Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095.
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1662.
Proc Natl Acad Sci U S A. 2018 May 22;115(21):E4815-E4822. doi: 10.1073/pnas.1802749115. Epub 2018 Apr 23.
The formation of identical twins from a single egg has fascinated developmental biologists for a very long time. Previous work had shown that blastulae bisected along the dorsal-ventral (D-V) midline (i.e., the sagittal plane) could generate twins but at very low frequencies. Here, we have improved this method by using an eyelash knife and changing saline solutions, reaching frequencies of twinning of 50% or more. This allowed mechanistic analysis of the twinning process. We unexpectedly observed that the epidermis of the resulting twins was asymmetrically pigmented at the tailbud stage of regenerating tadpoles. This pigment was entirely of maternal (oocyte) origin. Bisecting the embryo generated a large wound, which closed from all directions within 60 minutes, bringing cells normally fated to become Spemann organizer in direct contact with predicted ventral-most cells. Lineage-tracing analyses at the four-cell stage showed that in regenerating embryos midline tissues originated from the dorsal half, while the epidermis was entirely of ventral origin. Labeling of D-V segments at the 16-cell stage showed that the more pigmented epidermis originated from the ventral-most cells, while the less-pigmented epidermis arose from the adjoining ventral segment. This suggested a displacement of the organizer by 90°. Studies with the marker Chordin and phospho-Smad1/5/8 showed that in half embryos a new D-V gradient is intercalated at the site of the missing half. The displacement of self-organizing morphogen gradients uncovered here may help us understand not only twin formation in amphibians, but also rare cases of polyembryony.
从单个卵子中形成同卵双胞胎一直让发育生物学家着迷了很长时间。之前的工作表明，沿着背腹（D-V）中线（即矢状面）将囊胚二等分可以产生双胞胎，但频率非常低。在这里，我们通过使用睫毛刀和改变盐溶液来改进这种方法，达到了 50%或更高的双胞胎形成频率。这使得我们能够对双胞胎形成过程进行机制分析。我们出人意料地观察到，在再生蝌蚪的尾巴芽阶段，产生的双胞胎的表皮在尾部不对称地上色。这种色素完全来自母本（卵母细胞）。将胚胎二等分产生一个大伤口，该伤口在 60 分钟内从各个方向闭合，使通常注定成为 Spemann 组织者的细胞与预测的最腹侧细胞直接接触。在四细胞阶段进行的谱系追踪分析表明，在再生胚胎中，中线组织起源于背侧半，而表皮完全来自腹侧。在 16 细胞阶段对 D-V 节段进行标记显示，色素沉着较多的表皮起源于最腹侧的细胞，而色素沉着较少的表皮则起源于相邻的腹侧节段。这表明组织者发生了 90°的位移。用 Chordin 和磷酸化 Smad1/5/8 标记的研究表明，在一半胚胎中，缺失一半的部位插入了新的 D-V 梯度。这里揭示的自我组织形态发生素梯度的位移可能有助于我们不仅理解两栖动物中的双胞胎形成，还理解罕见的多胚胎形成。
