Successful implantation of the embryo requires a complex and ordered series of adhesive interactions occurring between the trophoblast cells of the blastocyst and the uterus. First, the blastocyst hatches from the acellular zona pellucida and attaches to the uterine epithelium. These initial adhesive interactions are probably transient since in mice and in humans the cells of the trophoblast outgrowth are also invasive. As a result, these cells rapidly penetrate the uterine epithelium and its associated basement membrane, then invade the endometrium where they contact decidual cells, each of which is surrounded by a specialized basement membrane collar (Wewer et al., 1985, 1986). Invasion stops once the trophoblast cells have penetrated the uterine arterioles and tapped the maternal blood supply (Ramsey et al, 1976). The result is formation of the hemochorial placenta, in which blood from the maternal circulation constantly bathes the fetal chorionic villi. Thus, both cell-cell and cell-matrix interactions are important in the adhesive interactions that occur during placental development.
Amino Acid Transport Regulates Blastocyst Implantation 1
Biology Of Reproduction. 2003. 69(4) pp. 1101-1108.
Martin, Patrick M, Ann E Sutherland, and Lon J Van Winkle
Mouse blastocyst outgrowth in vitro and probably implantation in vivo require amino acid signaling via the target of rapamycin (TOR) pathway. This signaling does not simply support protein synthesis and trophoblast differentiation. Rather, it regulates development of trophoblast protrusive activity and may act as a developmental checkpoint for implantation. Moreover, intracellular amino acids per se are insufficient to elicit TOR signaling. Instead, de novo transport of amino acids, and particularly of leucine, stimulate mTOR activity at the blastocyst stage. The activity of the broad-scope and yet leucine-selective amino acid transport system B0,+ could produce such increases in intracellular amino acid concentrations. For example, system B0,+ uses a Na+ gradient to drive amino acid uptake, and the Na+ concentration in uterine secretions increases by nearly two-fold about 18 h before implantation. The resultant mTOR signaling could trigger polyamine, insulin-like growth factor II, and nitric oxide production in blastocysts and the increased cell motility sometimes associated with synthesis of these bioactive molecules.
Amino acid-regulated development of trophoblast protrusive activity in mouse blastocysts.
Biology Of Reproduction. 2003. 68 pp. 340-340.
Van Winkle, LJ, AA Shah, AE Sutherland, PM Martin, and AL Campione
Analysis of compaction in the preimplantation mouse embryo
An SEM analysis of the effects of tunicamycin, cytochalasin B, and colcemid has yielded insights into the process of compaction in the early mouse embryo. All three reagents block or reverse compaction and decrease the number of microvilli (MV), although some MV polarization is permitted. In addition, tunicamycin is shown to lessen cell adhesion even in compacted embryos. Cytochalasin B causes the formation of MV clumps some of which are preferentially localized to the apex or lateral ring region. Colcemid reverses compaction and, coupled with Pronase treatment, completely blocks compaction of uncompacted 8-cell embryos. Observations also suggest that MV polarization can occur only once but compaction (the close adherance and flattening of blastomeres) can be reversed and reinduced. Evidence is consistent with a three-step compaction process involving (1) cell surface recognition and attachment of a ring of lateral microvilli to adjacent blastomeres, (2) subsequent microfilament shortening in these lateral MV, and (3) maintenance of the compacted and polarized state by microtubules.
Axial elongation in mouse embryos involves mediolateral cell intercalation behavior in the paraxial mesoderm
Biomedical Optics. 2006. pp. 60891K-60891K-9.
Yen, WeiWei, Carol Burdsal, Ammasi Periasamy, and Ann E Sutherland
The cell mechanical and signaling pathways involved in gastrulation have been studied extensively in invertebrates and amphibians, such as Xenopus , and more recently in non-mammalian vertebrates such as zebrafish and chick. However, because culturing mouse embryos extra-utero is very difficult, this fundamental process has been least characterized in the mouse. As the primary mammalian model for genetics, biochemistry, and the study of human disease and birth defects, it is important to investigate how gastrulation proceeds in murine embryos. We have developed a method of using 4D multiphoton excitation microscopy and extra-utero culture to visualize and characterize the morphogenetic movements in mouse embryos dissected at 8.5 days of gestation. Cells are labeled by expression of an X chromosome-linked enhanced green fluorescent protein (EGFP) transgene. This method has provided a unique approach, where, for the first time, patterns of cell behavior in the notochord and surrounding paraxial mesoderm can be visualized and traced quantitatively. Our observations of mouse embryos reveal both distinct differences as well as striking similarities in patterned cell motility relative to other vertebrate models such as Xenopus , where axial extension is driven primarily by mediolateral oriented cell behaviors in the notochord and paraxial somitic mesoderm. Unlike Xenopus, the width of the mouse notochord remains the same between 4-somite stage and 8-somite stage embryos. This implies the mouse notochord plays a lesser role in driving axial extension compared to Xenopus , although intercalation may occur where the anterior region of the node becomes notochordal plate. In contrast, the width of mouse paraxial mesoderm narrows significantly during this period and cells within the paraxial mesoderm are both elongated and aligned perpendicular to the midline. In addition, these cells are observed to intercalate, consistent with a role for paraxial mesoderm in driving convergence and extension. These cell behaviors are similar to those characterized in the axial mesoderm of frog embryos during convergence and extension[1], and suggests that tissues may play different roles in axial elongation between the frog and the mouse.
CXCR4 acts as a costimulator during thymic [beta]-selection
Nature Immunology. 2010. 11(2) pp. 162-170.
