In situ study of c-myc protein expression during avian development

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Abstract
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.