Functional Analysis Of TGF - beta Family Signaling

了解T淋巴细胞在胚胎发育过程中的细胞谱系决定机制，对于艾滋病的防治具有重要意义。T淋巴细胞来源于造血干细胞，然而，造血细胞是如何从未分化的中胚层组织中发育出来的仍然不清楚。本课题主要研究TGF-β超家族成员骨形态发生蛋白（BMPs）在小鼠发育过程中的作用。骨形成蛋白的功能，以指定的中胚层组织的命运更腹。造血细胞来源于最腹侧的中胚层，因此，BMPs被推测在造血组织中T淋巴细胞谱系的特化过程中发挥重要作用。研究小鼠发育的困难之一是胚胎发生在母体内部。为了克服这一问题，我们正在尝试建立完整胚胎的体外培养体系。项目ES 71004 -02中描述的组织特异性基因靶向方法是一种解决给定组织中给定基因功能的强大技术。然而，它仍然具有依赖于启动子表达Cre重组酶的特异性和强度的局限性。本项目的另一个目标是克服这一限制。其基本策略是利用全胚胎培养系统培养具有floxed等位基因的胚胎，然后显微注射重组腺病毒，使Cre重组酶在胚胎的感兴趣区域表达。通过这种方法，我们可以实现区域特异性的基因打靶，而不管启动子的特异性。首先，我们建立了早在5.5天胚胎的培养方法。我们保持了一个转基因小鼠系，表达lacZ依赖于DNA重组催化的Cre重组酶。作为模型系统，我们培养从该转基因系中取出的胚胎并注射表达Cre的腺病毒（Adex-Cre）。将Adex-Cre注入7.5天胚胎羊膜腔内进行lacZ染色。然而，表达lacZ的细胞群体非常低。我们正在优化实验条件。一旦建立了这种条件，我们将应用这种方法对BMP受体进行神经外胚层特异性破坏。除了Cre表达病毒的区域特异性注射之外，我们将病毒添加到培养基中以感染卵黄囊，即培养胚胎的最外层。卵黄囊是原始造血器官，从妊娠7.5天开始。有争议的是，确定性造血的起源开始于第9.5天，是来自卵黄囊。我们可以解决这个问题的中断BMPR在卵黄囊特异性的方式。通过本研究所获得的知识将使我们更好地了解原始和最终的造血，并可能导致艾滋病的预防和治疗的一些见解。

For prevention and cure of AIDS, it is important to know the mechanism of how cell lineage for T lymphocyte is determined during embryonic development. T lymphocytes are derived from hematopoietic stem cells, however, it is still unclear how hematopoietic cells are developed from undifferentiated mesodermal tissues. We are focusing on the function of Bone Morphogenetic Proteins (BMPs), the members of TGF-beta superfamily, during mouse development. BMPs have function to specify the fate of mesodermal tissues more ventral. Hematopoietic cells are derived from the most ventral mesoderm, therefore, BMPs are hypothesized to play important roles during specification of T lymphocyte lineage in the hematopoietic tissues. One of the difficulties in studying mouse development is that embryogenesis occurs inside of the mother. To overcome this problem, we are trying to establish in vitro culture system for whole embryos. The tissue-specific gene targeting method described in Project ES71004-02 is a powerful technique to address the function of the given genes in given tissues. However, it still has limitations dependent upon the specificity and strength of promoters to express Cre recombinase. Another goal of this project is to overcome this limitation. The basic strategy is to culture embryos that have floxed alleles using whole embryo culture system, and then to microinject recombinant Adenovirus to express Cre recombinase in the interested region of embryos. Through this method, we could achieve region specific gene targeting regardless of the specificity of promoters. First, we established the culture method for the embryos as early as day 5.5 embryos. We maintain a transgenic mouse lines that expresses lacZ depending on the DNA recombination catalyzed Cre recombinase. As a model system, we culture embryos taken from this transgenic line and inject Cre expressing Adenovirus (Adex-Cre). We injected Adex-Cre into the amniotic cavity of day 7.5 embryos to get lacZ staining. However, population of the cells that expressed lacZ was very low. We are underway to optimize the experimental condition. Once this condition is established, we will apply this method for neuroectoderm specific disruption of receptors for BMPs. In addition to the region specific injection of the Cre expressing virus, we will add the virus to the culture media to infect the yolk sac, the most outer layer of cultured embryos. The yolk sac is the organ for primitive hematopoiesis, starting at day 7.5 of gestation. There is debate whether the origin of definitive hematopoiesis starts at day 9.5 is derived from the yolk sac. We could address this question by the disruption of Bmpr in yolk sac-specific manner. The knowledge that will be acquired through this research may give us better understanding for the primitive and definitive hematopoiesis and that may lead some insights for prevention and cure of AIDS.