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.

A number of studies have focused upon the expression of protooncogenes during embryonic life. Presence of pp 60 c-src has been related to neuronal differentiation in various animal groups, including birds (Cotton & Brugge, 1983; Levy et al. 1984; Sorge et al. 1984), fish and frogs (Shartl & Barnekow, 1984) and Drosophila (Simon et al. 1985). Activity of the fos protooncogene is particularly well documented in the case of the mouse embryo. High levels of c-fos were detected in the late placenta (Müler et al. 1982; Adamson et al. 1985; Deschamp et al. 1985) and in growing bones (Müller & Verma, 1984; Dony & Gruss, 1987). Other protooncogenes may be expressed in one cell lineage at a specific time. This is the case for intl which was found in the neural tube of mouse embryos (Shackleford & Varmus, 1987; Wilkinson et al. 1987) and in postmeiotic mouse male germ cells (Shackleford & Varmus, 1987). A high expression of mos has also been found in mouse germ cells (Propst et al. 1987; Mutter & Wolgemuth, 1987). C-ras is widely expressed both during embryonic and adult life in mice and humans. Nevertheless its expression appears enhanced in a variety of tissues irrespective of their differentiated or proliferative state (Furth et al. 1987; Leon et al. 1987; Tanaka et al. 1987). We became interested in the expression of c-myc during development when we found that the MC29 virus containing the v-myc gene induced heart rhabdomyosarcomas in the young avian embryo (Saule et al. 1987; Al Moustafa et al. 1988). These tumours, which appear restricted to this particular target, develop only when the embryo is inoculated before E4. Knowing the time and tissue pattern of c-myc expression in normal embryos might help to understand the basis for this particular in vivo effect of v-myc.

The c-myc gene product is a 62 × 103 Mr polypeptide located in the cell nucleus (Alitalo et al. 1983; Persson & Leder, 1984; Beimling et al. 1985). Although DNA-binding properties of the protein have been docu-mented (Moelling et al. 1986; Studzinski et al. 1986), its functions remain unknown. Various reports have shown that c-myc increases after serum or growth-factor stimulation of quiescent cells (Kelly et al. 1984; Armelin et al. 1984; Campisi et al. 1984; Bravo et al. 1985; Dean et al. 1986). On the other hand, c-myc is down-regulated during induced differentiation in several cell types (Westin et al. 1982; Reitsma et al. 1983; Campisi et al. 1984; Gonda & Metcalf, 1984). Moreover, the introduction of an exogenous c-myc gene in a variety of cell types blocks terminal differentiation (Coppola & Cole, 1986; Denis et al. 1987; Maruyama et al. 1987). However, myc transcripts have also been detected in differentiated cells both in vivo (Ruppert et al. 1986) and in vitro (Thiele et al. 1985; Curran & Morgan, 1985; Dotto et al. 1986).

Myc protooncogene expression during development has been studied in human placenta (Pfeiffer-Ohlsson et al. 1984, 1985; Goustin et al. 1985) and in Xenopus embryos (Godeau et al. 1986; King et al. 1986; Taylor et al. 1986; Nishikura, 1987). Zimmerman et al. (1986) have described in mice a differential expression of the myc family members indicating that N- and L-myc expression is regulated during embryogenesis. These studies have been mainly carried out at the biochemical level, so that the expression of myc in specific cell types, tissues or organs, could not be defined precisely. Here we report an extensive immunohistological study bearing on the in situ distribution of the c-myc protein in chick and quail embryos. The transcription of several protooncogenes during chick development is being analysed in a parallel study using in situ hybridization (B. Vandenbunder et al. unpublished data). A few results from this study pertaining to c-myc mRNA in E3, E6 and E8 chick embryos will be described for comparison with the protein distribution. C-myc appears to be regulated at the transcriptional and translational levels according to specific patterns during the organogenesis period.

Staging and preparation of the embryos

White Leghorn chicken and Japanese quail(Coturnix coturnix japonica) embryos obtained from commercial sources were used for this study. The eggs were incubated at 38 ± 1 °C in a humidified air chamber. Embryos of both species from 1 to 17 days of development (El to El7, E0 being the first day of development) were staged according to Hamburger & Hamilton (HH) (1951). The embryos were routinely fixed either in 3·7% formaldehyde or 3·7% paraformaldehyde in sodium phosphate buffer (0·1 M-Na2HPO4, 0·1 M-NaH2PO4) for 1-4 h. After extensive washes in PBS they were embedded in a PBS-sucrose solution (15% wt/vol) overnight and frozen in Tissue Tek (Lab-Tek product) in liquid nitrogen. Cryostat sections (Bright Instrument Co. Ltd, Huntingdon, England) were cut at 3-4 µmin order to provide one layer of cell nuclei. They were collected on slides coated with gelatin according to the procedure of Lohman et al. (1981). The staining was performed either immediately after sectioning or after 12-14 h at -20 °C. Both procedures yielded identical results. Two other fixation procedures were also compared to those used routinely: embryos were either embedded in PBS-15% sucrose solution and sectioned as previously described prior to fixation either in 100% methanol or in 99% methanol-1% glacial acetic acid or were fixed unembedded. Table l summarizes the range and number of samples observed in this study. All the embryos were sectioned serially. For older embryos (HH 27 to HH 43, i.e. E8 to E14), in some samples only the heart was examined(* in the Table).

Dorsal root ganglia cell culture

Dorsal root ganglia were removed from E10 chick embryos over a length encompassing thoracic and lumbosacral levels of the rachis. Approximately 50 pooled ganglia were treated as described in Xue et al. 1985.

