Huszar D, Sharpe A, Jaenisch R
Whitehead Institute for Biomedical Research, Nine Cambridge Center, MA 02142.
Development. 1991 May;112(1):131-41. doi: 10.1242/dev.112.1.131.
Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.
通过聚集植入前胚胎产生的嵌合小鼠在哺乳动物毛色图案发育研究中发挥了重要作用。然而，这种方法无法对黑素母细胞的前体神经嵴细胞进行直接实验操作。我们设计了一种系统，该系统可以在体外对神经嵴细胞进行实验操作后，评估其在体内的发育潜能和迁移情况。将培养的C57Bl/6神经嵴细胞在子宫内显微注射到正在神经形成的Balb/c或W胚胎中，结果显示这些细胞能有效地对宿主动物的色素沉着做出贡献。然而，根据宿主胚胎的基因型，产生的神经嵴嵌合体表现出不同的被毛色素沉着模式。Balb/c神经嵴嵌合体中供体细胞对色素的贡献非常有限，主要局限于头部，而W突变体嵌合体则全身都有广泛的色素沉着，通常超过被毛的50%。与Balb/c嵌合体不同，在Balb/c嵌合体中供体黑素母细胞似乎主要沿特征性的背腹方向迁移，而在W突变体中，注入的细胞似乎沿纵向以及背腹方向迁移，就好像细胞在一个空旷的空间中扩散。这与W突变体中缺乏功能性内源性黑素母细胞群体一致，与之形成对比的是，Balb/c小鼠含有完整的黑素细胞。我们的结果表明，W突变会干扰内源性黑素母细胞的迁移和/或增殖。为了获得关于供体细胞克隆大小和混合程度的信息，将两个基因标记的神经嵴细胞群体混合并共同注射到W胚胎中。在一半的三色嵌合体中，未观察到供体嵴细胞的共定位，而在另一半中，供体来源的颜色出现了精细的混合。这些结果与以下假设一致，即嵌合体中的色素沉着区域可能来自少数供体克隆的广泛增殖，这些克隆能够在宿主胚胎中占据大片区域。我们还分析了体外神经嵴培养物中色素沉着形成过程，发现从携带野生型或弱W等位基因的胚胎中取出的神经管能产生有色素的黑素细胞，而更严重的W基因型与体外色素形成缺陷有关。
