Spatial-temporal misexpression of morphogenetic genes

我们建立了非洲爪蟾胚胎在神经板阶段将合成基因或mRNA递送到前景眼区域的非接触电穿孔方法（Genesis 33，81 -85和86- 96，2002），并将其应用于耳、鳃、水泥腺、肌肉和肢芽等多种组织器官。我们还将这种方法扩展到了日本蝾螈胚胎。这里我们重点关注原肠胚早期阶段将基因递送到组织者区域。由于背侧囊胚唇周围的细胞相当大且脆弱，即使使用我们温和的非接触电穿孔，它们也很容易受伤。一些技术上的改进，包括用mRNA或质粒填充卵黄蛋白空间，导致基因有效地转移到Spemann组织者或其他区域的细胞中。在第10-10.5阶段，将GFP mRNA电穿孔到组织者区后2-3小时，可检测到GFP蛋白沿着轴向中胚层的表达，并在第10-10.5阶段达到荧光峰值。 ...更多信息 在电分娩后12小时内。在早期原肠胚阶段非洲爪蟾组织者区域BMP 4的错误表达抑制了正常头部的形成。在第14阶段将BMP 4 mRNA电穿孔到头神经孔中抑制了某组神经嵴细胞的迁移。日本蝾螈胚胎的类似处理导致电穿孔一侧的鳃损失。我们还将BMP的拮抗剂Noggin引入相同的区域。将Noggin导入背唇中会产生发育不良的尾部表型，这与我们对大头胚胎的预期相反。我们正在进行的研究项目是早期原肠胚中被修饰为组成型活性和显性负性受体类型的受体基因的错误表达。组成型活性FGF受体对前后轴的形成具有后验效应，而组成型活性视黄酸受体则抑制了最前部结构如骨水泥腺的形成，因此，该技术可以拓展两栖动物胚胎特定区域或特定发育阶段的功能获得和丧失途径。它还使得能够在形态发生期间追踪特定组织和/或器官的细胞谱系。少

We have established the non-contact electroporation method for Xenopus embryos to deliver synthetic genes or mRNAs into the prospective eye region at the neural plate stage (Genesis 33,81-85 and 86-96,2002), and have expanded its application in various tissues and organs such as ear, gill, cement gland, muscle and limb bud. We have also expanded this method to Japanese newt embryos.Here we focus on the gene delivery into the organizer region at the early gastrula stage. As the cells around the dorsal blastopore lip are rather large and fragile, they are easily injured even with our mild non-contact electroporation. Some technical improvements including the filling up of vitellin space with mRNAs or plasmids led to the efficient gene transfer into the cells of Spemann organizer or other regions. The expression of GFP protein along with axial mesoderm was detectable 2-3 hr after electroporation of GFP mRNA into the organizer region at stage 10-10.5, and reached the peak of fluorescence w … More ithin 12 hr after the electric delivery. Misexpression of BMP4 at the Xenopus organizer region at the early gastrula stage suppressed normal head formation. The electroporation of BMP4 mRNA into the head neural hold at stage 14 inhibited the migration of a certain group of neural crest cells. The similar treatment of Japanese newt embryos resulted in the loss of gill on the side of the electroporation. We also introduced the antagonist of BMP, Noggin, into the same regions. The delivery of Noggin into the dorsal lip made the stunted tail phenotype, contrary to our expectation of a big-headed embryo. Our ongoing research project is the misexpression of the receptor genes modified as the constitutive active and dominant negative receptor types in the early gastrulae. The constitutive active FGF receptor showed a posteriorizing effect on the anterior-posterior axis formation, and the constitutive active retinoic acid receptor suppressed the formation of the anterior-most structures such as cement gland.As described above, this technique can expand the gain-and loss-of-function approaches at specific regions or certain developmental stages of amphibian embryos. It also makes enable to trace the cell lineage of a certain tissue and/or organ during morphogenesis. Less