Antibodies

The c-myc protein was detected by means of a polyclonal serum raised in rabbits against a bacterially expressed polypeptide corresponding to human exon 3 (Ferre et al. 1986). This antiserum specifically recognizes the human and avian c-myc proteins.

Control experiments were performed either with a preinoculated rabbit serum or with the anti-myc serum blocked by preincubation with an excess of the corresponding bacterially expressed polypeptide.

Premuscular or muscular cells were identified by means of a monoclonal antibody, 13F4, which recognizes avian myoblasts as soon as they become committed to the myogenic lineage within the somites and the heart(Rong et al. 1987).

The neuronal cells of the dorsal root ganglia were revealed by means of an anti-160xla3 Mr neurofilament monoclonal antibody(Amersham International).

Immunological procedures

The c-myc protein, the 13F4 antigen and cell nuclei were revealed by triple labelling on the same section. All the antibodies except the 13F4 culture supernatant were diluted in phosphate buffer. Incubations were performed in a moist chamber.

After cryostat sectioning, the slides were washed twice in PBS and placed in PBS-5% NCS (Newborn Calf Serum, Gibco Europe) for 30 min. From El to E3·5 the anti-myc antibody was used diluted 1/100. At that point, bound antiserum was revealed by incubation with the rhodamineconjugated goat anti-rabbit lg diluted 1/50(Nordic Laboratories) for 45 min. From E4 to El7, the anti-myc serum was diluted 1/50 and the streptavidin-biotin system (Amersham International) was used. In all cases, the procedure was identical: the slides were covered with a biotinylated sheep anti-rabbit lg diluted 1/50 for 45 min, rinsed twice in PBS, and treated with streptavidin-Texas Red diluted 1/100 for 45 min followed by two rinses in PBS. At that point, cells of the myogenic lineage were stained by incubating the sections for 45 min with 13F4 supernatant; after two rinses in PBS, FITC-conjugated goat anti-mouse lg (Biosys, France) diluted 1/50 was applied. Cell nuclei were stained by Hoechst 33258 (Serva) which binds DNA covalently(Hilwig & Groop, 1972). The staining solution was prepared by diluting 10 mg Hoechst 33258 in 250 ml PBS. The sections were immersed for 1 min in the Hoechst 33258 solution and rinsed 3 times in PBS. A few drops of PBS were added and the slides were covered with a glass coverslip which was sealed with nail polish.

The sections were observed on a Leitz photomicroscope equipped with epifluorescence, using a Ploemopak filter A for Hoechst stain and filter 12 for fluoresceine and filter N2 for Texas Red. Photographs were taken on T-Max 400 (Kodak).

In situ hybridization

Preparation of the RNA probe

The RNA probe used in this study was synthesized from a 0·6 kb Eco RI fragment of the 3’ part of the chicken c-myc gene (Saule et al. 1984) subcloned into the polylinker of pSP 64 and 65 (Promega Biotech) by standard methods.

RNA was transcribed from truncated plasmids in 20 µI reaction mixtures by the SP6 RNA polymerase (Promega Biotech) using 20 µ Ci [3 H]UTP and 20 µCi [3H]GTP (40 Ci mmole-, NEN) for in situ analysis. We checked by electrophoresis on 2·2 M-formaldehyde/2·2% agarose gels that the sizes of the sense and of the antisense labelled transcripts were identical. The probes for in situ hybridization were submitted to a limited alkaline hydrolysis according to Cox et al. (1984). After ethanol precipitation, 2 mm°-EDTA with 2 mg ml−1 the probes were dissolved in E. coli tRNA and stored at -80 C for a maximum of 6 months.

In situ hybridization

In situ hybridization was performed essentially by the method of Cox et al. (1984). After deparaffinization and hydration, slides were incubated in 0·l M-glycine, 0·2 m-Tris-HCl pH 7·4 during 10 min at room temperature, treated with proteinase K (1 µg m1−1 1 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 as described by Cox et al. (1984).

Immediately prior to hybridization, the probes were denatured at 80 °C for 3 min and diluted in the hybridization buffer (Cox et al. 1984). 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 in the hybridization buffer. Thereafter, the slides were washed in 4xSSC at room temperature and the sections treated with RNAse A (10µg ml−1, type III A, Sigma) for 30 min at 37 °C. The final washes were in 0·1xSSC at 60-70 °C during 10min. The slides were dehydrated and dipped in a nuclear track emulsion (Kodak NTB2 diluted 1: 1 with 0·6 M ammonium acetate). The exposure was routinely carried out during 3 months. After photographic development (Cox et al. 1984), the coverslips were mounted. The sections were examined under phase-contrast and dark-field illumination on a Zeiss microscope (IM35, Axioskop).

Immunological procedures and controls

Influence of the fixation procedure on the distribution of the signal within the cell nuclei

Sections of E3 chicken embryos were used to compare the signal obtained after different fixation procedures (see Material and methods). Fixation in 100% methanol resulted in an intense but diffuse staining of both nucleus and cytoplasm. 99% methanol-1% glacial acid acetic provided detectable staining in the trunk, where whole cell nuclei were stained. In contrast, no myc signal could be detected in the head, where the neural tissue exhibited a general diffuse fluorescence. In our hands, the most satisfactory nuclear staining was obtained in embryos fixed either with 3·7% paraformaldehyde or 3·7% formaldehyde. In this case, myc fluorescence appeared as small fluorescent granules excluded from the nucleolar areas. Such a distribution was described in MC29-infected cells with similar fixation procedures (Hann et al. 1983; Spector et al. 1987).

