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 confirm a requirement for npnt in forelimb formation, we attempted to phenocopy the mutation with antisense morpholino oligonucleotides directed against npnt message. Control morpholino injections did not affect forelimb formation (431/431 wild type), but a morpholino targeting the first exon splice donor injected into one cell of two-cell embryos resulted in a statistically significant frequency of one-armed metamorphosing tadpoles (3/98, p < 0.01) which like xdm lacked all forelimb skeletal elements on the injected side (Figs. 4A and B). A combination of a different splice-blocking morpholino (directed against the third intron splice donor) and a translation-blocking morpholino also resulted in one-armed metamorphosing larvae (2/47, p < 0.01); neither of these morpholinos injected singly gave significant effects. Injected morpholino oligonucleotides have been shown to be capable of blocking expression until at least stage 43 in X. tropicalis, with recovery of endogenous gene expression by stage 47 (Nutt et al., 2001). We tried to define a window during which npnt was required by evaluating the persistence of the first exon splice-blocking morpholino. RT-PCR using primers spanning exons 1 and 2 shows that correctly-spliced message is depleted by the morpholino at stages 24 and 37, with recovery underway at stage 42 and approaching normal levels by stage 45 (Fig. 4C). Only a small minority (< 4%) of MO-injected embryos display limb defects, so the requirement for npnt is likely to occur after splicing has recovered in the majority of embryos. Our results show npnt splicing remains blocked at stage 37, consistent with a later requirement for this gene in forelimb initiation. The lower level of npnt product in control MO-injected embryos at stages 42 and 45 is consistent with WISH analysis (see Fig. 6). Interpretation of this result is complicated by the low penetrance of the morpholino in blocking forelimb formation. Even at relatively low penetrance, it is striking that npnt morpholino injection at cleavage stages can specifically affect limb formation, as forelimb buds first appear in X. tropicalis 2–3 weeks post-fertilization.
Affiliation: Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.