Functional Analysis Of TGF - beta Family Signaling

对于艾滋病的预防和治疗，了解胚胎发育过程中T淋巴细胞的细胞谱系是如何确定的机制是很重要的。T淋巴细胞来源于造血干细胞，但目前尚不清楚造血细胞是如何从未分化的中胚层组织发育而来的。我们正在关注骨形态发生蛋白(BMPs)在小鼠发育过程中的作用，BMPs是转化生长因子-β超家族的成员。BMP更多地在腹侧起决定中胚层组织命运的作用。造血细胞来源于最腹侧的中胚层，因此，BMPs被认为在造血组织中T淋巴细胞谱系的确定中起着重要作用。研究老鼠发育的困难之一是胚胎发生在母亲体内。为了解决这一问题，我们正在尝试建立完整胚胎的体外培养系统。在项目ES71004-02中描述的组织特异性基因打靶方法是解决给定组织中给定基因功能的强大技术。然而，表达Cre重组酶仍有一定的局限性，这取决于启动子的特异性和强度。该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含有等位基因的胚胎，然后在胚胎的感兴趣区微量注射表达Cre重组酶的重组腺病毒。通过这种方法，我们可以实现区域特异性的基因打靶，而不受启动子的特异性影响。首先，我们建立了早在5.5天的胚胎的培养方法。我们维持了一个依赖于DNA重组催化的Cre重组酶来表达LacZ的转基因小鼠系。作为模型系统，我们培养了来自该转基因系的胚胎并注射了表达Cre的腺病毒(Adex-Cre)。我们将Adex-Cre注射到7.5天胚胎的羊膜腔内，进行LacZ染色。然而，表达LacZ的细胞数量很少。我们正在对实验条件进行优化。一旦这一条件确立，我们将应用这种方法对神经外胚层特异性破坏BMPs的受体。除了区域特异性注射表达Cre的病毒外，我们还将在培养基中加入病毒感染培养胚胎的最外层--卵黄囊。卵黄囊是原始造血的器官，从妊娠7.5天开始。到底是从9.5天开始的，还是从卵黄囊开始的，目前还存在争议。我们可以通过以卵黄囊特异的方式干扰Bmpr来解决这个问题。通过这项研究获得的知识可能会让我们更好地了解原始和确定的造血机制，这可能会对艾滋病的预防和治疗有一些启示。

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.