INTEGRIN RECEPTORS FOR EXTRACELLULAR MATRIX IN PERI-IMPLANTATION DEVELOPMENT

细胞外基质（ECM）受体在调节 发育过程中的形态发生。 整合素超家族 异二聚体跨膜受体包括ECM的两个家族 受体：β 1和α V整合素。 这些受体具有 多个重叠的配体偏好，表明功能性的 冗余或精确的特异性。 在目前的资助期内，我们 已经表明植入前小鼠胚胎表达至少两种α V， - 和两个β 1相关的整合素。 这两个家族的其他成员都在β- 着床期：到第7.5天，例如，滋养层（TB）来源的 外胎盘锥和次级巨细胞表达至少7个α V 和β 1整联蛋白复合物。 α V整合素存在于 在孵化的囊胚外，在一个位置上发挥作用， 初始植入，而α V和β 1整联蛋白都是 在胚胎的其他区域检测到。 我们消除了β 1整合素 在小鼠胚胎中的基因打靶。 β 1缺失胚胎形成a 正常的胚泡并开始着床 然而，他们死在 植入后早期。 Beta1-null的形态发生分析 体外胚胎研究提出了几种假设：a）α V和β 1 整联蛋白在TB和ICM分化中发挥独特的作用， 形态发生 B）β 1整联蛋白为细胞提供存活信号， ICM，导致ICM在围着床期死亡。 c）、 结核病最初并不依赖β 1生存，但未能发展 此外，在初始植入后，由于缺乏来自 ICM。 d）细胞外配体结合和细胞质信号 β 1整联蛋白亚基的转导结构域是 恢复正常的胚胎发育。 来验证这些假设 我们将：1）确定特定的alphaV与Beta1的功能 整合素在结核病形态发生中的体外和体内正常和β 1- 无效胚胎 2)确定β 1整合素在ICM中的功能 分化 实验将评估β 1整联蛋白在 ICM存活和分化为胚外内胚层， 原始胚胎外胚层谱系。 3)确定最大 体内β 1缺失TB和ICM谱系的分化能力， 在体外，使用含β 1嵌合体和无β 1嵌合体的胚胎，以及 β 1-null胚胎干细胞 4)确定在多大程度上生存和 通过重建β 1-null， 具有β 1亚基的胚胎，其中含有截短或改变的β 1 胞质结构域 这种方法应该恢复β 1-整联蛋白配体 结合并延长胚胎存活，但揭示了Beta1的独特特征 影响形态发生、组织特异性 分化和/或生长控制。 β 1缺失胚胎分析 形态发生将涉及与项目III的广泛互动， 六. 我们将扩展整合素表达的研究， 与项目II合作， 和VI. 这些研究应该可以增加对正常的理解。 围着床期发育，并显示细胞-ECM相互作用 有助于形成胚外谱系， 在怀孕期间成功的胎儿和母亲的沟通。

Extracellular matrix (ECM) receptors play critical roles in regulating morphogenesis during development. The integrin superfamily of heterodimeric transmembrane receptors includes two families of ECM receptors: the Beta1 and alphaV integrins. These receptors have multiple, overlapping ligand preferences, suggesting either functional redundancy, or exquisite specificity. In the present grant period, we have shown that preimplantation mouse embryos express at least two alphaV - and two Beta1-associated integrins from the outset of development. Additional members of both families are upregulated in the peri- implantation period: by d7.5, for example, the trophoblast (TB)-derived ectoplacental cone and secondary giant cells express at least 7 alphaV and Beta1 integrin complexes. alphaV integrins are present on the outside of the hatched blastocyst, in a position to play a role in initial implantation, whereas both alphaV and Beta1 integrins are detected in other areas of the embryo. We eliminated the Beta1 integrin family in mouse embryos by gene targeting. Beta1-null embryos form a normal blastocyst, and initiate implantation. However, they die in the early postimplantation period. Analysis of morphogenesis of Beta1-null embryos in vitro suggests several hypotheses: a) alphaV and Beta1 integrins play distinctive roles in TB and ICM differentiation and morphogenesis. b) Beta1 integrins provide a survival signal for the ICM, resulting in death of the ICM in the peri-implantation period. c) TB is not initially Beta1-dependent for survival, but fails to develop further after initial implantation due to the lack of signals from the ICM. d) Both the extracellular ligand binding, and cytoplasmic signal transducing domains of the Beta1 integrin subunit are required for the restoration of normal embryonic development. To test these hypotheses we will: 1) Determine the functions of specific alphaV vs. Beta1 integrins in TB morphogenesis in vitro and in vivo in normal and Beta1- null embryos. 2) Determine the functions of Beta1 integrins in ICM differentiation. Experiments will assess the role of Beta1 integrins in ICM survival, and differentiation to extraembryonic endoderm and primitive embryonic ectoderm lineages. 3) Determine the maximum differentiation capacity of Beta1 null TB and ICM lineages in vivo and in vitro, using chimeric Beta1-containing and Beta1-null embryos, and Beta1-null embryo stem cells. 4) Determine to what extent survival and further embryonic development are restored by reconstituting Beta1-null embryos with Beta1 subunits that contain a truncated or altered Beta1 cytoplasmic domain. This approach should restore Beta1-integrin ligand binding and extend embryo survival, but reveal unique features of Beta1 integrin signaling that affect morphogenesis, tissue specific differentiation and/or growth control. Analysis of Beta1-null embryo morphogenesis will involve extensive interactions with Projects III and VI. We will extend studies of integrin expression in the peri- implantation period to human embryos in collaboration with Projects II and VI. Together these studies should increase understandings of normal peri-implantation development and show how cell-ECM interactions contribute to forming the extraembryonic lineages that result in successful fetal-maternal communication during pregnancy.