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 granylocyte-specific mim-1 gene is regulated by c-myZ>-dependent and c-myh-independent mechanisms depending upon the environment in which granulocytic precursor cells differentiate.

The avian myeloblastosis virus AMV and the avian leukemia virus E26, which have independently trans-duced the nuclear oncogene v-myb (Roussel et al., 1979), induce myeloid leukemias in chickens and transform hemopoietic cells in vitro that have distinct myeloid phenotypes (for review see Moscovici and Gazzolo, 1982). The v-myb gene of E26 virus is fused to a second oncogene, v-ets, which confers upon it the ability to transform erythroid progenitor cells and to induce an erythroleukemia in chickens (Radke et al., 1982; Golay et al., 1988; Nunn and Hunter, 1989). The c-myb proto-oncogene from which the two forms of v-myb arose is expressed at high levels predominantly in immature hemopoietic cells including those of the erythroid, lymphoid and myeloid lineages (Gonda et al., 1982; Westin et al., 1982; Coll et al., 1983). The expression of c-myb declines as these cells mature (Craig and Block, 1984; Gonda and Metcalf, 1984; Duprey and Boettiger, 1985; Liebermann and Hoff-man-Lieberman, 1989). c-myb itself appears to control this differentiation process since the expression of exogenous c-myb or v-myb can block or reverse the differentiated phenotype of various hemopoietic cells (Ness et al., 1987; Clarke et al., 1988; Todokoro et al., 1988; McClinton et al., 1990). In addition, c-myb- specific antisense oligonucleotides have been used to inhibit the growth of hemopoietic progenitor cells (Gewirtz and Calabretta, 1988). Together these results suggest that the c- and v-myb genes encode proteins that regulate the phenotype of normal and transformed hemopoietic cells, respectively.

A number of shared biochemical properties have implicated the v-myb and the c-myb gene products as potential regulators of transcription. Both the v-myb and the c-myb proteins are localized in the nucleus (Boyle et al., 1984; Klempnauer et al., 1984) and bind DNA in vitro (Moelling et al., 1985). The DNA-binding domain of the v-myb proteins is composed of two imperfectly conserved 52 amino acid direct repeats located near the amino terminus (Gerondakis and Bishop, 1986; Rosson and Reddy, 1986; Klempnauer and Sippel, 1987). It corresponds to a truncated version of the one found in chicken c-myb and in several myb- related DNA-binding proteins isolated from species as diverse as mammals, insects and plants (for review see, Lüscher and Eisenman, 1991). A consensus DNA-binding sequence for v-myb protein was identified by comparing random fragments of cellular DNA that were bound by a bacterially synthesized form of the AMV v-myb protein (Biedenkapp et al., 1988). Since then, several laboratories have shown that concatemers of this consensus sequence can confer v-myb and c-myb- dependent inducibility to a variety of otherwise unres-ponsive test promoters (for review see Liischer and Eisenman, 1991 and also Klempnauer et al., 1989; Sakura et al., 1989; Weston and Bishop, 1989; Ibanez and Lipsick, 1990).

Recently Ness et al. (1989) identified a new gene mim-1 (for myb induced myeloid protein-1) which is directly regulated by the product of the E26 v-myb gene, mim-1 was isolated using a differential hybridiz-ation strategy in myeloid cells transformed by a temperature-sensitive mutant of the E26 virus (Beug et al., 1984, 1987; Frykberg et al., 1988). The promoter of mim-1 contains three v-myb responsive elements and can be transactivated by the E26 v-myb protein, mim-1 is specifically expressed in normal, immature granulo-cytes and encodes a 35 x 103MT secretable component of the granules found in such cells. When these promyelo-cytes undergo terminal differentiation into neutrophil granulocytes, the level of mim-1 protein decreases.

Since this former study was achieved with the E26 v-myb protein, it was essential to investigate the relation-ship between c-myb and mim-1. We demonstrate that c-myb can transactivate an exogenously added or the endogenous mim-1 promoter. Using in situ hybridiz-ation, we have also undertaken a comparative descrip-tion of the expression pattern of c-myb and mim-1 throughout the development of the chick embryo, c-myb and mim-1 are coexpressed during granulopoiesis in the pancreas and the spleen. Surprisingly, 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, indicating that factors other than c-myb might regulate mim-1 gene transcription.

Cotransfection and transactivation assays

Transfection of HD-11 cells with a plasmid containing 240 bp of the promoter plus the first exon of the mim-1 gene fused to the firefly luciferase reporter gene has been described previously (Ness et al., 1989), as well as the E26 v-myb expression vector (Introna et al., 1990). The expression vector for c-myb (kindly provided by J. Lipsick) was derived from pMAV-NEO (Ibanez and Lipsick, 1990) and will be described in detail elsewhere. Published procedures were used for assays of luciferase (De Wet et al., 1987) and β-galactosidase (Herbomel et al., 1984). For northern blots, total RNA from transfected HD-11 cells was prepared, fractionated on formaldehyde-agarose gels, transferred to Genescreen (NEN-Du Pont) and then hybridized to radiolabeled probes specific for mim-1 (exposure 3 days) (Ness et al., 1989) or β-actin (exposure 4 hours) (Kost et al., 1983) using standard methods (Manlatis et al., 1982; Feinberg and Vogelstein, 1983).

