Functional Analysis Of TGF - beta Family Signaling

了解胚胎发育过程中T淋巴细胞细胞系的形成机制对防治艾滋病具有重要意义。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而来的尚不清楚。我们关注的是tgf - β超家族成员骨形态发生蛋白（BMPs）在小鼠发育过程中的功能。bmp具有明确中胚层组织腹侧命运的功能。造血细胞来源于最腹侧的中胚层，因此，假设bmp在造血组织中T淋巴细胞谱系的规范中起重要作用。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，我们正在尝试建立全胚体外培养体系。ES71004-02项目中描述的组织特异性基因靶向方法是一种强大的技术，可以解决给定组织中给定基因的功能。然而，它仍然有局限性，这取决于启动子表达Cre重组酶的特异性和强度。这个项目的另一个目标是克服这个限制。其基本策略是利用全胚培养系统培养具有固定等位基因的胚胎，然后在胚胎感兴趣的区域微注射重组腺病毒表达Cre重组酶。通过这种方法，我们可以实现不考虑启动子特异性的区域特异性基因靶向。首先，我们建立了早于5.5天胚胎的培养方法。我们维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，我们培养了从该转基因系中取出的胚胎，并注射了表达腺病毒（Adex-Cre）的Cre。我们在7.5天胚胎的羊膜腔内注射Adex-Cre，进行lacZ染色。然而，表达lacZ的细胞数量非常少。我们正在进行优化实验条件的工作。一旦这个条件成立，我们将应用这种方法神经外胚层特异性破坏受体的bmp。除了区域特异性注射表达Cre的病毒外，我们还将病毒添加到培养基中感染培养胚胎的最外层卵黄囊。卵黄囊是从妊娠第7.5天开始的原始造血器官。有争议的起源确定的造血开始于第9.5天卵黄囊。我们可以通过卵黄囊特异性地破坏Bmpr来解决这个问题。通过这项研究将获得的知识可能使我们更好地了解原始和最终的造血，并可能为预防和治疗艾滋病提供一些见解。

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.