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.
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