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两个ECM家族 受体：β1和αV整合素。这些受体有 多个重叠的配基偏好，表明 冗余性，或精致的专一性。在目前的授权期内，我们 已经表明，植入前的小鼠胚胎至少表达两个字母V -和两个与Beta1相关的整合素，从发育开始。 这两个家族的额外成员在近期内都被上调了- 着床期：例如，到d7.5，滋养层细胞(TB)来源 外胎盘锥体和次级巨细胞表达至少7个αV 和β1整合素复合体。αV整合素存在于 在孵化的胚泡之外的，在其中起作用的 最初的植入，而αV和β1整合素都是 在胚胎的其他区域检测到。我们去除了Beta1整合素 通过基因打靶在小鼠胚胎中发现了一个家族。Beta1基因缺失的胚胎形成 正常胚泡，并开始着床。然而，他们死于 栽植后早期。Beta1基因缺失的形态发生分析 体外胚胎支持几种假说：a)αV和β1 整合素在TB和ICM分化中发挥独特作用 形态发生。B)β1整合素为 ICM，导致ICM围植入期死亡。c) 结核病最初并不依赖于Beta1生存，但未能发展 在初始植入之后，由于缺乏来自 ICM。D)胞外配基结合和胞质信号 β1整合素亚单位的转导结构域是 恢复正常的胚胎发育。为了检验这些假说 我们将：1)确定特定的αV和β1的功能 整合素在正常和β1-结核体内外形态发生中的作用 零胚胎。2)确定β1整合素在ICM中的作用 差异化。实验将评估Beta1整合素在 ICM存活和分化为胚胎外内胚层和 原始胚胎外胚层谱系。3)确定最大值 Beta1基因缺失的TB和ICM系在体内和体外的分化能力 在体外，使用含有Beta1的嵌合胚胎和Beta1缺失的胚胎，以及 β1缺失的胚胎干细胞。4)确定生存和生存的程度 通过重组Beta1-Null来恢复进一步的胚胎发育 含有截短或改变的Beta1亚基的胚胎 细胞质结构域。这种方法应该可以恢复β1整合素配体 结合并延长胚胎存活，但揭示了Beta1的独特功能 影响形态发生的整合素信号，组织特异性 分化和/或生长控制。β1缺失型胚胎的分析 形态形成将涉及与项目III和项目III的广泛互动 VI.我们将扩大整合素在围产期表达的研究 与项目II合作，人类胚胎的着床期 和VI。这些研究一起应该增加对正常的理解 围着床期发育并展示细胞-细胞外基质如何相互作用 有助于形成胚外血统，从而导致 在怀孕期间成功地进行胎儿与母亲的沟通。

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.