Control antibodies

Two controls were performed for this study: (1) preinoculated rabbit serum, (2) anti-myc serum blocked by preincubation with an excess of the corresponding bacterially expressed polypeptide. The controls were used undiluted in both cases and no signal could be detected on any section.

Influence of antibody dilution and time of contact on detection sensitivity

Two dilutions of the c-myc antibody have been used (see Material and methods). While c-myc was readily detected between El and E4 with antiserum diluted 1/100 followed by rhodamine-conjugated sheep antirabbit serum, the streptavidin-biotin Texas Red amplification system was nevertheless used routinely, since it provided both a brighter and a less diffuse signal. From E4 onwards, it was not possible to detect c-myc with the 1/100 dilution and the rhodamine conjugate. Consequently, the 1/50 dilution and the streptavidine-biotin amplification system were used from E4 to El7. From ES onwards, it was also necessary to increase the contact time between the sections and the serum from 1 to 2 h. With this protocol, the c-myc protein was revealed in a variety of tissues. When dilutions of 1/100 were applied to sections of E7 embryos, the only myc-positive nuclei were located in the mesenchyme of the head and the body wall. The technique used throughout this study did not permit quantification of myc protein. However, an order of magnitude could be inferred from the necessity of increasing several folds the sensitivity of the method after E6·5. Furthermore, in order to evaluate the ratio of c-myc-positive nuclei to total nuclei, double labelling with Hoechst nuclear stain was carried out.

Tissue distribution of the c-myc protein during avian development

Early development (El to E2·5)

The c-myc protein can be detected in the avian embryo at El, the earliest time we investigated. At that time, the germ layer is composed of two zones: the area opaca which will form the yolk sac and the area pellucida where the embryo develops. The area opaca was studied from El to E2·5. A strong signal was present in endodermal cell nuclei during this period. In comparison, ectodermal and mesodermal cells displayed a weaker signal. This particular pattern was maintained during the development of the blood islands. In the embryonic area proper, some myc protein was also detected in the cephalic neural folds (Fig. 1). Myc protein subsequently appeared in all tissues of the head at the 8-15 pairs of somites stage. Nevertheless the signal was higher in the pharyngeal epithelium than in the neural tube, which generally was weakly stained.

Fig. 1

Transverse section through the posterior neural plate of a 2-somite-stage chick embryo. Double labelling with anti-myc (A) and Hoechst nuclear stain (B). All the cells express the c-myc protein except those dividing in the neural plate (arrowheads). np, neural plate; ch, chorda; m, mesoderm; e, endoderm. Bar, 50 µm.

Fig. 1

Transverse section through the posterior neural plate of a 2-somite-stage chick embryo. Double labelling with anti-myc (A) and Hoechst nuclear stain (B). All the cells express the c-myc protein except those dividing in the neural plate (arrowheads). np, neural plate; ch, chorda; m, mesoderm; e, endoderm. Bar, 50 µm.

Newly formed somites and the segmental plate expressed low levels of c-myc protein. The signal remained weak in the somites until they organized into an epithelial structure. From that time, somitic cells began to express the c-myc protein more strongly. No change in c-myc expression was observed when the dermomyotomal structure appeared. In the segmental plate and the forming somites, a few randomly distributed cells displayed a strong myc signal. At the level of segmented somites, myc protein was ubiquitous in the ectoderm, mesoderm and endoderm. In the neural tube, c-myc protein expression was low until E2 (Fig. 2). We noted an increase in the signal of neural crest cells at the onset of their migration (Fig. 3).

Fig. 2

Transverse section through the pharyngeal level of an 8-somite-stage chick embryo. At this stage, the neural tube displays little myc signal. Anti-myc immunofluorescence (IF). ao, aorta; ch, chorda; e, endoderm; ec, ectoderm; me, myocardium; nt, neural tube; ph, pharynx. Bar, 50 μm.

Fig. 2

Transverse section through the pharyngeal level of an 8-somite-stage chick embryo. At this stage, the neural tube displays little myc signal. Anti-myc immunofluorescence (IF). ao, aorta; ch, chorda; e, endoderm; ec, ectoderm; me, myocardium; nt, neural tube; ph, pharynx. Bar, 50 μm.

Fig. 3

Transverse section through somites 14-15 of an 18-somite-chick embryo. Double labelling with anti-myc (A) and Hoechst nuclear stain (B). Migrating neural crest cells (arrows) have brighter nuclei than the adjacent tissues. Nore rhe presence of a positive red cell nucleus in the aorta (arrowhead). Cytoplasmic positivity in this cell is due to nonspecific yellow autofluorescence characteristic of red cells. Bar, 50 µ m.

Fig. 3

Transverse section through somites 14-15 of an 18-somite-chick embryo. Double labelling with anti-myc (A) and Hoechst nuclear stain (B). Migrating neural crest cells (arrows) have brighter nuclei than the adjacent tissues. Nore rhe presence of a positive red cell nucleus in the aorta (arrowhead). Cytoplasmic positivity in this cell is due to nonspecific yellow autofluorescence characteristic of red cells. Bar, 50 µ m.

Organogenesis period

We place the beginning of this period at E3·5 (Hamilton, 1952). The general features can be summarized as follows. Myc was widely distributed among mesoderm-derived tissues. The ectoderm showed a transient increase in expression during palate and feather follicle formation. The endoderm did not express a detectable level of the protein during the early organogenesis period.

