Ann E. Sutherland

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (α1β1γ1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin γ1 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 lamininα 1 and laminin β1, 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 (α5β1γ1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reicherts membrane, which was absent, and the embryos died by E7. Overexpression of laminin α5 from a transgene improved the phenotype of Lama1-/- embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reicherts 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.

CXCR4 acts as a costimulator during thymic [beta]-selection

Passage through the β-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-β gene (Tcrb) and formation of a pre-TCR on the surface of CD4−CD8− thymocytes. How other receptors influence ββ-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 β-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 β-selection.

Amino Acid Transport Regulates Blastocyst Implantation

Abstract 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.

PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation

Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. Here we show, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior. The initial events in the onset of CE are mediolateral elongation, alignment and orientation of mesoderm cells as they exit the primitive streak. This cell shape change occurs prior to, and is required for, the subsequent onset of mediolaterally polarized protrusive activity. In embryos mutant for PTK7, a novel cell polarity protein, the normal cell elongation and alignment upon leaving the primitive streak, the subsequent polarized protrusive activity, and CE and axial elongation all failed. The mesoderm normally thickens and extends, but on failure of convergence movements in Ptk7 mutants, the mesoderm underwent radial intercalation and excessive thinning, which suggests that a cryptic radial cell intercalation behavior resists excessive convergence-driven mesodermal thickening in normal embryos. When unimpeded by convergence forces in Ptk7 mutants, this unopposed radial intercalation resulted in excessive thinning of the mesoderm. These results show for the first time the polarized cell behaviors underlying CE in the mouse, demonstrate unique aspects of these behaviors compared with those of other vertebrates, and clearly define specific roles for planar polarity and for the novel planar cell polarity gene, Ptk7, as essential regulators of mediolateral cell intercalation during mammalian CE.

Distinct apical and basolateral mechanisms drive planar cell polarity-dependent convergent extension of the mouse neural plate.

The mechanisms of tissue convergence and extension (CE) driving axial elongation in mammalian embryos, and in particular, the cellular behaviors underlying CE in the epithelial neural tissue, have not been identified. Here we show that mouse neural cells undergo mediolaterally biased cell intercalation and exhibit both apical boundary rearrangement and polarized basolateral protrusive activity. Planar polarization and coordination of these two cell behaviors are essential for neural CE, as shown by failure of mediolateral intercalation in embryos mutant for two proteins associated with planar cell polarity signaling: Vangl2 and Ptk7. Embryos with mutations in Ptk7 fail to polarize cell behaviors within the plane of the tissue, whereas Vangl2 mutant embryos maintain tissue polarity and basal protrusive activity but are deficient in apical neighbor exchange. Neuroepithelial cells in both mutants fail to apically constrict, leading to craniorachischisis. These results reveal a cooperative mechanism for cell rearrangement during epithelial morphogenesis.

Inner Cell Allocation in the Mouse Morula: The Role of Oriented Division during Fourth Cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.

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.

Mechanisms of implantation in the mouse: differentiation and functional importance of trophoblast giant cell behavior

