Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter.

The 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 alpha subunit of the platelet integrin alpha IIb beta 3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alpha IIbtk) 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.

Identification of homeobox-containing genes expressed in hematopoietic blast cells.

Among putative candidates involved in commitment and differentiation of hematopoietic cells are homeoproteins, a large family of transcription factors playing a major role during development. Using a polymerase chain reaction (PCR)-derived protocol, we have investigated for Antennapedia-like homeobox-containing (HOX) gene expression in an enriched population of human hematopoietic progenitors. Nine members of HOX 1 and HOX 2 loci were isolated. Together with recent studies using established cell lines, this indicates a large representation of HOX genes in the hematopoietic compartment and suggests a participation of this class of nuclear proteins to early steps of hematopoiesis.

Lineage and stage specific expression of HOX 1 genes in the human hematopoietic system.

Recent observations have demonstrated the expression of several members of the homeobox-containing (HOX) gene complexes within the hematopoietic compartment. We have analyzed the expression pattern of the entire HOX 1 locus in a panel of leukemia-derived human cell lines representing various blood phenotypes. The expression of the eleven HOX 1 genes is lineage-restricted and these genes are predominantly detected within cells of myelomonocytic origin. This is in strong contrast with the erythro-megakaryocytic specific expression of HOX 2 genes. Furthermore, we have observed that the expression of three HOX 1 genes within B lymphoid lineages is stage-related and that the expression of several of them is switched off during TPA-induced differentiation of Kg1 and U937. These observations suggest that HOX 1 homeoproteins could be regulators of lineage determination during hematopoiesis.

Dissecting megakaryocytopoiesis in vivo with toxigenes.

The genetic programs that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage remain poorly defined and require appropriate in vivo models. Using a cell-specific obliteration technique, a transgenic mouse model was produced where perturbations of megakaryocytopoiesis and platelet production may be induced on demand. This was achieved by targeting the expression of the herpes virus thymidine kinase (HSV-tk) to megakaryocytes using the regulatory regions of the gene coding for the alphaIIb gene, an early marker of megakaryocytopoiesis, which encodes the alpha subunit of the platelet integrin alphaIIb beta3. The HSV-tk gene is not toxic by itself, but sensitizes the target cell to the effect of ganciclovir (GCV), leading to the inhibition of DNA synthesis in dividing cells. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alphaIIb-tk) with GCV. After 10 days of treatment, the platelet number was reduced by greater than 96.5% and megakaryocytes were not detectable in the bone marrow (BM). After discontinuing GCV, BM was repopulated with megakaryocytes, and the platelet count was restored within seven days. The recovery was accelerated by the administration of interleukin 11. 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 bipotent progenitor cells. The reversibility and facility of this system provide 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.

[Homeoproteins: participation in hematopoietic processes?]. / Les homéoprotéines: participation aux processus hématopoïétiques?

The molecular basis of commitment and differentiation of hematopoietic cells remain poorly understood at the genetic level. Among putative candidates involved in these processes are homeoproteins, a large family of transcription factors which play a major role during development. Using a strategy based on the polymerase chain reaction (PCR) we have isolated nine different Antennapedia-like homeobox (HOX) genes from purified human hematopoietic precursors. Their expression patterns, analyzed with a panel of leukemia-derived cell lines representing various blood cells phenotypes, appears to be lineage-restricted. Extending our study to all the known members of the HOX 1 and HOX 2 clusters, we found that HOX 1 genes are predominantly detected within cell of myelomonocytic origin whereas HOX 2 genes transcripts are principally expressed in erythro-megakaryocytic cell lines. Furthermore, we have observed that the expression of three HOX 1 genes within B lymphoid lineages is stage-related and that the expression of several of them is switched off during TPA-induced differentiation of KG1 and U937 cells. These observations support the idea that homeoproteins could be regulators of lineage determination during hematopoiesis.

Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment.

To analyze the transcriptional activity of the gene encoding the alpha subunit of the platelet integrin alpha(IIb)beta(3) during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore the knock-in of the tk gene into the alpha(IIb) locus resulted in the knock-out of the alpha(IIb )gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the alpha(IIb) glycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from alpha(IIb)-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet alpha-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia. (Blood. 2000;96:1399-1408)

A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.

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 alpha subunit of the platelet integrin alphaIIb beta3 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 alphaIIb 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 alphaIIb 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 alphaIIb 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.

Ultrastructural analysis of bone marrow hematopoiesis in mice transgenic for the thymidine kinase gene driven by the alpha IIb promoter.

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the alphaIIb murine promoter of the gene encoding the alphaIIb-subunit of the platelet integrin alphaIIbbeta3 (Tropel et al, Blood 90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1(+) and CD34(+) cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the alphaIIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic alphaIIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.

Role of vascular endothelial-cadherin in vascular morphogenesis.

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.

Gene regulation in megakaryocyte.

Analysis of the mechanisms which control the differentiation of the megakaryocytic lineage is a major commitment to understand the production of circulating blood platelets. One way to approach this question is to examine the promoter domain of a marker gene which is expressed exclusively in the megakaryocytic lineage and at an early stage of the differentiation process. For this purpose the gene coding for the platelet specific glycoprotein IIb was isolated and its promoter was analysed. This promoter contains positive and negative DNA responsive elements that are responsible for the cell specific expression of the gene. With this promoter region it is now possible to direct the expression of heterologous genes in vivo using the transgenic approach.