Yuan S, Darnell D K, Schoenwolf G C
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City 84132, USA.
Dev Genet. 1995;17(1):38-54. doi: 10.1002/dvg.1020170106.
The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate.
在鸟类和哺乳动物发育的原肠胚晚期/神经胚早期，通常注定要形成脊索的细胞占据原条头端的一个区域。如果在培养中将这些细胞从鸟类胚胎中手术移除，在大多数情况下仍会形成脊索。此前尚未对这个重建脊索的起源进行研究，而这正是本研究的目的。培养处于原肠胚晚期/神经胚早期的鸡胚，切除包括亨氏结在内的原条头端，并用鹌鹑上胚层的非节点细胞进行替换，以确保正常注定要成为脊索的细胞缺失，并确保胚盘能够愈合。让胚胎发育24小时，使用针对脊索或鹌鹑细胞的抗体评估脊索的存在及其起源（宿主或移植）。通常会发育出两条脊索；两者几乎完全来自宿主。从另一组胚胎中移除原条，在某些情况下还移除相邻组织，以试图估计形成重建脊索所需细胞的中外侧位置和范围。对有或没有移植的其他实验胚胎在不同的头尾水平进行横切，以试图估计形成重建脊索所需细胞的头尾范围。最后，将原条的不同水平部分置于中性环境（生殖细胞新月区）中，或移植到节点的位置。所有实验的综合结果表明，原条头端部分外侧的区域对于重建脊索的形成至关重要，估计其头尾跨度小于500微米，中外侧范围小于250微米。对该区域的命运图谱绘制和组织学检查确定了4种可能的前体细胞群体。正在进行进一步研究，以确定这4种可能的前体细胞类型中哪一种形成或诱导了重建脊索，以及哪些组织相互作用是这种细胞命运变化的基础。