On the whole, the pattern of development detailed below holds true for both quail and chick species, with the expected delay in the developmental pattern in the chick. Other differences will be noted as they occur in each tissue.

Somites

The myotome was the first tissue in which c-myc protein expression appeared developmentally regulated. High amounts of the protein were present in most myotomal cell nuclei as soon as the myotomal structure emerged from the dermomyotome, i.e. as soon as cells could be marked by monoclonal antibody 13F4 (Fig. 4A,B). This strong labelling persisted during the whole period of myotomal bud.migration (Christ et al. 1983), (Fig. 4C,D) and ceased as the myotomal cells became organized into premuscular masses (i.e. at E4·5-5). The c-myc protein then decreased to a level similar to that of adjacent mesenchymal tissues (Fig. 4E,F). In contrast, during the whole period described, the signal in the dermatomal and the sclerotomal cell nuclei appeared similar to that in other embryonic tissues.

Fig. 4

Developmental modulation of the c-myc protein signal in chick myotomal cell nuclei. A,C,E: anti myc IF; B,D,F: 13F4 IF. Bar, 100 µ m. (A,B) E2·5: the myc signal (A) is very bright in myotomal cell nuclei (arrowheads). m, myotome; d, dermatome: s, sclerotome. 13F4 immunofluorescence delineates myotomal cells (B). (C,D) E3 chick embryo. Myc expression is maximal in myotomal cells. (E,F) E4 chick embryo. Myc decreases in migrating myotomal cells.

Fig. 4

Developmental modulation of the c-myc protein signal in chick myotomal cell nuclei. A,C,E: anti myc IF; B,D,F: 13F4 IF. Bar, 100 µ m. (A,B) E2·5: the myc signal (A) is very bright in myotomal cell nuclei (arrowheads). m, myotome; d, dermatome: s, sclerotome. 13F4 immunofluorescence delineates myotomal cells (B). (C,D) E3 chick embryo. Myc expression is maximal in myotomal cells. (E,F) E4 chick embryo. Myc decreases in migrating myotomal cells.

Endoderm

Low amounts of c-myc protein were detected during morphogenetic movements of the gut. During the whole period of organogenesis, endodermderived epithelia such as the oesophageal, tracheal or broncheal endoderm did not express detectable levels of c-myc protein, even when numerous mitotic figures were visible after Hoechst 33258 staining. The c-myc protein was never detected in quail hepatocytes, though it was present along the external border of the organ (i.e. in mesothelial cells) (Fig. 5C). In the chick, myc staining was detected in a few cell nuclei of the liver as organogenesis occurred. In contrast, in situ hybridization technique revealed numerous mRNA copies in the liver at E3 (compare Fig. 5A and C,D). The mRNA signal decreased at E6 but still remained detectable at E8, though during the whole period no myc protein could be detected.

Neural primordia

The c-myc protein was expressed in all cells of the central nervous system (i.e. the brain and the spinal cord) until E5. From E5 to E7, the signal disappeared in most cell nuclei of the dorsal plate of the prosencephalon. It remained present in most nuclei of the floor plate at least until E7. In the floor of the diencephalon, we detected clearly delineated paired groups of cells where most nuclei were positive when compared to those of adjacent cell groups. These groups of cells presumably represent ventral nuclei of the diencephalon. In the other parts of the brain, the signal did not appear restricted to particular groups of cells. No significant modulation of the c-myc signal was present in the otic vesicle which remained positive during the entire period examined until E14. The optic vesicle, on the other hand, exhibited a very precise pattern of c-myc expression in the course of development. The optic vesicle and, subsequently, the eye are composed of four main tissues; the cornea and the lens have a placodal origin, the pigmental retina and the neuroretina originate from the diencephalon. It is generally thought that the central zone of the retina matures more rapidly than the lateral zones (Romanoff, 1960). At E3, most cells of the optic vesicle displayed a myc protein signal (Fig. 6B). Thereafter, in the central zone of the neuroretina, the distribution of c-myc changed from E4·5 to E12; while the protein was present in most nuclei at E4·S, it became progressively restricted up to E7. Fig. 6E shows this central zone of quail retina at E6·5. At this stage in this zone, though most cells of the neuroretina expressed c-myc, the signal was higher in the ganglionar layer. In contrast, in the lateral zones of the neuroretina, all cells displayed a similar signal. Most cell nuclei became positive in the ganglionar layer at E9 (Fig. 6H). In addition, at Ell, some nuclei appeared positive in the future inner horizontal layer (Fig. 6K). At El7, these cells were still positive. In the pigmental retina, myc protein was detected in all nuclei on day 6. One day layer, only some cells remained positive in the central zone (which synthesize large amounts of pigments), while all cells in the external zones (which synthesize little pigment) still expressed c-myc.

Fig. 5

Myc expression in the liver. Comparison of RNA and protein. (A) in situ hybridization; (B,E) Hoechst nuclear stain; (C,D) anti-myc IF. Bars, 200 µm in A; 100 µm in B-E. (A) Transverse section of an E3 chick embryo at the level of the liver. g, gut; h, heart; I, liver. (B,C) Liver of an E3 chick embryo. Note that mesothelial cells (thin arrows) and red blood cells (arrowheads) are positive. dv, ductus venosus. (D,E) Liver of an E4 quail embryo. Whereas the RNA signal is strong in the liver (arrows in A), the protein signal is undetectable at all stages.

