| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Research Abstract |
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
|