Probes

We attempted first to detect c-myb transcripts using a 350 bp EcoRI-Sa/I fragment containing the major part of v-myb from a molecularly cloned E26 provirus (Leprince et al., 1983). However, unless otherwise stated, all the results presented in this paper have been obtained with a full-lengh (3 kb) chicken c-myb cDNA cloned in the plasmid pSG3 (Gerondakis and Bishop, 1986). The mim-1 probe derives from a 869 bp mim-1 partial cDNA fragment cloned in Bluescript SK minus (Stratagene).

The specificity of the probes was demonstrated using northern blot analysis of total RNA from different organs (yolk sac, bursa of Fabricius, liver, thymus, spleen and gut) of E10 embryos. Although both c-myb probes used in this study encompass the DNA-binding domain which is well conserved in the myb gene family, under stringent conditions they hybridize only with the 4.0 kb mRNA species which correspond to the expected size for c-myb transcripts (Coll et al., 1983) (data not shown). Hybridization of probes with tissue sections has been always carried out under stringent conditions, at 60°C, followed by RNAase treatment which removes mismatched hybrids. On northern blots, the mim-1 probe hybridizes with a major band of 1.0 kb (Ness et al., 1989) (data not shown).

RNA probes were transcribed from truncated plasmids by the SP6, T7 or T3 RNA polymerase (Promega Biotec) using α-35S-CTP (∽1300 Ci/mmol NEN).

Tissue preparation

The eggs of brown Leghorn chicken were incubated at 37°C in a humidified air chamber. The age of the embryos is indicated as El, E2 etc…, E0 being the first day of incubation. Embryos or dissected organs were fixed at 4°C for 16 h in 4% paraformaldehyde in PBS containing 5 mM MgC12, dehy-drated and embedded in paraffin. 4 μm thick transverse sections were transferred to either poly-L-lysine or 3-aminopropyltriethoxysilane (TESPA; Aldrich)-coated slides and dried at 42°C for two days. Slides may be stored at 4°C for up to 1 year before hybridization.

In situ hybridization

Prehybridization treatments were performed essentially by the method of Cox et al. (1984). After deparaffinization and hydration, slides were incubated in 0.1 M glycine, 0.2 M Tris-HC1, pH 7.4 for 10 min at room temperature, treated with 1 μg ml−1 proteinase K (Boehringer-Mannheim) for 15 min at 37°C and further fixed in 4% paraformaldehyde in PBS. The slides were subsequently washed in PBS, acetylated and dehydrated.

Prior to hybridization, the probes were diluted in the hybridization buffer [50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.9), 5 mM EDTA, 10% dextran sulfate, 1× Denhardt’s solution, 0.5 mg ml−1E. coli tRNA and 100 mM dithiothreitol] and denaturated at 80°C for 2 min. A low level of background and a highly specific signal were obtained when the hybridization was performed at 60°C for 16 h with a probe concentration of 50 pg μl−1. Thereafter, the slides were washed in 4×SSC, 10 mM dithiothreitol for one hour, and in 50% formamide, 0.15 M NaCl, 20 mM Tris-HCl (pH 7.9), 5 mM EDTA, 100 mM dithiothreitol at 65°C for 30 min as described in Angerer et al. (1987). The sections were subsequently treated with 20 μg ml−1 RNAase A (type III A, Sigma) for 30 min to one hour at 37°C in 0.4 M NaCl, 10 mM Tris-HCl (pH 7.4), 0.05 M EDTA, incubated 15 min at 60°C in 2xSSC and 15 min at 60°C in 0.l×SSC. The slides were dehydrated and dipped in a nuclear track emulsion (Kodak NTB2) diluted 1:1 with 0.6 M ammonium acetate.

The slides were exposed at 4°C for one to three weeks and developed according to the recommendations of Cox et al. (1984). Labeling with the Hoechst dye 33258 (bisbenzimidine) was performed to visualize the nuclei. The sections were examined under dark-field and epifluorescence illumination with a Zeiss microscope (IM35). We have always used on serial sections in parallel with the antisense probes c-myb and mim-1 sense probes that never gave any signal.

The mim-1 promoter can be transactivated by the product of the c-myb gene

To determine whether the c-myb gene product can activate the mim-1 promoter in chicken myeloid cells, we used a transient expression assay in the chick macrophage cell line HD11 (Ness et al., 1989). Cotransfection of plasmids expressing c-myb as well as E26 v-myb proteins greatly stimulated the luciferase activity in cells transfected by the mim-1 promoter-luciferase constructs (Fig. 1A), but had little or no effect on control plasmids lacking mim-1 promoter sequences (data not shown).

Fig. 1.