Fig. 5

Myc expression in the liver. Comparison of RNA and protein. (A) in situ hybridization; (B,E) Hoechst nuclear stain; (C,D) anti-myc IF. Bars, 200 µm in A; 100 µm in B-E. (A) Transverse section of an E3 chick embryo at the level of the liver. g, gut; h, heart; I, liver. (B,C) Liver of an E3 chick embryo. Note that mesothelial cells (thin arrows) and red blood cells (arrowheads) are positive. dv, ductus venosus. (D,E) Liver of an E4 quail embryo. Whereas the RNA signal is strong in the liver (arrows in A), the protein signal is undetectable at all stages.

Fig. 6

Myc protein expression in the retina. (A,D,G,J) Phase contrast; (B,E,H,K) anti-myc IF; (C,F,I,L) Hoechst nuclear stain. Bar, SO µm. (A,B,C) Optic vesicle of an E3 chick embryo. All the cells express the c-myc protein. n, neuroretina; pr, pigmental retina. (D,E,F) Central zone of the retina of an E6·5 chick embryo. Most cell nuclei express c-myc. Note that nuclei of the internal ganglionar layer (igl) appear brighter (arrowheads). (G,H,I) Central zone of the retina of an ES chick embryo. Few pigment cells are visible in the pigmental retina (black arrowheads in G). Most cells of the internal ganglionar layer are positive (arrowheads). (J,K,L) Central zone of the retina of an ElO chick embryo. Pigment accumulates in the pigmental retina (black arrowheads in J). Cells of the internal ganglionar layer (white arrowheads in K and L) display c-myc. Note that some of the precursor cells in the internal horizontal layer are beginning to express c-myc (thin arrows in K) and that the internal ganglionar layer (white arrowheads in L) has separated from the other layers.

Fig. 6

Myc protein expression in the retina. (A,D,G,J) Phase contrast; (B,E,H,K) anti-myc IF; (C,F,I,L) Hoechst nuclear stain. Bar, SO µm. (A,B,C) Optic vesicle of an E3 chick embryo. All the cells express the c-myc protein. n, neuroretina; pr, pigmental retina. (D,E,F) Central zone of the retina of an E6·5 chick embryo. Most cell nuclei express c-myc. Note that nuclei of the internal ganglionar layer (igl) appear brighter (arrowheads). (G,H,I) Central zone of the retina of an ES chick embryo. Few pigment cells are visible in the pigmental retina (black arrowheads in G). Most cells of the internal ganglionar layer are positive (arrowheads). (J,K,L) Central zone of the retina of an ElO chick embryo. Pigment accumulates in the pigmental retina (black arrowheads in J). Cells of the internal ganglionar layer (white arrowheads in K and L) display c-myc. Note that some of the precursor cells in the internal horizontal layer are beginning to express c-myc (thin arrows in K) and that the internal ganglionar layer (white arrowheads in L) has separated from the other layers.

In the spinal cord, the signal, widely distributed from E2·S to E4, persisted until E13, becoming progressively restricted to some neurones of the ventral and dorsal horns; it disappeared thereafter. Ependymal cell myc protein expression decreased to low levels at this same age. The c-myc protein is developmentally regulated in the cranial ganglia as early as E3·5. From that stage, only a few nuclei remained positive until Ell. These large nuclei, faintly stained by Hoechst 33258, probably represent a population of precursor cells (Kahn, 1973). Trunk dorsal root ganglia cells displayed the same pattern from E4 onwards. This pattern persisted during development: embryos at E9 and E14 still exhibited similar staining. Fig. 7B shows a dorsal root ganglion of an E13 chick embryo where bright neuronal nuclei are clearly present. In contrast, glial cell nuclei in nerve fascia of the trunk were never positive after c-myc antibody treatment. In the quail, the pattern was similar except that it was not possible to detect a signal at E14. The presence of a strong c-myc signal was also observed in cultures of ElO ganglionar cells in a pattern similar to those observed in situ (Fig. 7E).

Fig. 7

Myc protein expression in neurones. (A,B,C) Section through a cervical dorsal root ganglia of an E13 chick embryo. (A) Hoechst nuclear stain. (B) Anti-myc IF. (C) Phase contrast. Note that neuronal cells display weaker Hoechst staining than non-neuronal cells. A subpopulation of neuronal cells display a strong nuclear signal (arrowheads). nf, nerve fascia. (D,E,F) Neurone in a culture of ElO chick embryo dorsal root ganglia. (D) Anti-neurofilament IF. (E) Anti-myc IF. (F) Phase contrast. Note the strong signal displayed by the neurone (arrowhead). Non-neuronal cells also display a signal. Bar, 50 µm.

Fig. 7

Myc protein expression in neurones. (A,B,C) Section through a cervical dorsal root ganglia of an E13 chick embryo. (A) Hoechst nuclear stain. (B) Anti-myc IF. (C) Phase contrast. Note that neuronal cells display weaker Hoechst staining than non-neuronal cells. A subpopulation of neuronal cells display a strong nuclear signal (arrowheads). nf, nerve fascia. (D,E,F) Neurone in a culture of ElO chick embryo dorsal root ganglia. (D) Anti-neurofilament IF. (E) Anti-myc IF. (F) Phase contrast. Note the strong signal displayed by the neurone (arrowhead). Non-neuronal cells also display a signal. Bar, 50 µm.

To summarize, myc was expressed in all cells of the neural primordia, then appeared to become restricted to neuronal precursors. It remained expressed in the nuclei of a large proportion of postmitotic neurones as late as E14. Extinction occurred progressively thereafter.

