Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium.

To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase (Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the beta-galactosidase (LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds.

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.

Intravascular coagulation activation in a murine model of thrombomodulin deficiency: effects of lesion size, age, and hypoxia on fibrin deposition.

We consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM-/-) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P <.005) in radiolabeled cross-linked fibrin in TM-/- chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM-/- regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 micrometer2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in beta-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.

Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.

The present study was undertaken to define the 5' and 3' regulatory sequences of human von Willebrand factor gene that confer tissue-specific expression in vivo. Transgenic mice were generated bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon fused in-frame to the Escherichia coli lacZ gene. In situ histochemical analyses in independent lines demonstrated that the von Willebrand factor promoter targeted expression of LacZ to a subpopulation of endothelial cells in the yolk sac and adult brain. LacZ activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart, and aorta, as well as in megakaryocytes. In contrast, in mice containing the lacZ gene targeted to the thrombomodulin locus, the 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside reaction product was detected throughout the vascular tree. These data highlight the existence of regional differences in endothelial cell gene regulation and suggest that the 733-bp von Willebrand factor promoter may be useful as a molecular marker to investigate endothelial cell diversity.

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

We have targeted the murine thrombomodulin (TM) gene in embryonic stem cells and generated embryos as well as mice with TM deficiency. The heterozygous TM-deficient (TM+/-) mice as compared to wild-type (TM+/+) littermates exhibit 50% reductions in the levels of TM mRNA and TM protein. However, TM+/- mice appear normal and are free of thrombotic complications. The homozygous TM-deficient (TM-/-) embryos die before embryonic day 9.5. An overall retardation in growth and development of TM-/- embryos is first evident on embryonic day 8.5 (8-12 somite pairs). However, no specific pathologic abnormalities are observed. These initial changes take place at a time when TM is normally expressed in the parietal yolk sac. The removal of embryonic day 7.5 TM-/- embryos from maternal decidua and their subsequent culture in vitro allow development to proceed to stages not observed in vivo (13-20 somite pairs) with the appearance of normal blood vessels in the visceral yolk sac and embryo. The results of our studies suggest that the failure of TM-/- embryos to survive at mid-gestation is a consequence of dysfunctional maternal-embryonic interactions caused by the absence of TM in the parietal yolk sac and demonstrate that the receptor is necessary for normal embryonic development in utero.

Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice.

We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.

Development of the fast, transient outward K+ current in embryonic sympathetic neurones.

Voltage-activated outward potassium (K+) currents in developing sympathetic neurones, dissociated from the rat superior cervical ganglion (SCG), were studied using the whole-cell patch clamp recording technique. In voltage-clamped neonatal SCG cells, two voltage-dependent K+ currents were measured: the fast, transient K+ current, IA; and, the slower activating, non-inactivating delayed rectifier, IK. Only IK, however, appeared to be present in SCG neurones isolated from early embryonic (E14.5-16.5) rat pups; IA was not observed in these cells. When these embryonic neurones were maintained in cell culture, IA developed over a time course (approximately 4-6 days) similar to that seen in vivo. IA, therefore, which appears to facilitate the fast repolarization phase of the action potential in rat SCG neurones, is the last voltage-activated current to develop in these cells.