The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis.
Bottom Line: While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans.Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis.Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.
Affiliation: Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.Show MeSH
Mentions: To characterize the transgene locus, we used Ligation-Mediated PCR (LM-PCR) with primers specific to both ends of the transgene to locate the integration at bp 91378–91352 in scaffold_111 of the JGI X. tropicalis genome assembly (http://www.genome.jgi-psf.org/Xentr4/Xentr4.home.html) in the third intron of the nephronectin (npnt) gene (Fig. 3A). Concatameric transgene insertions often result when Xenopus eggs are injected with sperm nuclei incubated with linear DNA constructs (Kroll and Amaya, 1996). Only 26 bp of endogenous npnt intron sequence is missing in the mutant allele, consistent with a relatively clean transgene insertion (Figs. 3A and S1).
Affiliation: Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.