Blood cells and vessels

With the hypersensitive streptavidin-biotin method, it was possible to observe the c-myc protein signal in the nuclei of all circulating red blood cells from E2 to E3·5. Thereafter, the proportion of myc-positive cells in the blood progressively decreased becoming almost null at E5·5. Myc was detected in the diffuse intraembryonic hemopoietic foci at E5-E6·5 (Fig. 8B). These foci, identified previously by various cytochemical techniques are aggregates of erythropoietic or granulopoietic cells (Dieterlen-Lievre & Martin, 1981). Two days later, no signal could be detected although these cells were clearly recognizable (Fig. 8D,F). In quail embryos, cell nuclei in the wall of arteries displayed a strong signal from E6 to E8. No such expression was detected in the veins at any time during embryonic life. In contrast, no myc signal was detected in the arterial walls of chicks from ES to ElO while veins displayed some staining.

Fig. 8

Myc protein expression in intraembryonic hemopoietic foci. (A,D) Hoechst nuclear stain; (B,E) anti-myc IF; (C,F) phase contrast. (A,B,C) Intraembryonic foci in an E6 quail embryo. Note strong specific nuclear stain of the hemopoietic cells (arrowheads) (compare with adjacent tissues). ao, aorta. The hemopoietic cells appear refractile in phase contrast. (D,E,F) lntraembryonic hemopoietic cells in an E9 quail embryo. The hemopoietic focus is clearly visible (arrowheads). Maturing blood cells have become negative; mesenchymal cells display a weak signal. ic, internal carotid artery. Bar, 50 µm.

Fig. 8

Myc protein expression in intraembryonic hemopoietic foci. (A,D) Hoechst nuclear stain; (B,E) anti-myc IF; (C,F) phase contrast. (A,B,C) Intraembryonic foci in an E6 quail embryo. Note strong specific nuclear stain of the hemopoietic cells (arrowheads) (compare with adjacent tissues). ao, aorta. The hemopoietic cells appear refractile in phase contrast. (D,E,F) lntraembryonic hemopoietic cells in an E9 quail embryo. The hemopoietic focus is clearly visible (arrowheads). Maturing blood cells have become negative; mesenchymal cells display a weak signal. ic, internal carotid artery. Bar, 50 µm.

Hearts

In both species, all cell nuclei in the three germ layers, pericardium, myocardium and endocardium, expressed the c-myc protein from the tubular stage to the completion of organogenesis (E2 to E4). The myc signal was slightly brighter in the endocardium than in the other layers and remained unchanged until E6·5. At that time, myc negative areas distributed at random were found in the external ventricular wall (Fig. 9B). The c-myc protein was particularly well expressed in the forming heart valves (not shown). At E8 and E10, large

Fig. 9

Myc expression in the heart. (A,F) 13F4 IF; (B,D) anti-myc IF; (C,E) Hoechst nuclear stain. (A,B,C) Section through the ventricle of an E6·5 chick embryo. Some myocardial cell nuclei appear positive (arrowheads). Compare to the regular distribution of nuclei visible with Hoechst stain (C). my, myocardium; en, endocardium; ep, epicardium. (D,E,F) Section through the ventricle of an E10 quail embryo. The leftside half of the section is photographed with filter A (Hoechst stain, E), the right side is photographed with filter I2 (13F4 IF, F). The external myocardium is negative. The circular orientation of external myocardium fibres is especially visible in E. Bar, 50 μm.

Fig. 9

Myc expression in the heart. (A,F) 13F4 IF; (B,D) anti-myc IF; (C,E) Hoechst nuclear stain. (A,B,C) Section through the ventricle of an E6·5 chick embryo. Some myocardial cell nuclei appear positive (arrowheads). Compare to the regular distribution of nuclei visible with Hoechst stain (C). my, myocardium; en, endocardium; ep, epicardium. (D,E,F) Section through the ventricle of an E10 quail embryo. The leftside half of the section is photographed with filter A (Hoechst stain, E), the right side is photographed with filter I2 (13F4 IF, F). The external myocardium is negative. The circular orientation of external myocardium fibres is especially visible in E. Bar, 50 μm.

c-myc protein negative areas were present in the external ventricular myocardium (Fig. 9D). The extent of these areas varied from one embryo to another, covering the whole external ventricular wall in some cases while the inner ventricular myocardium never exhibited such a phenomenon. No such negative areas were found in E13-14 hearts. From E6·5 to El4 both endothelial and epicardial cells expressed the c-myc protein. To summarize a patchy expression seems characteristic in the myocardium between E6·5 and E10.

Chondrogenic tissues

The c-myc signal was particularly intense in comparison with surrounding tissues in prechondrogenic condensations at E6. This was true both at the cranial and trunk levels. In the head, prechondrogenic areas displayed higher amounts of c-myc protein than adjacent neural tissues. This situation persisted until the basal chondrocranium was almost complete (i.e. E7). The c-myc signal was particularly bright in the sclerotomal, branchial and appendicular prechondrogenic condensations. This pattern was transient and the levels of c-myc decreased on E7.

