ACTIVATION OF HEMATOPOIESIS IN THE MOUSE EMBRYO

哺乳动物胚胎卵黄囊中的造血和血管发生是在原肠胚形成期间开始的过程，并且首先需要中胚层的诱导。 当胚外中胚层被诱导分化时，第一批血液和血管内皮细胞形成。 然而，对胚胎发育过程中参与这些过程的分子知之甚少。 为了研究上皮-间充质相互作用在小鼠卵黄囊造血和血管发生中发挥重要作用的可能性，我们设计了一种新的转基因胚胎外植体培养系统。 将在血液形成之前收获的转基因胚胎剥离其周围的原始内胚层（上皮），并单独或与分离的内胚层组织一起在胶原液滴培养物中生长。 使用这个系统，我们已经证明，原始（内脏）内胚层信号是必不可少的原始造血和胚胎血管发生的激活。 这些信号是短程的、可扩散的和阶段依赖性的，在原肠胚形成后期变得不那么有效，最终检测不到。 值得注意的是，原始内胚层信号传导可以重新指定组织中的造血和血管发生，这些组织注定不会形成血液或血管细胞，基本上使A-P轴复极化。 这个建议的目标是阐明内胚层信号的来源和分子后果，并确定一些参与这些关键发育过程的分子。 在第一个目标的建议，我们将问是否重新指定的前外胚层（注定形成neurectoderm）涉及诱导中胚层第一和激活造血和血管生成第二。 我们将确定在发育后期如何通过原始内胚层信号实现前外胚层的复极化，以及非造血组织响应原始内胚层信号的能力有多普遍。 在第二个目标的建议，我们将使用外植体培养测定开始，以确定内胚层信号分子参与诱导造血和血管生长。 最后，我们将集中在造血发育外植体问干/祖细胞是否内胚层信号的目标，以及是否确定造血细胞在这些文化的形式。这些研究可能不仅对我们理解正常发育具有重要意义，而且可能为起源于干/祖细胞水平的人类疾病的机制提供见解。 他们还可能为造血和血管疾病提出新的和更有效的基于干细胞的治疗方法。

Hematopoiesis and vasculogenesis in the yolk sac of the mammalian embryo are processes that begin during gastrulation and first require the induction of mesoderm. The first blood and vascular endothelial cells form when the extraembryonic mesoderm is induced to differentiate. However, little is known about the molecules involved in these processes during embryonic development. To examine the possibility that epithelial- mesenchymal interactions play an important role in yolk sac hematopoiesis and vasculogenesis in the mouse, we devised a novel transgenic embryo explant culture system. Transgenic embryos harvested prior to the formation of blood are stripped of their surrounding primitive endoderm (epithelium) and grown in collagen drop cultures alone or together with the isolated endoderm tissue. Using this system we have demonstrated that primitive (visceral) endoderm signaling is essential for activation of primitive hematopoiesis and embryonic vasculogenesis. These signals are short-range and diffusible and stage-dependent, becoming less potent around late gastrulation and eventually undetectable. Remarkably, primitive endoderm signaling can respecify hematopoiesis and vasculogenesis in tissue that is not fated to form blood or vascular cells, essentially repolarizing the A-P axis. The goal of this proposal is to elucidate the sources and molecular consequences of endodermal signaling and to identify some of the molecules involved in these critical developmental processes. In the first aim of the proposal, we will ask whether respecification of anterior ectoderm (fated to form neurectoderm) involves the induction of mesoderm first and activation of hematopoiesis and vasculogenesis second. We will determine how late in development repolarization of anterior ectoderm by primitive endoderm signals can still be achieved and how general is the ability of non-hematopoietic tissue to respond to primitive endoderm signaling. In the second aim of the proposal we will use the explant culture assay to begin to identify endodermal signaling molecules involved in induction of hematopoiesis and vascular growth. Finally, we will focus on hematopoietic development in explants by asking whether stem/progenitor cells are targets of endodermal signaling and whether definitive hematopoietic cells form in these cultures. These studies may have important implications not only for our understanding of normal development but may provide insights into mechanisms underlying human diseases originating at the level of stem/progenitor cells. They may also suggest new and more effective stem cell-based therapeutic approaches for hematopoietic and vascular diseases.