The Notophthalmus viridescens radical fringe cDNA, molecular cloning and spatiotemporal expression in forelimb development and adult forelimb regeneration

grantor: University of TorontoThe 'Notophthalmus viridescens radical fringe' cDNA was isolated, cloned and sequenced. It codes for a 396 amino acid protein with a predicted N-terminal signal sequence. Newt 'radical fringe' shows high homology to the 'radical fringe' genes of ' Gallus gallus' (chick), 'Xenopus laevis' (frog) and ' Mus musculus' (mouse). There is 77% identity between newt and chick genes in the C-terminal 270 amino acids of the protein (89% when conserved changes are considered). During forelimb development, newt 'radical fringe' is first expressed in the limb field, with intense expression as the forelimb bud develops. Homogeneous 'radical fringe' gene expression spanning both the dorsal and ventral halves of the developing forelimb is detected at this stage. 'radical fringe' is exclusively expressed in the mesenchyme regions of the developing forelimb bud. Gene expression diminishes with advanced digit development. 'radical fringe' is re-expressed in the mesenchymal tissues during epimorphic regeneration of the adult newt forelimb. Weak 'radical fringe' expression is first seen in the two-week forelimb regenerate. The expression signal intensifies in subsequent weeks as the regeneration bud (blastema) increases in size. A very intense signal is seen in the region immediately flanking the differentiating cartilagenous, tissues. This gene appears to be highly expressed in regions of active cell division. The highest expression pattern is seen at the early four digit stage where the signal seems to be localised in the interdigital mesenchyme of the regenerating forelimb. As the regenerate progresses past the advanced digit stage, newt 'radical fringe' expression diminishes and eventually disappears. Overall, the results obtained suggest that 'radical fringe' might be involved in both forelimb development as well as in adult forelimb regeneration in the newt, 'Notophthalmus viridescens'.Ph.D