SummaryThe mechanisms that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage are largely unknown. Using a molecular approach to the study of megakaryocytopoiesis and platelet production, mice in which thrombocytopoiesis could be controlled were produced by targeting the expression of the herpes simplex virus thymidine kinase toxigene to megakaryocytes using the regulatory region of the gene encoding the c~ subunit of the platelet integrin cdIbB3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (odlbtk) with the antiherpetic drug ganciclovir (GCV). After 10 d of treatment, the platelet number was reduced by >94.6%. After discontinuing GCV, the bone marrow was repopulated with megakaryocytes and the platelet count was restored within 7 d. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. The reversibility and facility of this system provides a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.
Vascular endothelial (VE)-cadherin is an adhesive transmembrane protein specifically expressed at interendothelial junctions. Its extracellular domain exhibits Ca2+-dependent homophilic reactivity, promoting cell-cell recognition. Mice deficient in VE-cadherin die at mid-gestation resulting from severe vascular defects. At the early phases of vascular development (E8.5) of VE-cadherin-deficient embryos, in situ differentiation of endothelial cells was delayed although their differentiation program appeared normal. Vascularization was defective in the anterior part of the embryo, while dorsal aortae and vitelline and umbilical arteries formed normally in the caudal part. At E9.25, organization of endothelial cells into large vessels was incomplete and angiogenesis was impaired in mutant embryos. Defects were more severe in extraembryonic vasculature. Blood islands of the yolk sac and clusters of angioblasts in allantois failed to establish a capillary plexus and remained isolated. This was not due to defective cell-cell recognition as endothelial cells formed intercellular junctions, as shown by electron microscopy. These data indicate that VE-cadherin is dispensable for endothelial homophilic adhesion but is required for vascular morphogenesis.
The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the α subunit of the platelet integrin αIIbβ3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the αIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5′-flanking region of the murine αIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the αIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.
In order to address the molecular signature of human glioma, we investigated the polymorphism of 5'UTR of the mRNA of Contactin, an adhesion molecule which plays a role in the invasive behavior of these tumors. Contactin mRNA is identified by RT-PCR and a strategy based on rapid amplification of cDNA ends (RACE) reveals different 5'UTRs resulting from both an alternative use of two types of leader exons and a splicing mechanism within the 5'UTR. The spliced exon is an Alu-containing element specific to the primate lineage, thus indicating a recent evolution of regulatory processes specific to the simian line that occurs on this gene. Each 5'UTR exhibits different transcription/translation efficiencies and contains features that allow translation to occur independently of the classic cap-dependent mechanism. These data illustrate the complex regulation of Contactin expression in human brain tumors occurring at both transcriptional and translation levels. The different 5'UTRs are differentially expressed in diverse types of human tumors. Thus, the polymorphism occurring within the non-coding part of the Contactin mRNA reveals new opportunities to explore deregulation that occurs during the oncogenic process.
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