Induction and Specification of Hematopoietic Mesoderm

这是一个修订的竞争性更新的赠款，研究激活小鼠胚胎造血。在之前的资助期间，我们使用了一种新的转基因胚胎外植体培养系统，以表明上皮-间充质相互作用在小鼠卵黄囊造血和血管发育中起着重要作用。来自内脏内胚层的可扩散信号介导这些相互作用，并且可以重新编程组织（前上胚层）中的造血，该组织注定不能形成血细胞。我们确定了两个造血诱导VE信号，印度刺猬（Ihh）和骨形态发生蛋白（BMP）-2，并发现他们上调BMP 4在外植体培养。在青蛙中，成对型同源结构域转录因子XMix.1由BMP 4诱导。它在整个胚胎中的异位表达将背侧中胚层转化为腹侧命运，导致大量血细胞的形成。我们克隆了非洲爪蟾和斑马鱼Mix/Bix基因家族的小鼠亲属（mMix或Mixl），并产生了许多独特的遗传模型，用于分析其在发育过程中的功能。单个小鼠Mix基因在原肠胚形成之前在后部VE中表达，随后在原肠胚形成胚胎的原条和新生中胚层中表达。尽管先前在小鼠胚胎中的研究已经指出mMix在原肠胚形成中的关键作用，但其在中胚层衍生物发育中的功能仍不清楚。在分化的胚胎干（ES）细胞中已经鉴定出造血缺陷，其中mMix被遗传灭活。我们最近发现，在ES细胞衍生的胚状体中条件诱导mMix导致中胚层发育程序的加速。从这项工作中出现的一个主要发现是，中胚层，成血管细胞和造血祖细胞的数量增加的形式响应于mMix的过早激活。我们推测，小鼠MX功能的早期招募和/或扩大中胚层祖细胞的血管母细胞和造血谱系。在本申请中，我们将：（1）鉴定mMix可调节造血干/祖细胞形成时中胚层发育程序中的关键点;（2）评估mMix表达细胞的发育潜力并更好地确定造血谱系的中胚层祖细胞的表面抗原;（3）检查在体内造血谱系的中胚层祖细胞中缺失mMix功能的后果。中胚层干/祖细胞群体的表征和阐明胚胎与成人造血和血管发育的共同和区别特征将具有根本的重要性，并且还可能提高我们调节干细胞自我更新/分化以用于治疗目的的能力。

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.