The mim-1 gene can be activated by either v-myb or c-myb. (A) The mim-1 promoter-luciferase reporter plasmid construct was cotransfected into HD-11 cells without (open bars) or with either 200 or 600 ng (hatched and shaded bars, respectively) of plasmids that encode either E26 v-myb or c-myb as indicated. In each case, the pRSV-β-gal plasmid was included as an internal control. Luciferase activities shown are the means from assays of duplicate plates, normalized to β-galatosidase levels. (B) Northern blot analysis of HD-11 cells transfected with lane 1, pRSV-β-gal only; lane 2, as lane 1 plus the E26 v-myb expression plasmid; lane 3, as lane 1 plus the c-myb expression plasmid. Each lane contained 10 μg of total RNA. The blot was hybridized first with the radiolabeled mim-1 cDNA, then stripped and rehybridized with a probe specific for βactin.

Fig. 1.

The mim-1 gene can be activated by either v-myb or c-myb. (A) The mim-1 promoter-luciferase reporter plasmid construct was cotransfected into HD-11 cells without (open bars) or with either 200 or 600 ng (hatched and shaded bars, respectively) of plasmids that encode either E26 v-myb or c-myb as indicated. In each case, the pRSV-β-gal plasmid was included as an internal control. Luciferase activities shown are the means from assays of duplicate plates, normalized to β-galatosidase levels. (B) Northern blot analysis of HD-11 cells transfected with lane 1, pRSV-β-gal only; lane 2, as lane 1 plus the E26 v-myb expression plasmid; lane 3, as lane 1 plus the c-myb expression plasmid. Each lane contained 10 μg of total RNA. The blot was hybridized first with the radiolabeled mim-1 cDNA, then stripped and rehybridized with a probe specific for βactin.

In the same HD11 cell line, we have also investigated whether the c-myb gene product could activate the endogenous (chromosomal) mim-1 gene. Two days after transfection, total cellular RNA was purified and analyzed after northern blotting. The cells transfected with plasmids expressing the c-myb or the E26 v-myb protein did express mim-1 (Fig. 1B). In contrast no mim-1 mRNA was detected in untransfected HD-11 cells or in cells transfected with control plasmids. These results indicated that c-myb as well as E26 v-myb can transactivate the mim-1 promoter. Both with the plasmid containing mim-1 promoter and with the chromosomal mim-1 gene, E26 v-myb worked better than c-myb. This may be due to a difference in the efficiency of expression of the two myb proteins in transfected cells because the two expression vectors were different. Alternatively, the E26 v-myb protein may be a better transactivator than the c-myb protein. In any case, this difference was not due to the presence of v-ets in E26 since an E26 construct with v-ets deleted or with the AMV construct worked as well as the wild-type E26 (data not shown).

These experiments have been made with the E26 form of v-myb. The AMV form activates the luciferase construct, but is unable to turn on the endogenous mim-1 gene (Ness et al., 1989). This suggests that the regulation of the endogenous mim-1 gene is complex, and dependent on v-myh-specific factors. To contribute towards a better understanding of the role of the c-myb protein in regulating mim-1 expression, we have undertaken a comparative study of the pattern of expression of c-myb and mim-1 during the chick ontogeny.

The expression of c-myb precedes the expression of mim-1 during the early stages of hemopoiesis

At the beginning of the development of the chick embryo, the first blood cells belong to the erythroid lineage and are produced in the yolk sac. There, aggregates of cells called blood islands are found attached within the lumen of blood vessels and give rise to the first generation of erythroblasts between day 2 and 5 of incubation-(Dieterlen-Lièvre, 1988). In the chick blastoderm from E2 to E6, a few cells within these blood islands expressed c-myb (data not shown). No signal was observed with the mim-1 probe on these structures until E3. Later, some isolated cells labeled with the mim-1 probe were found in the yolk sac (data not shown).

In the 3- to 4-day avian embryo, after the first wave of erythropoiesis which occurs in the yolk sac, multipotent hemopoietic progenitor cells emerge in the ventrolateral wall of the aorta (Dieterlen-Lièvre and Martin, 1981; Dieterlen-Lièvre, 1984). The c-myb probe detected a high signal on these intraembryonic hemo-poietic cells in the periaortic clusters (Fig. 2A). In contrast, these clusters were negative with the mim-1 probe (Fig. 2B) and only a few wandering cells dispersed in the embryo displayed an intense labeling at E3 (data not shown).

Fig. 2.

c-myb and mim-1 in periaortic clusters at E3. (Bar: 50 μm). Transverse sections from E3 embryos at trunk level (a, aorta). (A) c-myb expression is restricted to hemopoietic clusters, on the ventrolateral wall of the aorta (arrows). (B) No labeling is observed with the mim-1 probe on the next section. Circulating erythrocytes in the aorta display a non-specific light scattering.

