Detection of BMP Signaling In Pre-Implantation Mouse Embryos

描述（由申请人提供）：哺乳动物的发育始于受精，随后是多个细胞分裂事件和从母体转录到合子转录的过渡。所有这些事件协同工作，建立第一个，因此非常重要的发展事件，谱系分化。在植入前胚胎中建立不同谱系的第一步产生滋养外胚层（TE），其是胚外结构如胎盘和卵黄囊的前体，以及内细胞团（ICM），其成为胚胎本身。虽然转录因子如Oct 3/4、Nanog、Sox 2、Tead 4和Cdx 2的调控相互作用和形态学贡献已被广泛研究，但令人惊讶的是，关于分泌的生长因子在细胞生长过程中的作用知之甚少。 这些早期的发展阶段。骨形态发生蛋白（BMP）在这些早期小鼠胚胎阶段的存在表明可能的功能作用，BMP信号在谱系分化。本项目将通过阐明BMP信号的时空出现和功能作用来确定BMP信号在植入前阶段胚胎中的意义。此外，该项目旨在确定BMP和谱系特异性转录因子之间的分子相互作用，这对第一谱系分化至关重要。通过高灵敏度的图像分割定量方法，磷酸化Smad 1/5/8的免疫荧光检测和定量将指示植入前胚胎中活跃的BMP信号传导。功能丧失实验将用于确定BMP信号传导活性是否影响在胚泡形成期间控制谱系特化的两个主要转录网络：Oct 4/Nanog/Sox 2的多能调节基序和Tead 4、Cdx 2和Gata 6的TE提交网络。BMP信号在植入前哺乳动物胚胎发育中的生物学作用的直接证据为理解转录调节因子和信号形态发生素之间的新型调节相互作用提供了基础。这种理解有利于多个健康相关学科，如干细胞治疗学，癌症生物学和生殖医学。

DESCRIPTION (provided by applicant): Mammalian development begins with fertilization, followed by multiple cell cleavage events and the transition from maternal to zygotic transcription. All these events work in concert to establish the first and thus highly significant developmental event, lineage differentiation. The first step in the establishment of different lineages in the pre-implantation embryo gives rise to the trophectoderm (TE), the precursor for extraembryonic structures such as the placenta and yolk sac, and the inner cell mass (ICM), which becomes the embryo proper. While the regulatory interactions and morphological contributions of transcription factors such as Oct3/4, Nanog, Sox2, Tead4, and Cdx2 have been extensively studied, surprisingly little is known about the roles of secreted growth factors during these early stages of development. The presence of bone morphogenetic proteins (BMPs) in these early mouse embryonic stages suggests likely functional roles for BMP signaling in lineage differentiation. This project will determine the significance of BMP signaling in pre- implantation stage embryos by elucidating the spatiotemporal emergence and functional role of BMP signaling. Also, this projects aims to identify molecular interactions between BMPs and lineage specification transcription factors vital for the first lineage differentiation. Through a highly sensitive image segmentation quantification method, immunofluorescence detection and quantification of phosphorylated Smad1/5/8 will denote active BMP signaling in pre-implantation embryos. Loss of function experiments will be used to determine if BMP signaling activity impinges on two major transcriptional networks that control lineage specification during blastulation: the pluripotent regulatory motifs of Oct4/Nanog/Sox2 and the TE committing networks of Tead4, Cdx2, and Gata6. Direct evidence for the biological role of BMP signaling in pre-implantation mammalian embryonic development provides a foundation for understanding novel regulatory interactions between transcriptional regulators and signaling morphogens. This understanding benefits multiple health related disciplines such as stem cell therapeutics, cancer biology, and reproductive medicine.