Skin

At the same time (i.e. E5 to E7), the skin pattern of c-myc expression also varied. The protein was found in the ectoderm at early stages of development; thereafter, its expression progressively decreased becoming almost null on E6. The epitrichial cell layer, however, constantly expressed detectable levels of c-myc from E4 to Ell. No particular pattern of expression was observed in the dermis until the beginning of feather formation. At that time (E5), strongly positive c-myc areas were detected in groups of mesodermal cells where the first feather buds were going to appear, i.e. the thigh and breast (Romanoff, 1960), while the signal persisted in mesodermal nuclei; ectodermal nuclei also became positive for c-myc protein at E6·5, at the time when epitheliomesenchymal interactions occur in the skin. The myc-positive ectodermal areas were clearly restricted to the myc-positive mesodermal areas. A similar pattern of c-myc mRNA distribution was observed with the in situ hybridization method in feather buds of the spinal pteryla at E8. Neither cellular condensation nor mitoses were observed with Hoechst 33258 in these areas at E8. Other c-myc-protein-positive areas appeared according to the spatial sequence of feather bud formation. Positivity progressively reappeared in the whole ectoderm from E9. When feather buds grew in the pterylae, the c-myc signal was most often restricted to the mesoderm while positivity was weak in the ectoderm. In contrast, ectoderm in the apteral zones expressed the c-myc protein. Myc was preferentially located in the pulp of formed feathers while ectodermal structures displayed low fluorescence.

Immunocytological staining has enabled us to identify the cell types whose nuclei store detectable c-myc protein and to describe the variations of the c-myc content in these cell types during development of the avian embryo. Adamson et al. (1985) studied the effects of the fixation procedure upon the c-fos signal conservation and demonstrated that some procedures resulted in a strong nonspecific fluorescence. Performing a study on c-fos protein expression during murine development, they demonstrated that embryonic mouse tissues expressed varying levels of c-fos protein depending on the organs examined. In late embryonic or adult tissues, fos could be detected only by increasing either the antibody titre or the time of contact. Our results concerning the c-myc protein are similar. In E2 to E4 embryos, c-myc could be detected in all tissues by using a rhodamine-conjugated goat anti-rabbit as second antibody. After E4 (i.e. at the onset of organogenesis), a decrease in c-myc expression occurred and the protein could only be revealed with the streptavidin-biotin amplification method. Thereafter, three patterns of c-myc positivity could be distinguished.

  1. Site-specific time-restricted expression related to organogenetic processes. This pattern was found in prechondrogenic condensations, intraembryonic hemopoietic cell foci, and presumptive feather buds. In prechondrogenic tissues and hemopoietic cell foci, myc was transiently expressed at a comparatively high level between E5·5 and E7. The signal decreased until E9 in prechondrogenic tissues, and was no longer detected in hemopoietic cell foci at E9. In birds, diffuse intraembryonic foci are the first site where hematopoiesis occurs in the embryo proper (Dieterlen-Lievre & Martin, 1981), prior to the initiation of this function in the spleen or in the bone marrow. The blood cell precursors, which multiply actively and differentiate, are the progeny of the cells arising in the periaortic region at E2 and E3 (Dieterlen-Lievre, 1984; Cormier et al. 1986; Cormier & Dieterlen-Lievre, 1987).

    Transient variations in c-myc mRNA expression have been detected previously during DMSO-induced differentiation of murine erythroleukemia cells (Lachman & Skoultchi, 1984), in the F9 teratocarcinoma cell line induced to differentiate (Dony et al. 1985) and in myocytes during myogenesis (Endo & Nadal-Ginard, 1986). It is commonly thought that down regulation of the c-myc gene occurs as differentiation begins; however, some authors have observed a rapid biphasic change in c-myc expression (i.e. overexpression, sudden decrease and reexpression) in some differentiating cells (Lachman et al. 1985). It is possible that such variation acts as a commitment signal and that we detected in vivo a similar phenomenon.

    More often, the c-myc expression is reported to be switched off during terminal differentiation in a variety of cell types. This was the case for retinoic-acid-induced (Campisi et al. 1984) or vitamin D-induced F9 cells (Reitsma et al. 1983) and for differentiating murine myeloid erythroleukemia cells (Gonda & Metcalf, 1984).

  2. Permanent absence of expression in the endodermal lineage. The myc protein was not detected in proliferating endoderm-derived tissues under our experimental conditions. In contrast, in situ hybridization detected a strong signal in the liver at E3 and E6. A similar situation has been described by Blanchard et al. (1985) for G0 arrested cells which transcribed high rates of c-myc mRNA while no c-myc protein could be detected, suggesting a control at the post-transcriptional level. Mitosis and meiosis in mouse male germ cells have been shown to occur with very few myc transcripts present (Stewart et al. 1984). Thus, in endoderm-derived cells, proliferation or differentiation may occur with very low levels of c-myc protein present.

  3. Continued expression at the time of differentiation. In dorsal root ganglia, the birthdate of neurones is E8 (McMillan-Carr & Simpson, 1978). Tritiated thymidine incorporation experiments have shown that these cells are postmitotic after E7 when we still detect c-myc (Fujita, 1962; Fujita & Horri, 1963; Kahn, 1973). We found the c-myc signal at E13 in the chick dorsal root ganglia neurones. No observations were carried out in the quail at comparable stages.

    Ruppert et al. (1986) have also found high amounts of c-myc transcripts in Purkinje cells of the mouse cerebellum during their dendritic arborization. Thiele et al. (1985) demonstrated that the levels of c-myc mRNA were constant either in proliferating or in retinoic-acid-induced neuroblastoma cells while, at the same time, N-m ye dramatically decreased. Curran & Morgan (1985) reported that rat pheochromocytoma cells (PC12), when induced to differentiate with NGF and stimulated with benzodiazepine treatment, showed very little changes in the amounts of c-myc mRNA, even when neurite outgrowth occurred. Persistent high levels of c-myc mRNA were also detected in mouse primary keratinocytes irrespective of their proliferation or differentiation state (Dotto et al. 1986).