Fig. 2.

c-myb and mim-1 in periaortic clusters at E3. (Bar: 50 μm). Transverse sections from E3 embryos at trunk level (a, aorta). (A) c-myb expression is restricted to hemopoietic clusters, on the ventrolateral wall of the aorta (arrows). (B) No labeling is observed with the mim-1 probe on the next section. Circulating erythrocytes in the aorta display a non-specific light scattering.

At E6, erythroid and myeloid progenitors become numerous in the dorsal mesentery and they assemble into dense foci around blood vessels (Dieterlen-Lièvre and Martin, 1981). Various patterns of expression of c-myb and mim-1 were observed over these hemopoietic foci, presumably depending on the commitment of the hemopoietic cells and on the progression of these cells into a differentiation pathway. Erythrocytes, which could be easily recognized by their lenticular shape and their réfringent cytoplasm, were not labeled. Fig. 3A illustrates a section through a cluster of hemopoietic cells that hybridized with the c-myb probe. On the neighboring section, only a few cells were intensely labeled with the mim-1 probe (Fig. 3B). A detailed examination of numerous hemopoietic foci revealed that mim-1 displayed two levels of expression, faint all over the foci and high in some isolated cells (Fig. 3D,E,F).

Fig. 3.

c-myb and mim-1 in hemopoietic clusters at E6 (Bar: 50 μm). (A) c-myb transcripts are detected all over an hemopoietic cluster in the vicinity of carotid (c, carotid). The intensity of the c-myb signal is lower in this experiment because the probe used was synthesized with a shorter 350 bp v-myb fragment. (B) On the neighbouring section, only a few cells with an intense labeling for the mim-1 probe are quite noticable in this cluster and its surrounding. (C,D,E) Serial transverse sections through an E6 embryo hybridized with the mim-1 probe (p, pharynx). Near the pharynx a cluster of cells is weakly labeled with mim-1 (C). The sense probe hybridized on a next section gave no signal above background, indicating that the signal with the antisense probe is specific (data not shown). On the next section, one cell appears more intensively labeled (D). On another section, four cells display a strong signal (E).

Fig. 3.

c-myb and mim-1 in hemopoietic clusters at E6 (Bar: 50 μm). (A) c-myb transcripts are detected all over an hemopoietic cluster in the vicinity of carotid (c, carotid). The intensity of the c-myb signal is lower in this experiment because the probe used was synthesized with a shorter 350 bp v-myb fragment. (B) On the neighbouring section, only a few cells with an intense labeling for the mim-1 probe are quite noticable in this cluster and its surrounding. (C,D,E) Serial transverse sections through an E6 embryo hybridized with the mim-1 probe (p, pharynx). Near the pharynx a cluster of cells is weakly labeled with mim-1 (C). The sense probe hybridized on a next section gave no signal above background, indicating that the signal with the antisense probe is specific (data not shown). On the next section, one cell appears more intensively labeled (D). On another section, four cells display a strong signal (E).

In some places where the cells were more loosely arranged, there was a good correlation between the expression of c-myb and mim-1. Figs 4A and B illustrate cells expressing both c-myb and mim-1.

Fig. 4.

At E6 in the mesentery ventral to aorta, the patterns of expression of c-myb (A) and mim-1 (B) obtained on neighboring sections superimpose (a, aorta). (Bar: 50μm).

Fig. 4.

At E6 in the mesentery ventral to aorta, the patterns of expression of c-myb (A) and mim-1 (B) obtained on neighboring sections superimpose (a, aorta). (Bar: 50μm).

These intraembryonic precursor cells probably emi-grate into the rudiments of the hemopoietic organs (yolk sac, spleen, thymus, bursa of Fabricius) where they initiate hemopoiesis (Dieterlen-Lièvre, 1988). After 9 days of incubation, the decrease of diffuse intramesodermal hemopoiesis follows the initiation of the blood-forming activity in the hemopoietic organs.

In the yolk sac the c-myb and mim-1 transcripts are not expressed in the same cells

The haemopoietic stem cells that emerge in the ventrolateral wall of the aorta within the embryo at E3 later colonize the yolk sac where they form secondary blood islands. There they give rise to the definitive strain of erythroblasts and to myeloid cells (Dieterlen-Lièvre, 1988). Three stages of development have been investigated, E10, E12 and E14, which gave similar results. The c-myb proto-oncogene was transcribed within the bulk of some blood islands in dividing cells which had characteristic small, round and strongly fluorescent nuclei upon staining with the intercalating dye bisbenzimide (Fig. 5A). In contrast mim-1 tran-scripts were detected extravascularly, outside of these blood islands, in more mature myeloid cells character-ized by a large polymorphic and weakly fluorescent nucleus (Fig. 5B). Therefore, surprisingly, we detected mim-1 transcripts in promyelocytes which were nega-tive with the c-myb probe. Whether these promyelo-cytes arise from a common erythroid and myeloid precursor present in the blood island or emerge extravascularly is still an open question. However, we showed here that the onset of mim-1 transcription did not coincide with the transcription of c-myb during hemopoiesis in the yolk sac. This prompted us to study the expression of c-myb and mim-1 in the intraembryo-nic hemopoietic organs where the location of the major hemopoietic events has been more precisely delineated.