Trampont, Paul C, Annie-Carole Tosello-Trampont, Yuelei Shen, Amanda K Duley, Ann E Sutherland, Timothy P Bender, Dan R Littman, and Kodi S Ravichandran
Passage through the ?-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-? gene (Tcrb) and formation of a pre-TCR on the surface of CD4?CD8? thymocytes. How other receptors influence ??-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced ?-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during ?-selection.
Cell-matrix interactions in the early mouse embryo
Proceedings Of The Annual Meeting-electron Microscopy Society Of America. 1992. pp. 600-600.
Sutherland, AE, PG Calarco, and CH Damsky
Cell–cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development
Developmental Dynamics. 2011. 240(11) pp. 2505-2519.
Watson, Erica D, Martha Hughes, David G Simmons, David RC Natale, Ann E Sutherland, and James C Cross
The focus of this study is to evaluate how the TANGO1 protein contributes to trophoblast differentiation in mutated and normal trophoblast cells. These cells are the primary and structural elements of the placenta. Trophoblast cells originate from the trophectoderm layer of the blastocyst, and differentiate to form the mature placenta. Failure of trophoblast cells to properly differentiate can lead to placental dysfunction, which directly affects the growth and development of the fetus, resulting in fetal death or abnormal growth. Previous inquiry has shown that mice homozygous for the Xst199 mutation develop a placenta that is poorly structured and lacks fetal blood vessels, consequently leading to fetal death. In order to investigate the function of the protein TANGO1 in placental development, mouse embryos isolated at E6.5, E7.5, and E9.5 heterozygous for the Xst199 mutation were stained to localize the expression of the gene trap construct that carries the Mia3 gene. Results show that in embryos dating E6.5 and E7.5 the Mia3 gene is localized in the placental precursors, namely the ectoplacental cone and the chorion regions of the embryos. Future studies will examine the expression of trophoblast marker genes to understand the function of the Mia3 gene and the TANGO1 protein in trophoblast differentiation. Knowing how the TANGO1 protein is involved the trophoblast differentiation process will allows us to more thoroughly understand how these factors regulate the behavior of trophoblast cells during placental morphogenesis.
Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation
Development. 2004. 131(10) pp. 2247-2256.
Miner, Jeffrey H, Cong Li, Jacqueline L Mudd, Gloriosa Go, and Ann E Sutherland
Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (?1?1?1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin ?1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1-/- embryos die within a day of implantation. We report the generation of mice lacking laminin? 1 and laminin ?1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1-/- embryos are similar to Lamc1-/- embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1-/- embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (?5?1?1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin ?5 from a transgene improved the phenotype of Lama1-/- embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.
Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.
Genes & Development. 1995. 9(15) pp. 1883-1895.
Stephens, Laurie E, Ann E Sutherland, Irina V Klimanskaya, Annie Andrieux, Juanito Meneses, Roger A Pedersen, and Caroline H Damsky
Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.
Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.
Genes & Development. 1995. 9(15) pp. 1883-1895.
Stephens, Laurie E, Ann E Sutherland, Irina V Klimanskaya, Annie Andrieux, Juanito Meneses, Roger A Pedersen, and Caroline H Damsky
Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.
Developmental expression of Tiam-1 in preimplantation mouse embryo
Developmental Biology. 2000. 222(1) pp. 283-283.
Martin, PM, and AE Sutherland
Developmental regulation of integrin expression at the time of implantation in the mouse embryo
The trophectoderm layer of the mouse blastocyst differentiates at the late blastocyst stage to form the invasive trophoblast that mediates implantation of the embryo into the uterine wall. The first sign that trophoblast cells have developed an invasion-specific cell behavior appears about 10–15 hours after the embryo hatches from the zona pellucida, when the quiescent, non-adherent trophectoderm cells initiate protrusive activity and become adhesive to extracellular matrix. Our previous findings that trophoblast outgrowth on extracellular-matrix-coated substrata involves the integrin family of adhesion receptors (Sutherland, A. E., Calarco, P. G. and Damsky, C. H., 1988, J. Cell Biol. 106, 1331–1348), suggested that the onset of trophoblast adhesive and migratory behavior at the time of implantation may be due to changes in expression or distribution of integrin receptors. We have thus examined the mRNA and protein expression of individual integrin subunits during pre- and periimplantation development (E0-E7.5). A basic repertoire of integrins, including receptors for fibronectin (alpha 5 beta 1), laminin (alpha 6B beta 1) and vitronectin (alpha v beta 3), was expressed continuously throughout this period, whereas the expression of five other integrin subunits was developmentally regulated. The mRNA for three of these (alpha 2, alpha 6A and alpha 7) was first detected in the late blastocyst, coincident with endoderm differentiation and development of attachment competence. The mRNA for another (alpha 1) was not detected until after trophoblast outgrowth had begun, suggesting that its expression may be induced by contact with matrix. At E7.5, three of the temporally regulated integrins (alpha 1, apha 6A, alpha 7), all of which can form receptors for laminin, were detected only in the ectoplacental cone (differentiating trophoblast), and may thus play specific roles in trophoblast adhesion and/or differentiation. Because laminin expression is upregulated in decidualized uterine stroma in response to the implanting embryo, we examined trophoblast-laminin interactions, using laminin fragments and integrin antibodies to determine which integrin receptors were involved. Trophoblast cells attached and spread on both the E8 and P1? fragments of laminin; however, the P1? binding site was cryptic in intact laminin. Interaction with P1? was RGD- and alpha v beta 3-dependent, whereas outgrowth on E8 was RGD-independent and not inhibited by antibodies to the laminin receptor alpha 6 beta 1, suggesting that alpha 7 beta 1 is the major trophoblast integrin E8 receptor.