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https://doi.org/10.1016/0012-1606(68)90064-x
Copy DOIJournal: Developmental Biology | Publication Date: Mar 1, 1968 |
Citations: 25 |
Neural crest explants from different anteroposterior levels of axolotl ( Ambystoma mexicanum) embryos were grown in a modified hanging drop system. The cultures were treated with 3,4-dihydroxyphenyl-alanine-α-β- 3H (DOPA- 3H) at various ages, and tritium incorporation was determined by radioautography. By day 7 after explantation, all the potential melanophores in both trunk and cranial cultures have synthesized sufficient melanin to make their positive identification possible. When treated with DOPA- 3H on day 7, 8, or 9, only cells that contain melanin incorporate labeled DOPA, and all cells that contain melanin are also radioactive. This demonstrates that definitive melanoblasts selectively incorporate DOPA, and that DOPA serves as a specific melanin precursor in these cells. Since DOPA can arise only from tyrosine, it is concluded that a tyrosine-DOPA-melanin pathway is operative during melanogenesis in amphibian pigment cells. When treated with DOPA- 3H on day 3 after explantation, one-fourth of the anterior trunk outgrowths are composed almost entirely of radioactive cells. In the remaining 3-day anterior trunk cultures, and in all the 3-day posterior trunk, anterior cranial, and posterior cranial cultures, none of the cells of any of the outgrowths are labeled. Prior to the third day, there is no incorporation of DOPA. When treated on the fourth day, or thereafter, trunk outgrowths are composed almost exclusively of radioactive cells. In the cranial outgrowths, the labeled cell population does not exceed one-third of the total. The trunk and cranial regions of neural crest are also significantly different from each other with respect to pigment cell potential in vitro. The trunk segments give rise to outgrowths which consist almost exclusively of melanophores, and the cranial segments give rise to outgrowths in which melanophores comprise less than one-third of the total cell population. The close correspondence between the frequencies of radioactive cells and the frequencies of pigment cells indicates that melanoblasts, prior to visible differentiation, selectively incorporate DOPA. The time at which presumptive pigment cells are first able to utilize exogenously administered DOPA coincides with the time at which migration ceases, indicating that the capacity to synthesize melanin is not acquired until migration has ended. The incorporation of label by the presumptive pigment cells contained in cultures 3, 4, 5, and 6 days old permits the identification of melanoblasts earlier than has previously been possible and demonstrates that these cells have developed a highly specialized biochemical system before any outward signs of it are manifest. It is clear, moreover, that amphibian melanoblasts can utilize exogenous substrate in spite of their ample internal reserves.
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