Fig. 5.

c-myb and mim-1 in the yolk sac at E10. (Bar: 25 μm). (A) The c-myb proto-oncogene is expressed only on the bulk of some blood islands (arrows). (B) In contrast mim-1 transcripts are detected in promyelocytes, which differentiate extravascularly. No signal is detected on blood islands.

Fig. 5.

c-myb and mim-1 in the yolk sac at E10. (Bar: 25 μm). (A) The c-myb proto-oncogene is expressed only on the bulk of some blood islands (arrows). (B) In contrast mim-1 transcripts are detected in promyelocytes, which differentiate extravascularly. No signal is detected on blood islands.

In the pancreas and in the spleen c-myb and mim-1 are specifically expressed during granulopoiesis

Between the seventh and the ninth day of incubation, granulopoiesis occurs transiently into the pancreas (Dieterlen-Lièvre, 1965). Granulocytes are the only hemopoietic cells to differentiate in this organ. They form aggregates in the vicinity of blood vessels where they are easily recognized by their réfringent cyto-plasm. In the E8 pancreas, these promyelocytes expressed both c-myb and mim-1 transcripts (Fig. 6A,B). Before the onset of granulopoiesis or later in the pancreas, no signal was obtained either with the c-myb or with the mim-1 probe (data not shown).

Fig. 6.

c-myb (A) and mim-1 (B) in the pancreas at E8. (Bar: 50 μ m) Promyelocytes located in the vicinity of blood vessels express both c-myb and mim-1 transcripts.

Fig. 6.

c-myb (A) and mim-1 (B) in the pancreas at E8. (Bar: 50 μ m) Promyelocytes located in the vicinity of blood vessels express both c-myb and mim-1 transcripts.

During the spleen ontogeny, three hemopoietic lineages are observed in different overlapping phases (Yassine et al., 1989). Beginning at E10 the spleen is first erythropoietic. From E13 to hatching granulopoie-sis is active, becoming the dominant process at E15 as erythropoiesis decreases. During the last days of incubation, the lymphocytes start immigrating into the spleen. As early as Ell granulocytic precursors cells, which are confined by a network of stromal cells, expressed both c-myb and mim-1 (Fig. 7A,B). Their number increased until they reached a peak at E15 when the signal either with the c-myb or the mim-1 probe was at its maximum (data not shown). At hatching granulocytes begin to leave the spleen. One week after hatching when granulopoiesis is over in the spleen, no signal was observed with the c-myb probe (Fig. 7C) and only a few cells still expressed mim-1 (Fig. 7D). Thus c-myb and mim-1 were coexpressed through-out the ontogeny of the pancreas and the spleen, which suggests that the c-myb gene product might be a transcriptional activator of the mim-1 gene during granulopoiesis in these organs.

Fig. 7.

c-myb and mim-1 in the spleen. (Bar: 50 μ t). (A,B) Serial sections through the spleen at Ell. (A) Granulocytic precursor cells are labeled with the c-myb probe. (B) An intense level of mim-1 mRNA is found in the same population of cells. (C,D) Serial sections through the spleen, six days after hatching. At this time granulopoiesis is over in the spleen. (C) No signal is observed with the c-myb probe. (D) Only a few scattered cells still express mim-1.

Fig. 7.

c-myb and mim-1 in the spleen. (Bar: 50 μ t). (A,B) Serial sections through the spleen at Ell. (A) Granulocytic precursor cells are labeled with the c-myb probe. (B) An intense level of mim-1 mRNA is found in the same population of cells. (C,D) Serial sections through the spleen, six days after hatching. At this time granulopoiesis is over in the spleen. (C) No signal is observed with the c-myb probe. (D) Only a few scattered cells still express mim-1.

In the thymus, c-myb does not induce the expression of mim-1

Previous reports on the expression of c-myb in the thymus have described a higher level of c-myb mRNA found in immature thymocytes than in fully differen-tiated T-cells in vivo and in vitro (Sheiness and Gardinier, 1984; Thompson et al., 1986). Our own results corroborated these studies. The c-myb probe revealed a higher expression in the cortical immature and proliferative lymphocytes than in the medullary lymphocytes (Fig. 8A). In contrast, a few scattered cells expressed mim-1 in the thymus of the chick embryo (Fig. 8B), corresponding to a few granulocytic foci, which have been previously reported (Dieterlen-Lièvre, 1988). Thus in lymphoid cells the expression of c-myb does not induce the transcription of mim-1.

Fig. 8.

c-myb and mim-1 in the thymus at E15 (c: cortex, m: medulla). (Bar: 100 μ m). (A) A higher signal is obtained with the c-myb probe in the cortical thymocytes than in the medullar thymocytes. (B) On the neighbouring section only a few scattered cells express mim-1.

Fig. 8.

c-myb and mim-1 in the thymus at E15 (c: cortex, m: medulla). (Bar: 100 μ m). (A) A higher signal is obtained with the c-myb probe in the cortical thymocytes than in the medullar thymocytes. (B) On the neighbouring section only a few scattered cells express mim-1.

