Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation.

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd-/-) mice to term. Half of the Thbd-/- offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd-/- animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd-/- mice. However, Thbd-/- females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.

Derivation and genetic modification of embryonic stem cells from disease-model inbred rat strains.

The lack of rat embryonic stem cells (ESCs) and approaches for manipulation of their genomes have restricted the ability to create new genetic models and to explore the function of a single gene in complex diseases in the laboratory rat. The recent breakthrough in isolating germline-competent ESCs from rat and subsequent demonstration of gene knockout has propelled the field forward, but such tools do not yet exist for many disease-model rat strains. Here we derive new ESCs from several commonly used rat models including the Dahl Salt Sensitive (SS), the sequenced Brown Norway (BN), and Fischer (F344) rat and establish the first germline-competent ESCs from a hypertension disease model strain, the Fawn Hooded Hypertensive (FHH) rat. Genetic manipulations including transgenesis mediated by lentivirus, routine homologous recombination, and homologous recombination mediated by zinc-finger nucleases (ZFNs) were performed effectively in FHH rat ESCs. Our results showed these rat ESC lines, isolated from inner cell masses using mechanical splitting, had germline competency; the Pparg gene locus and homologous genomic region to the mouse Rosa26 locus can be targeted effectively in these rat ESCs. Furthermore, our results also demonstrated that ZFNs increased the efficiency of proper homologous recombination in FHH rat ESCs using targeting vectors with short homology arms. These rat ESC lines and advancements in genetic manipulation pave the way to novel genetic approaches in this valuable biomedical model species and for exploration of complex disease in these strains.

Knockout rats via embryo microinjection of zinc-finger nucleases.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells.

Humans and rodents exhibit a peculiar type of placentation in which zygote-derived trophoblast cells, rather than endothelial cells, line the terminal maternal vascular space. This peculiar aspect of the placental vasculature raises important questions about the relative contribution of fetal and maternal factors in the local control of hemostasis in the placenta and how these might determine the phenotypic expression of thrombophilia-associated complications of pregnancy. Using genomewide expression analysis, we identify a panel of genes that determine the ability of fetal trophoblast cells to regulate hemostasis at the fetomaternal interface. We show that spontaneous differentiation of trophoblast stem cells is associated with the acquisition of an endothelial cell-like thromboregulatory gene expression program. This program is developmentally regulated and conserved between mice and humans. We further show that trophoblast cells sense, via the expression of protease activated receptors, the presence of activated coagulation factors. Engagement of these receptors results in cell-type specific changes in gene expression. Our observations define candidate fetal genes that are potential risk modifiers of maternal thrombophilia-associated pregnancy complications and provide evidence that coagulation activation at the fetomaternal interface can affect trophoblast physiology altering placental function in the absence of frank thrombosis.

The thrombomodulin-protein C system is essential for the maintenance of pregnancy.

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.