Induction and Specification of Hematopoietic Mesoderm

这是对一项研究小鼠胚胎造血激活的资助的竞争性更新的修订。在之前的资助期间，我们使用了一种新型的转基因胚胎外植体培养系统来证明上皮-间充质相互作用在小鼠卵黄囊的造血和血管发育中起着重要的作用。来自内脏内胚层的可扩散信号介导了这些相互作用，并可以在注定不会形成血细胞的组织(前上胚层)中重新编程造血。我们鉴定了两个造血诱导的VE信号，印度刺猬(IHH)和骨形态发生蛋白(BMP)-2，并发现它们在外植体培养中上调Bmp4。在青蛙中，成对的同源结构域转录因子XMix.1是由BMP4诱导的。它在整个胚胎中的异位表达使背侧中胚层转变为腹侧命运，导致大量血细胞的形成。我们克隆了非洲爪哇和斑马鱼Mix/Bix基因家族的一个小鼠亲属(MMIX或Mix1)，并建立了一些独特的遗传模型来分析其在发育过程中的功能。单一的小鼠混合基因在原肠胚胎的后部VE中表达，之后在原肠胚胎的原始条纹和新生中胚层中表达。尽管之前在小鼠胚胎中的研究已经指出MMIX在原肠形成中起着关键作用，但它在中胚层衍生物发育中的作用仍然不清楚。在MMIX基因失活的分化胚胎干细胞(ES)中，已发现存在造血细胞缺陷。我们最近发现，在ES细胞来源的类胚体中有条件地诱导MMIX导致中胚层发育程序的加速。这项工作的一个主要发现是，中胚层、血管母细胞和造血祖细胞的数量增加是对MMIX过早激活的反应。我们假设小鼠MX在中胚层祖细胞向血管母细胞和造血祖细胞的募集和/或扩展过程中起早期作用。在这一应用中，我们将：(1)确定中胚层发育计划中MMIX调控造血干/祖细胞形成的关键点；(2)评估表达MMIX的细胞的发育潜力，并更好地定义中胚祖细胞的表面抗原；(3)检测缺失中胚祖细胞的MMIX功能对体内造血祖细胞的影响。中胚层干细胞/祖细胞群体的特征和阐明胚胎和成人造血和血管发育的共同和区别特征将是至关重要的，并可能提高我们为治疗目的调节干细胞自我更新/分化的能力。

This is a revised competitive renewal of a grant to study activation of hematopoiesis in the mouse embryo. During the previous funding period, we used a novel transgenic embryo explant culture system to show that epithelial-mesenchymal interactions play an important role in yolk sac hematopoiesis and vascular development in the mouse. Diffusible signals from visceral endoderm mediate these interactions and can reprogram hematopoiesis in a tissue (anterior epiblast) that is not fated to form blood cells. We identified two hematopoietic-inducing VE signals, Indian hedgehog (Ihh) and Bone Morphogenetic Protein (BMP)-2, and found that they upregulate Bmp4 in explant culture. In the frog, the paired-type homeodomain transcription factor XMix.1 is induced by BMP4. Its ectopic expression in whole embryos transforms dorsal mesoderm to a ventral fate, resulting in formation of large numbers of blood cells. We cloned a mouse relative (mMix or Mixl) of the Xenopus and zebrafish Mix/Bix gene family and have generated a number of unique genetic models for analysis of its function during development. The single mouse Mix gene is expressed in the posterior VE prior to gastrulation and later in the primitive streak and nascent mesoderm in the gastrulating embryo. Although previous studies in the mouse embryo have pointed to a critical role for mMix in gastrulation, its function in the development of mesodermal derivatives remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem (ES) cells in which mMix was genetically inactivated. We have recently discovered that conditional induction of mMix in ES cell-derived embryoid bodies results in acceleration of the mesodermal developmental program. A major finding to emerge from this work is that increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. We hypothesize that mouse MX functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages. In this application, we will: (1) identify the critical points in the mesoderm developmental program when mMix can regulate formation of hematopoietic stem/progenitor cells; (2) assess the developmental potentials of mMix- expressing cells and better define the surface antigens of the mesodermal progenitors for the hematopoietic lineage; (3) examine the consequences of deleting mMix function in mesodermal progenitors for the hematopoietic lineage in vivo. Characterization of mesodermal stem/progenitor cell populations and elucidation of the common as well as the distinguishing features of embryonic versus adult hematopoietic and vascular development will be of fundamental importance and may also advance our ability to modulate the self-renewal/differentiation of stem cells for therapeutic purposes.