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The v-myb oncogene of the acute avian leukemia virus E26 encodes a transcription factor that directly regulates the promyelocyte-specific mim-1 gene (Ness, S.A., Marknell, A. and Graf, T. Cell, 59, 1115-1125). We have investigated the relationship between the c-myb proto-oncogene and the transcription of the mim-1 gene both in vitro and in vivo. We demonstrate that the c-myb protein can transactivate the transcription of mim-1 in a transient transfection assay. In the chick embryo, we confirm that mim-1 is specifically expressed during granulopoiesis and we show that the expression of c-myb and mim-1 are perfectly correlated in the granulocytic spleen and pancreas. However we suggest that mim-1 is efficiently transcribed in the absence of c-myb in the yolk sac and in the promyelocytes at the onset of the colonization of the bursa of Fabricius. On the other hand c-myb transcripts detected in the early hemopoietic progenitor cells, in lymphoid cells and in proliferative epithelia are never associated with mim-1 transcription. We conclude that the granulocyte-specific mim-1 gene is regulated by c-myb-dependent and c-myb-independent mechanisms depending upon the environment in which granulocytic precursor cells differentiate.

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.

The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-κB family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its IκBα inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and IκBα are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis.

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase typeplasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.

Both 1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP bind at the AMP site of phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Etheno-AMP induces the same activation as AMP, about 30-fold higher than the activation induced by etheno-dAMP. The fluorescence of etheno-AMP and etheno-dAMP is associated with the base moiety; therefore, when free in solution, the two derivatives have identical fluorescence properties. However, when bound to phosphorylase, the fluorescence of etheno-AMP is quenched more efficiently than the fluorescence of etheno-dAMP. This difference between the fluorescence properties of the bound nucleotides suggests that a modification in the ribose ring affects the position of the adenine in the AMP site of phosphorylase b. The observed quenching may be due to a stacking interaction between an aromatic residue and the base moiety of the bound nucleotide.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5'-distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3' region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.

We have used in situ hybridization to study the spatial and temporal distribution of the transcription of three cellular oncogenes encoding DNA-binding proteins, c-ets 1, c-myb and c-myc during the development of the chick embryo. c-ets 1 mRNA expression appears linked to the mesodermal lineage and is strongly expressed in early endothelia; it subsequently becomes restricted to small vessel endothelia. Hemopoietic cells in extraembryonic blood islands at E2 express c-ets 1, while intraembryonic hemopoietic cells in aortic clusters (E3) and paraaortic foci (E6) express c-myb. c-myc transcripts are detected in cells undergoing hemopoiesis in both these extraembryonic and intraembryonic sites. Outside the blood forming system, c-myc is transcribed in a large variety of cells; c-ets 1 displays tissue-specific expression in groups of mesodermal cells engaged in morphogenetic processes and appears excluded from all epithelia; finally the expression of c-myb is the most tightly linked to hemopoietic cells. In any case, it is clear that these three oncogenes display complementary expression in endothelial and hemopoietic cells where their patterns are modulated in relationship to multiplication and differentiation.

The distribution of the c-myc protein was studied in the developing embryo from the two-somite stage to embryonic day 17 (E17). A triple labelling method was used, with a polyclonal serum recognizing the human and avian c-myc proteins as the first marker followed by Hoechst 33258 for nuclear staining and the monoclonal antibody 13F4 which reveals the avian myogenic lineage. In situ hybridization was carried out at three selected stages (E3, E6 and E8), in order to compare the distribution of myc mRNA and myc protein. The c-myc protein signal was barely detectable in blastodisc nuclei during the period of somlte formation, after which it became ubiquitous in the embryonic body until E4. Myotomal cell nuclei displayed a strong signal until their organization Into premuscular masses. On day 4, the level of c-myc protein decreased In all embryonic tissues. By doubling the antibody titre and amplifying the signal by means of the streptavidin-blotin method, c-myc could still be detected in nuclei of defined groups of cells. Such was the case in some mesenchyme-derived tissues at critical periods of or-ganogenesis, for instance in prechondrogenic condensations or hemopoietic cell foci at E6, the latter becoming negative at E9. The heart ventricle displayed a patchwork of positive and negative nuclei from E6 to E10. A myc signal restricted to the quail species was found In the wall of the carotid arteries. Cell nuclei in the nervous system displayed a detectable signal which became restricted to postmitotic neurones. In the ectoderm, the c-myc protein was generally not present after E4, except In presumptive feather buds at the time of epitheliomesenchymal interactions. Endodermal cells (such as hepatocytes, oesophageal and tracheal epithelia) did not express detectable levels of c-myc at any time. Our results reveal a time- and tissue-specific expression of c-myc during avian development. It is noteworthy that the expression of the c-myc protein often appears dissociated from cell proliferation as shown by the absence of the signal in endodennal cells at E3-E13 as well as its presence in postmitotic neurones. Finally, although RNA and protein are simultaneously detected in some structures such as presumptive feather buds, their expression is dissociated in endodermal tissues, notably hepatocytes, where in situ hybridization detects a large number of RNA copies with no detectable protein signal.

Le travail en équipe est défini par la multidisciplinarité : il regroupe des personnes de catégories socioprofessionnelles, formations et origines différentes, réunies pour atteindre un objectif commun. Nous questionnons dans cet article la notion du « faire-équipe » dans le domaine de la recherche en sciences de la vie et de la santé, domaine ayant subi de profonds changements au cours des dernières années. En effet, la diminution des ressources financières récurrentes, les pressions à la productivité, la précarisation de l’emploi et les échéances à court terme réduisant les délais d’exécution des tâches interviennent à un moment où les institutions de recherche insistent sur la valorisation des travaux effectués ; introduisant une forme d’injonction contradictoire. Dans ce contexte, la place de chacun au sein de l’équipe (responsabilités, prérogatives), les aspirations professionnelles (engagement, reconnaissance/visibilité, projection dans l’avenir) et la qualité des interactions humaines (confiance, solidarité, échanges) apparaissent déstabilisées et mouvantes, affectant le sens même du métier de chercheur. Nous proposons une vision éthique du « faire-équipe » qui pourrait permettre de retrouver le partage de visions convergentes au sein de l’équipe de recherche en limitant les effets néfastes résultant des processus organisationnels actuels. En redonnant du sens au travail mené par chacun des membres de l’équipe, une réflexion éthique sur le « faire-équipe » suscitera un mode de fonctionnement plus harmonieux de la recherche dans le domaine des sciences de la vie et de la santé.

Transcription factors of the Rel/NF-κB family are widely involved in the immune system. In this study, we investigate the in vivo expression of the avian protein c-Rel in the T-cell lineage during thymus development. The majority of thymocytes do not express the c-Rel protein. However, lymphocyte precursor cells that colonize the thymus express the c-Rel protein shortly after their homing in the organ and before they begin to differentiate, c-Rel is also detected in different subsets of,antigen-presenting cells such as epithelial cells, dendritic cells, and macrophages. In vitro studies have shown that Rel/NF-κB proteins are sequestered in an inactive form in the cytoplasm by interaction with the IκBα inhibitory protein. By immunocytochemistry, we show that in vivo c-Rel is localized in the cytoplasm of antigen-presenting cells but in both the cytoplasm and nucleus of lymphocyte precursor cells. The cytoplasmic localization of c-Rel in antigen-presenting cells correlates with a high expression of IκBα, whereas the nuclear localization of c-Rel in lymphocyte precursor cells correlates with a much lower expression of IκBα. These results suggest that c-Rel might be constitutively activated in lymphocyte precursor cells.