延时成像，将确定激活的时间，并使用已知标志物的共染色来鉴定细胞类型。Wnt和BMP信号通路对心脏发育很重要，将检查它们对CRE活性的影响。基序搜索和足迹分析将确定候选转录因子（TF）结合位点和诱变将确定其功能的重要性。此外，重要的TF位点将通过使用CRISPR方法进行编辑。最后，克雷斯是否在物种间功能保守，将在非洲爪蟾以及转基因胚胎和蝌蚪中使用显微注射进行测试。

The cardiovascular system forms early in development to support growth and survival of the embryo. This project aims to identify transcriptional mechanisms governing early steps of progenitor cell specification using an avian model of heart development. Evolutionary conservation of these mechanisms will be assessed in the amphibian, Xenopus laevis.Cis-regulatory elements (CRE) associated with Nkx2.5 and Cer1, two genes that are essential for normal heart formation, have previously been identified. Pilot experiments, using Citrine-fluorescent reporters, show that the CREs become active early - in prospective cardiovascular cell lineages that will contribute to the heart and vessels.To identify upstream regulators of early fate decisions, the project will involve characterizing these CREs in vivo. Electroporation of Citrine-reporters followed by time-lapse imaging will determine the timing of activation and cell types will be identified using co-staining with known markers. The Wnt and BMP signaling pathways are important for cardiac development and their effect on CRE activity will be examined. Motif search and footprint analysis will identify candidate transcription factor (TF) binding sites and mutagenesis will determine their functional importance. Furthermore, important TF sites will be edited by using CRISPR approaches. Finally, whether CREs are functionally conserved across species will be tested using microinjections in Xenopus as well as transgenic embryos and tadpoles.