At this point it is worth noticing that c-myb transcripts were also detected in proliferating epithelia either from ectodermal (epidermis), neuroectodermal or from endodermal origin (oesophagus, gizzard, intestine, cloaca; Fig. 9). Likewise in these cells c-myb transcripts were not associated with mim-1 transcripts (data not shown).

Fig. 9.

c-myb transcripts in the epithelium of the gut at E8. The regions of growing epithelium where mitotic divisions are more frequent (arrows) are labeled with the c-myb probe. (Bar: 50 μ m).

Fig. 9.

c-myb transcripts in the epithelium of the gut at E8. The regions of growing epithelium where mitotic divisions are more frequent (arrows) are labeled with the c-myb probe. (Bar: 50 μ m).

During the ontogeny of the bursa of Fabricius mim-1 transcription precedes c-myb transcription

In the bursa of Fabricius, hemopoietic differentiation is dependent on the colonization of the bursal rudiment by two distinct extrinsic hemopoietic precursors. Ac-cording to Houssaint (1987), at E9 the first cells belonging to myelomonocytic lineage enter. About one day later the bursal rudiment is colonized by lymphoid precursors. The seeding of both lineages takes place first in the mesenchyme where these hemopoietic stem ceils are located until Ell. As shown in Fig. 10 (A, C, D), at Ell c-myb transcripts were only detected in the proliferative epithelium of the bursa. Surprisingly, however, many cells in the mesenchyme of the bursa already expressed mim-1 (Fig. 10B, E). After E12 the first myelomonocytic cells migrate and start to colonize the epithelium where they differentiate in cells belong-ing to the macrophage and dendritic lineage. These cells initiate the formation of epithelial buds, which are later colonized by the B lymphoid precursor cells. The cells remaining in the mesenchyme differentiate into granulocytes (Houssaint, 1987). At E13 c-myb was still expressed in the bursal epithelium but a few réfringent cells in the mesenchyme were weakly labeled by the c-myb probe, mim-1 transcripts were abundant in scattered cells in the central part of the mesenchyme (data not shown). At E15 the signal detected with the c-myb probe decreased in the densified epithelium. Numerous cells at the periphery of the mesenchyme are labeled with the c-myb probe. At this stage in the mesenchyme the distribution of c-myb and mim-1 transcripts superimposed (Fig. 10F,G).

Granulopoiesis in the bursa of Fabricius disappears around hatching. In the one-day-old chick, lymphoid follicles had greatly developed and expressed neither c-myb nor mim-1. The mesenchymal component of the bursa was reduced to a few cells between the follicles. No cell in these sites expressed c-myb and only a weak signal was detected with the mim-1 probe (data not shown).

Fig. 10.

c-myb and mim-1 in granulopoietic cells of the bursa of Fabricius. (e, epithelia, m, mesenchyme) (A-E) Serial sections through the bursa of Fabricius at Ell. (A) c-myb transcripts are only detected in the proliferative epithelium (arrows). (B) Numerous cells in the mesenchyme already express high levels of mim-1 mRNAs. (C) A control section hybridized with the c-myb sense probe reveals that cells that are positive for mim-1 mRNA are not labeled with the c-myb probe. These cells contain cytoplasmic granules which diffuse the light and give them the same aspect with a dark-field illumination on the section hybridized with the c-myb sense probe and on the section hybridized with the c-myb antisense probe. At a higher magnification these granules appear as white spots within the cells (D; Section hybridized with the c-myb antisense probe) which are readily distinguishable from the tiny white dots above the cells which are the silver grains (E; section hybridized with the mim-1 antisense probe). (F, G) Serial sections through the bursa of Fabricius at E15. (F) c-myb transcripts are now detected in the mesenchyme as well as in the epithelia. (G) The mesenchymal cells that express c-myb also express mim-1. (A, B, C, F, G - Bar: 100 μm). (D, E - Bar: 25 μ m).

Fig. 10.

c-myb and mim-1 in granulopoietic cells of the bursa of Fabricius. (e, epithelia, m, mesenchyme) (A-E) Serial sections through the bursa of Fabricius at Ell. (A) c-myb transcripts are only detected in the proliferative epithelium (arrows). (B) Numerous cells in the mesenchyme already express high levels of mim-1 mRNAs. (C) A control section hybridized with the c-myb sense probe reveals that cells that are positive for mim-1 mRNA are not labeled with the c-myb probe. These cells contain cytoplasmic granules which diffuse the light and give them the same aspect with a dark-field illumination on the section hybridized with the c-myb sense probe and on the section hybridized with the c-myb antisense probe. At a higher magnification these granules appear as white spots within the cells (D; Section hybridized with the c-myb antisense probe) which are readily distinguishable from the tiny white dots above the cells which are the silver grains (E; section hybridized with the mim-1 antisense probe). (F, G) Serial sections through the bursa of Fabricius at E15. (F) c-myb transcripts are now detected in the mesenchyme as well as in the epithelia. (G) The mesenchymal cells that express c-myb also express mim-1. (A, B, C, F, G - Bar: 100 μm). (D, E - Bar: 25 μ m).

