Tgif1 and Tgif2 Repress Expression of the RabGAP Evi5l.

Mouse embryos conditionally lacking Tgif1 and Tgif2 have holoprosencephaly and defects in left-right asymmetry. To identify pathways affected by loss of Tgif function during embryogenesis, we performed transcriptome profiling on whole mouse embryos. Among the genes with altered expression in embryos lacking Tgifs were a number with links to cilium function. One of these, Evi5l, encodes a RabGAP that is known to block the formation of cilia when overexpressed. Evi5l expression is increased in Tgif1; Tgif2-null embryos and in double-null mouse embryo fibroblasts (MEFs). Knockdown of Tgifs in a human retinal pigment epithelial cell line also increased EVI5L expression. We show that TGIF1 binds to a conserved consensus TGIF site 5' of the human and mouse Evi5l genes and represses Evi5l expression. In primary MEFs lacking both Tgifs, the number of cells with primary cilia was significantly decreased, and we observed a reduction in the transcriptional response to Shh pathway activation. Reducing Evi5l expression in MEFs lacking Tgifs resulted in a partial restoration of cilium numbers and in the transcriptional response to activation of the Shh pathway. In summary, this work shows that Tgifs regulate ciliogenesis and suggests that Evi5l mediates at least part of this effect.

Genetic and Molecular Analyses indicate independent effects of TGIFs on Nodal and Gli3 in neural tube patterning.

Holoprosencephaly (HPE) is a prevalent craniofacial developmental disorder that has both genetic and environmental causes. The gene encoding TG-interacting factor 1 (TGIF1) is among those that are routinely screened in HPE patients. However, the mechanisms by which TGIF1 variants cause HPE are not fully understood. TGIF1 is a transcriptional repressor that limits the output of the Transforming Growth Factor ß (TGFß)/Nodal signaling pathway, and HPE in patients with TGIF1 variants has been suggested to be due to increased Nodal signaling. Mice lacking both Tgif1 and its paralog, Tgif2, have HPE, and embryos lacking Tgif function do not survive past mid-gestation. Here, we show that in the presence of a Nodal heterozygous mutation, proliferation defects are rescued and a proportion of embryos lacking all Tgif function survive to late gestation. However, these embryos have a classic HPE phenotype, suggesting that this is a Nodal-independent effect of Tgif loss of function. Further, we show that the Gli3 gene is a direct target for repression by Tgifs, independent of TGFß/Nodal signaling, consistent with Tgif mutations causing HPE via Nodal-independent effects on the Sonic Hedgehog (Shh) pathway. Based on this work, we propose a model for distinct functions of Tgifs in the Nodal and Shh/Gli3 pathways during forebrain development.

Tissue morphodynamics shaping the early mouse embryo.

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo.

Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh null embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor ß/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.

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.

Cell-cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development.

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj(-/-) chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and ß-catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj(-/-) trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E-cadherin misexpression or ECM disorganization. However, plating Mrj-deficient cells on exogenous laminin-511 normalized their cell behavior. Lastly, we show that Mrj(-/-) chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj.

Tgif1 and Tgif2 regulate Nodal signaling and are required for gastrulation.

Tgif1 and Tgif2 are transcriptional co-repressors that limit the response to TGFbeta signaling and play a role in regulating retinoic-acid-mediated gene expression. Mutations in human TGIF1 are associated with holoprosencephaly, but it is unclear whether this is a result of deregulation of TGFbeta/Nodal signaling, or of effects on other pathways. Surprisingly, mutation of Tgif1 in mice results in only relatively mild developmental phenotypes in most strain backgrounds. Here, we show that loss-of-function mutations in both Tgif1 and Tgif2 result in a failure of gastrulation. By conditionally deleting Tgif1 in the epiblast, we demonstrate that a single wild-type allele of Tgif1 in the extra-embryonic tissue allows the double null embryos to gastrulate and begin organogenesis, suggesting that extra-embryonic Tgif function is required for patterning the epiblast. Genetically reducing the dose of Nodal in embryos lacking all Tgif function results in partial rescue of the gastrulation defects. Conditional double null embryos have defects in left-right asymmetry, which are also alleviated by reducing the dose of Nodal. Together, these data show that Tgif function is required for gastrulation, and provide the first clear evidence that Tgifs limit the transcriptional response to Nodal signaling during early embryogenesis.

CXCR4 acts as a costimulator during thymic beta-selection.

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

Maternal Tgif is required for vascularization of the embryonic placenta.

The mammalian placenta is the site of exchange of nutrients and waste between mother and embryo. In humans, placental insufficiency can result in intrauterine growth retardation, perinatal death and spontaneous abortion. We show that in C57BL/6J mice a null mutation in the gene encoding the transcriptional corepressor, Tgif, causes placental defects. The major defects are decreased vascularization of the placenta, due to a decrease in the fetal blood vessels, and decreased expression of the gap junction protein Gjb2 (Cx26). These defects result in severe growth retardation in a proportion of Tgif null embryos in Tgif heterozygous mothers, and an overall growth delay in Tgif null animals. Placental defects are much more severe if the mother also completely lacks Tgif function, and placentas from heterozygous Tgif embryos are defective in a Tgif null mother. Embryo transfer experiments show that even the placenta from a wild type embryo is compromised in the absence of maternal Tgif. These results demonstrate that Tgif functions in the normal development of the placenta, and suggest a role for maternal factors in regulating the morphogenesis of embryonically-derived placental tissues.

Trophoblast cells exhibit differential responses to laminin isoforms.

Extracellular matrix (ECM) has specific effects on cell behavior that influence many aspects of early development. In the early postimplantation mouse embryo the ECM component laminin promotes polarization and survival of the embryonic ectoderm and formation of Reichert's membrane. In addition, dynamic patterns of laminins 1 and 10/11 expression in the embryo and the uterus correlate with the progression of implantation. In the implanting blastocyst, laminin 1 is strongly expressed in the trophectoderm basement membrane, whereas laminin 10/11 is expressed only in the inner cell mass and polar trophectoderm. In the uterus, laminin 10/11 is strongly expressed in the decidualizing matrix of the stroma. We show here that laminins 1 and 10/11 have distinct effects on trophoblast cell behavior that influence the process of implantation. Laminin 1 promotes random migration and decreases spreading, whereas laminin 10/11 promotes both spreading and persistent migration. When presented as adjacent substrates, cells stop at the boundary and do not enter the region containing laminin 1. Laminin 1 also affects cell-cell adhesion through changes in the localization of vascular endothelial (VE) cadherin. Cultured cells and primary trophoblast explants become single cells or very small groups on laminin 1 and VE-cadherin localization at regions of cell-cell contact decreases dramatically. In contrast, trophoblast cells maintain strong cell-cell contacts on substrates of laminins 10/11, and exhibit strong staining of VE-cadherin in all regions of cell-cell contact. These effects, and the localization of laminin 1 in Reichert's membrane and laminin 10/11 in the surrounding decidual matrix, suggest that these laminin isoforms influence the direction and quality of invasion of trophoblast cells during implantation, and provide epigenetic cues that drive the morphogenesis of the yolk sac placenta.

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 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 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 alpha1 and laminin beta1, 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 (alpha5beta1gamma1) 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 alpha5 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.

Amino acid transport regulates blastocyst implantation.

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.