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。该项目旨在利用心脏发育的禽类模型确定控制祖细胞指定的早期步骤的转录机制。这些机制的进化保守性将在两栖动物非洲爪哇中进行评估。与NKX2.5和Cer1相关的顺式调节元件(CRE)是正常心脏形成所必需的两个基因，此前已被鉴定。使用Citine荧光记者的初步实验表明，Cres在预期的心血管细胞谱系中早期变得活跃，这将对心脏和血管做出贡献。为了识别早期命运决定的上游调节因素，该项目将涉及在体内表征这些Cres。电穿孔的柑橘碱-记者，随后的时间推移成像将确定激活的时间和细胞类型将使用共染色与已知的标志物。Wnt和BMP信号通路对心脏发育很重要，我们将研究它们对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，突变将决定它们的功能重要性。此外，重要的信托基金网站将使用CRISPR方法进行编辑。最后，将通过在非洲爪哇以及转基因胚胎和蝌蚪中进行微量注射来测试Cres是否在功能上跨物种保守。

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.