Open AccessIntroduction of a retinoid reporter gene into the urodele limb blastema.
Author(s) -
Jeremy P. Brockes
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.23.11386
Subject(s) - blastema , retinoic acid , biology , hindlimb , reporter gene , transfection , regeneration (biology) , retinoid , progenitor cell , microbiology and biotechnology , sox9 , anatomy , limb development , limb bud , gene expression , cell culture , gene , stem cell , embryo , genetics
After amputation of the limb of an adult urodele amphibian at any point along the proximodistal axis, blastemal cells (the progenitor cells of the regenerate) give rise only to the missing structures. Retinoic acid (RA) is able to respecify the positional identity of the blastema to a more proximal value, thus raising the possibility that the RA response system is activated during limb regeneration. Cultured newt (Notophthalmus viridescens) limb cells were transfected by nuclear microinjection of plasmids which provided RA-sensitive reporter activity that could be normalized for differences in cell recovery and transfection efficiency. Such cells showed a dose-dependent response to RA in culture, and this required a functional RA response element. The cells were implanted under the wound epidermis of newt hindlimb blastemas. After injection of a proximalizing dose of RA there was a significant difference in the level of reporter activity dependent on a functional response element. When cells were implanted into contralateral proximal and distal hindlimb blastemas the proximal-to-distal ratio for activation of the reporter through the response element was approximately 3.5-fold, suggesting that a gene whose expression is regulated by RA could be differentially activated along the proximodistal axis during limb regeneration.