Other protooncogenes implicated in vitro in proliferation display continued expression during or after differentiation processes. The c-src gene provides the best documented example. The pp 60 c-src is particularly well expressed in the central nervous system of developing and adult fish and frogs (Shartl & Bamekow, 1984), and in the brain and heart of chick and human embryos (Levy et al. 1984). This protein is also developmentally regulated in the chick neuroretina (Cotton & Brugge, 1983; Sorge et al. 1984; Vardimon et al. 1986) and is found in postmitotic, fully differentiated neurones (Sorge et al. 1984). Interestingly, the latter authors demonstrated specific c-src expression restricted to the neurones of the inner surface of the neuroretina at the onset of their differentiation, the time when we find c-myc protein in the same cells.

A few studies have focused on c-myc gene expression during embryonic and postembryonic life in Xenopus, mouse and man. In Xenopus, mycmRNA accumulates from maternal transcripts during oogenesis (Godeau et al. 1986; Taylor et al. 1986). The c-myc protein is detected in the nuclei of somatic follicular cells and in the germinal vesicle of stage VI oocytes (Godeau et al. 1986). It reaches a maximum level in late oocytes (Taylor et al. 1986). In adult Xenopus, it is mainly detected in the skin (King et al. 1986).

In the mouse, a study of myc family transcripts has shown that the levels of c-myc mRNA were constant at least in the brain and kidney from embryonic day 15 to birth. Organ- and stage-specific expressions were found for N- and L-myc in the brain, kidney and lung (Zimmerman et al. 1986). Recently, Mugrauer et al. (1988) have applied in situ hybridization to the study of N-mycmRNA in the mouse fetus from E6·5 to El5. They found that mRNA storage seems to be a feature of early differentiation stages in kidney, brain and hair follicles, regardless of the proliferative status of the cell. While no myc gene family has been reported in birds, our results are very similar to these, in particular where neuronal cells are concerned. We cannot exclude that the c-myc probes used in this study cross-hybridized with other known members of the myc family. The two probes recognized the exon 3 product respectively at mRNA or protein levels (see Materials and methods). This exon was reported to be highly conserved during evolution from trout to man (Van Beneden et al. 1986). C-myc shares important homologies in exon 3 with N-myc (Stanton et al. 1986; Kohl et al. 1986; DePinho et al. 1986) and L-myc (Legouy et al. 1987). Thus, it is possible that the signal observed in some cell types resulted from the expression of one or the other or several of these myc genes and that the use of differential probes would further define the patterns that we observed.

In humans, Pfeiffer-Ohlsson et al. (1984) described high amounts of MYC transcripts in patches of cytotro-phoblastic cells during the first trimester of pregnancy. MYC expression in this case was closely related to DNA replication demonstrated by tritiated thymidine labelling. This distribution was confirmed by immuno-cytological detection of the MYC protein during the period of placental development (Maruo & Mochizuki, 1987). Furthermore, MYC is found coexpressed with the CIS protooncogene (PDGF f3 chain) in cytotro-phoblastic cells (Goustin et al. 1985). Less-clear-cut results were obtained in early human embryos using in situ hybridization (Pfeiffer-Ohlsson et al. 1985). Early embryos had very low MYC expression. While MYC transcripts were abundant in the skin or the gut, few transcripts were found in the liver, the brain or the cartilage, tissues that multiplied actively during this period.

Our study has allowed us to compare the detection of c-myc mRNA and protein in three selected stages of the chick embryo, E3, E6 and E8. A similar pattern for the distribution of the c-myc mRNA and protein appears in some tissues such as hemopoietic cell foci at E6 and the dermis prior to feather bud formation. By contrast, abundant mRNA transcripts were observed in the liver, the gut and the bronchi at E3 while no protein could be revealed at the same time or later during development in the tissues suggesting a control at a post-transcriptional level. An extensive analysis of c-myc mRNA expression is underway (Vandenbunder et al. unpublished data).

The present study was undertaken as part of an attempt to understand the genesis of MC29-induced cardiac rhabdomyosarcomas (Saule et al. 1987; Al Moustafa et al. 1988). We have previously reported low c-myc synthesis detected by metabolic labelling in the E3 chick embryo heart (Saule et al. 1987). In contrast, in the present work, no down-regulation of the protein stored in myocardiac nuclei could be detected at E3. In E6 to E10 heart cell nuclei, we did detect modulation in the c-myc protein expression. During this period, the heart tissue appeared as a patchwork of myc+ and myc-cell nuclei. In this regard, it may be significant that external cardiac myofibrils appear c-myc negative in E6 to E10 myocardium, since tumours induced in the chick embryo by MC29 are usually located towards the outside of the ventricle. The drawback of the in situ methods is that it is not easily amenable to quantification. We plan to remedy this imprecision by evaluating quantitatively the level of myc protein in various types of nuclei and the density of nuclei per surface unit using morphometric techniques.

We thank Dr D. Stehelin for his support and interest in this project. We are grateful to Dr S. Saule for providing the anti-myc serum and for helpful suggestions and discussions throughout this work. Y. Rantier and B. Henri contributed excellent photographic assistance. S. Schwartz’s efficient participation to IF work and M. Mirabel efficient help with in situ hybridization experiments are acknowledged gratefully. We also thank Z.-G. Xue for his advice and help in ganglion cell culture. Financial support was provided by the Centre National de la Recherche Scientifique and by the Association pour la Recherche contre le Cancer and by the Fondation pour la Recherche Medicate. T.J. is a recipient of a fellowship from the Ministere de la Recherche et de la Technologie.

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