According to Lewis Wolpert, “It is not birth, marriage or death, but gastrulation which is truly the most important time in your life” (quoted in Slack, 1983). While it is certainly true that gastrulation is a vitally important morphogenetic event, the argument can be made for us, and for all placental mammals, that implantation is an equally important event in our lives. If implantation fails, so does gastrulation and all subsequent developmental events. Unlike most vertebrate embryos, mammalian embryos require an external source of nutrients to grow and develop. The mouse embryo and those of humans and other primates accomplish this by forming contacts with the maternal blood supply, leading to the formation of a hemochorial placenta (Mossman, 1937; Perry, 1981). Two events are critical to the success of placentation: implantation and the formation of vascular connections. Implantation begins with the apposition and attachment of the embryo to the uterine epithelium, followed by displacement of this epithelium and invasion of the underlying stromal compartment. After implantation has begun, an anastomotic network of blood spaces is formed surrounding the embryo that is continuous with maternal capillaries (Cross et al., 1994; Welsh and Enders, 1987). These blood-filled spaces provide nutrients and oxygen to the embryo during the period before the definitive chorioallantoic placenta is formed, allowing the embryo to grow and differentiate. Both implantation and early vasculogenesis are mediated by trophoblast giant cells, which arise from the epithelial trophectoderm layer of the blastocyst. Trophoblast cell differentiation represents the first divergence of embryonic lineage, and comprises many of the cellular processes seen in other developmental events: cell polarization, epithelialization, and epithelial–mesenchymal transition. The process of transformation of trophectoderm into invasive trophoblast, and then into a vascular network, has many aspects in common with other developmental events, and with gastrulation in particular. Understanding implantation is thus important not only from a clinical standpoint, but unraveling the complex mechanisms that establish and regulate the behavior of this unique extra-embryonic lineage will complement and extend our understanding of the cellular processes that shape the embryo proper. Until recently, most of our knowledge of trophoblast giant cell differentiation and function was morphological, and little was known of the underlying molecular or cellular events. The development of in vitro model systems, the Rcho-1 and trophoblast stem (TS) cell lines (Faria and Soares, 1991; Tanaka et al., 1998), and the ability to delete relevant genes in the mouse have facilitated the analysis of the molecular mechanisms regulating trophoblast giant cell differentiation (reviewed in Cross, 2000; Rinkenberger et al., 1997). However, exploration of the temporal and causal relationships between molecular differentiation and functional cell behavior is just beginning. This review will focus on the cellular aspects of trophoblast giant cell differentiation and their correlation with known molecular aspects of differentiation, as a means of promoting the synthesis of these two approaches.

Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo

Uterine implantation is a critical element of mammalian reproduction and is a tightly and highly coordinated event. An intricate and reciprocal uterine-embryo dialog exists to synchronize uterine receptivity with the concomitant activation of the blastocyst, maximizing implantation success. While a number of pathways involved in regulating uterine receptivity have been identified in the mouse, less is understood about blastocyst activation, the process by which the trophectoderm (TE) receives extrinsic cues that initiate new characteristics essential for implantation. Amino acids (AA) have been found to regulate blastocyst activation and TE motility in vitro. In particular, we find that arginine and leucine alone are necessary and sufficient to induce TE motility. Both arginine and leucine act individually and additively to propagate signals that are dependent on the activity of the mammalian target of rapamycin complex 1 (mTORC1). The activities of the well-established downstream targets of mTORC1, p70S6K and 4EBP, do not correlate with trophoblast motility, suggesting that an independent-rapamycin-sensitive pathway operates to induce trophoblast motility, or that other, parallel amino acid-dependent pathways are also involved. We find that endogenous uterine factors act to induce mTORC1 activation and trophoblast motility at a specific time during pregnancy, and that this uterine signal is later than the previously defined signal that induces the attachment reaction. In vivo matured blastocysts exhibit competence to respond to an 8-hour AA stimulus by activating mTOR and subsequently undergoing trophoblast outgrowth by the morning of day 4.5 of pregnancy, but not on day 3.5. By the late afternoon of day 4.5, the embryos no longer require any exposure to AA to undergo trophoblast outgrowth in vitro, demonstrating the existence and timing of an equivalent in vivo signal. These results suggest that there are two separate uterine signals regulating implantation, one that primes the embryo for the attachment reaction and another that activates mTOR and initiates invasive behavior.

Role of multiple β1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

Abstract ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the α6β1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the α9β1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express α6β1 and α9β1 integrins at their surfaces. Antibodies to the α6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human α9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes α4β1 and α9β1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the α9 integrin subunit in mouse eggs. In contrast to our ability to detect α6β1, we were unable to convincingly detect α9β1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its ∼IC50 for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect α9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the α6 and α7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the α4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.