The mim-1 gene has been identified as a target of activation by the product of the E26 v-myb oncogene (Ness et al., 1989). We show here that c-myb can transactivate an exogenously added as well as the endogenous mim-1 promoter. The mim-1 protein was shown to be expressed in normal immature granulo-cytes from chicken bone marrow and to be down-regulated during the terminal differentiation of granu-locytes (Ness et al., 1989). Our in situ experiments confirm that mim-1 is specifically expressed during granulopoiesis. An extensive survey throughout the development of the chick embryo shows that mim-1 transcripts are found in granulopoietic organs, at the time and in the place where granulopoiesis occurs. Therefore, to our knowledge, mim-1 is the first described marker for the cells that are differentiating into the granulocytic lineage. We describe here a spatial and a temporal correlation between the transcription of c-myb and mim-1 in some hemopoietic foci in the dorsal mesentery at E6, in the pancreas and in the spleen. Providing that translational controls, which have been documented in rare occasions for gene regulatory proteins (Dollé et al., 1990), do not delay the c-myb protein expression, our data suggest that c-myb triggers mim-1 in these situations.

Quite unexpectedly however, both in the yolk sac and in the bursa of Fabricius at Ell, we detect a high level of mim-1 transcripts in cells where c-myb transcripts are not detected. The interpretation of this result indeed relies upon the presumption that the lack of signal signifies no expression. Assuming 100% hybridization efficiency (optimistic) and 2% detection efficiency (pessimistic) with a 3kb 35S-labeled c-myb probe, one mRNA molecule will give one silver grain after two weeks of exposure, which is the actual duration of the experiments presented here. Further, for the yolk sac and the bursa of Fabricius at Ell, after one month exposure, no hybridization signal is observed with the c-myb probe. Therefore these estimates suggest that one single copy of c-myb mRNA could be detected by in situ hybridization under the conditions used in this study. The sensitivity of the in situ hybridization can be also evaluated by the detection of a specific c-myb signal in proliferating epithelia (Fig. 9) where the abundance of c-myb transcripts is more than ten times lower than in hemopoietic cells (Thompson et al., 1986). The absence of c-myb transcripts accumulation cannot be readily ascertained by an independent method such as the reverse transcriptase-polymerase chain reaction be-cause, within the yolk sac or in the bursa of Fabricius at Ell, there are together mim-1 -positive c-wyb-negative cells and mim-1 -negative c-myb-positive cells. The absence of c-myb transcripts in mim-1 -positive cells may be related to a different turnover for the c-myb and the mim-1 mRNAs. Thus, after the induction of mim-1 transcription by c-myb, one may speculate that c-myb transcripts are rapidly degraded whereas mim-1 tran-scripts remain stable during several days in promyelo-cytes. According to this hypothesis, promyelocytes in the yolk sac and in the bursa of Fabricius at Ell would be more mature than promyelocytes in the pancreas or in the spleen when both c-myb and mim-1 transcripts are detected. However, in the spleen where we have described the entire process of granulopoiesis from E10 to hatching, we have not observed cells where mim-1 transcripts accumulate without a simultaneous tran-scription of c-myb. In the yolk sac, c-myb is transcribed within the bulk of some blood islands present in the vicinity of blood vessels. In contrast, mim-1 transcripts are detected extravascularly in promyelocytes. The origin of these promyelocytes is still debated. If they originate from blood islands one may argue that the c-myb protein, which is expressed in blood islands is still present in promyelocytes where mim-1 is transcribed. In our hands, all the attempts to detect the c-myb protein by immunocytochemistry gave inconclusive results. However, this hypothesis seems unlikely be-cause the half-life of the c-myb protein is short (Klempnauer et al., 1986; Lüscher et al., 1988). Alternatively cells expressing c-myb and cells express-ing mim-1 in the yolk sac may not belong to the same hemopoietic lineage. In any case, the onset of mim-1 transcription and the expression of c-myb seem unre-lated in the yolk sac. We also found mim-1 transcripts in the bursa of Fabricius at Ell, two days before appearance of the first c-myb transcripts. These results suggest that granulopoiesis in the yolk sac as well as in the bursa at Ell is independent of c-myb, and that the expression of mim-1 in this context is not induced by the c-myb protein. This hypothesis fits well with in vitro experiments showing that the myb protein-binding sites in the mim-1 promoter bind a number of cellular proteins which are immunologically distinct from v-myb or c-myb proteins (S.A. Ness unpublished result). Thus other proteins, including products of other members of the Myb family are potential candidates for the regulation of mim-1 expression in the yolk sac and the bursa.

