Spatial-temporal misexpression of morphogenetic genes

我们已经建立了非洲爪蟾胚胎非接触式电穿孔方法，在神经板阶段将合成基因或mrna传递到预期的眼区（Genesis 33,81-85和86-96,2002），并扩大了其在耳、鳃、水泥腺、肌肉和肢体芽等各种组织器官中的应用。我们还将这种方法扩展到日本蝾螈胚胎。在这里，我们关注的是基因在原肠早期阶段传递到组织者区域。由于背囊胚唇周围的细胞较大且脆弱，即使采用轻微的非接触式电穿孔也容易损伤。一些技术改进，包括用mrna或质粒填充卵磷脂空间，导致基因有效地转移到Spemann组织者细胞或其他区域。在10-10.5期，GFP mRNA电穿孔进入组织者区2-3小时后，沿轴向中胚层可检测到GFP蛋白的表达，并在12小时内达到荧光峰值。爪蟾原肠期早期组织区BMP4的错误表达抑制了正常头部的形成。在第14期，BMP4 mRNA电穿孔进入头神经孔，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丢失。我们还在相同的区域引入了BMP的拮抗剂Noggin。Noggin在背唇的分娩导致了发育不良的尾巴表型，这与我们预期的大头胚胎相反。我们正在进行的研究项目是受体基因的错误表达，修饰为早期原肠胚中构成活性和显性负受体类型。组成性活性FGF受体对前后轴的形成具有后置作用，组成性活性维甲酸受体抑制水泥腺等最前端结构的形成。如上所述，这项技术可以在两栖动物胚胎的特定区域或特定发育阶段扩展功能获得和功能丧失的方法。它还使我们能够在形态发生过程中追踪某个组织和/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