What is the physiological significance of different regulatory pathways for the transcription of mim-1? The inducibility of mim-1 by different factors may result from different environmental conditions. Hemopoiesis in vivo requires a framework of stromal cells which provide the necessary environment for self-renewal, differentiation and maturation of the hemopoietic stem cells (for review see Dexter et al., 1984). Thus it is tempting to speculate that the differentiation of granu-locytic precursor cells in different tissues involves different signaling molecules which in turn induce the transcription of mim-1 through different regulatory pathways.

The amount of mim-1 transcripts in promyelocytes is another point that suggests different steps in the regulation of mim-1. The high level of the hybridization signal with the mim-1 probe in granulopoietic organs indicates that the mim-1 mRNAs are very abundant in promyelocytes. Indeed western blot analysis indicated that the mim-1 gene encodes one of the most abundant proteins found in these cells (Ness et al., 1989). However we show here that this high rate of transcrip-tion is not achieved in a single step in the hemopoietic foci (Fig. 3). A quantification of the number of transcripts per cell has not been possible because the signal obtained with the mim-1 probe is very intense and because the average thickness of one cell is bigger than 4 μ m, the thickness of each serial section. Nevertheless the observation of cells expressing a low level of mim-1 mRNA suggests that different regulatory elements cooperate to achieve the high level of transcription for mim-1.

The comparison between the expression patterns of c-myb and mim-1 also provides new insights into the roles of c-myb. c-myb transcripts present in lymphoid or in epithelial cells seem to be merely associated with proliferation. Previous studies have indicated that c-myb is expressed during proliferation in hemopoietic cells as well as in chicken embryo fibroblasts. In MSB-1, a transformed T cell line, the induction of c-myb expression occurs not only during the initial activation of cells to proliferate, but also in subsequent cell cycles during exponential growth. Similarly the expression of c-myb is high in immature thymocytes and decreases during T-cell differentiation in vivo (Thompson et al., 1986). In related studies, we have shown that c-myb transcripts accumulate in proliferating epidermal cells of feather outgrowth (Desbiens et al., 1991). We show here that c-myb is expressed more generally in proliferative areas of developing epithelia from endo-dermal, ectodermal and neural origin. In all these situations c-myb transcripts are never associated with the transcription of mim-1.

Beside its role in proliferation, c-myb appears to have a specific function during myeloid cell differentiation. In regard to the transformation properties of v-myb, c-myb is usually considered as a master gene of myeloid differentiation. A particularly interesting feature of v-myb is that it does not simply block differentiation, but it dictates the differentiation phenotype of the myeloid cells that it transforms. Thus, when introduced into normal or v-myc transformed macrophages, both AMV and E26 are capable of causing a “dedifferentiation” through the dominant induction of phenotypic changes characteristic of immature cells (Durban and Boettiger, 1981; Beug et al., 1987; Ness et al., 1987). By contrast, the cellular counterpart of v-myb is expressed in various cell lines and seems to act in the myeloid lineage as a specialized factor involved in a single pathway of differentiation. It seems unlikely that c-myb is respon-sible for the commitment and differentiation of granu-locytic precursor cells by activating mim-1 transcrip-tion. Rather we suggest that c-myb plays a role in regulating mim-1 transcription in cells triggered to differentiate by the action of other factors that are located in granulocytic organs. Moreover, strict pro-myelocyte-specific expression of mim-1 cannot be attributed only to the binding of c-myb to the mim-1 promoter. Indeed several recent studies, including our results, suggest that the c-myb protein does not act alone and cooperates with other tissue-specific factors (Klempnauer et al., 1989; Ibanez and Lipsick, 1990). In this regard, it is interesting that the myfc-related BAS1 protein of 5. cerevisiae activates transcription of the HIS4 gene only in conjunction with BAS2, a homeo-domain protein (Tice-Baldwin et al., 1989).

Ness et al. (1989) have previously demonstrated that E26 v-myb activates the transcription of the promyelo-cyte-specific gene mim-1, suggesting that c-myb is part of the network of interacting transcription factors that regulates mim-1 transcription. One may assume that each of these factors plays a specific role either in one tissue or at one stage of differentiation. The description of the spatial and temporal relationship between the transcription of mim-1 and c-myb has allowed us to identify cells where c-myb could trigger the transcrip-tion of mim-1. We have also found cells where mim-1 is transcribed in the absence of c-myb. These cells in the bursa of Fabricius at Ell or in the yolk sac should be amenable to a molecular analysis that will allow the identification of other transcription factors that regulate the expression of mim-1.

We would like to thank Drs F. Dieterlen, M. Fauquet, E. Houssaint, J. Coll, V. Laudet, D. Leprince and O. Albagli for critical reading of the manuscript, J. Lipsick for providing the 3 kb c-myb probe and the c-myb expression vector. We thank M.A. Mirabel, M.B. Raes and A. Marknell for excellent technical assistance, N. Devassine and M.C. Bouchez for patient typing. This work was supported by the Institut Pasteur de Lille, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique and Association pour la Recherche sur le Cancer. C.Q. is a recipient of a fellowship from the Ministère de la Recherche et de la Technologie.

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