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???displayArticle.abstract??? Bone morphogenetic protein (Bmp) signalling has been implicated in setting up dorsoventral patterning of the vertebrate limb and in its outgrowth. Here, we present evidence that Bmp signalling or, more precisely, its inhibition also plays a role in limb and fin bud initiation. Temporary inhibition of Bmp signalling either by overexpression of noggin or using a synthetic Bmp inhibitor is sufficient to induce extra limbs in the Xenopus tadpole or exogenous fins in the Danio rerio embryo, respectively. We further show that Bmp signalling acts in parallel with retinoic acid signalling, possibly by inhibiting the known limb-inducing gene wnt2ba.
Fig. 1. Extra limb phenotype in the Xenopus N1 line. (A)Over-expression of noggin at stage 48 leads to inhibition of limb outgrowth (arrowhead) with occasional formation of a secondary limb. (B)Over-expression of noggin at stage 49 leads to induction of extra limbs in about 30% of the tadpoles. Endogenous limb is developmentally more advanced than the extra limb. (C)Over-expression of noggin at stage 50 or later leads to malformation of the limb. (D-F) Variability of the extra limb phenotype. Induction of full extra limbs (D). Partial duplications of distal elements with split in antero-posterior (E) or dorso-ventral (F) axis. (G,H) Alizarin Red staining for bone and Alcian Blue staining for cartilage in pre-metamorphotic tadpole limbs (G) and a froglet (H). Endogenous limbs look almost normal during development but protrude at different angles after metamorphosis. (I)Duplication of a forelimb. Arrowheads indicate endogenous limbs; arrows indicate extra limbs. Red arrowheads and arrows indicate forelimbs.
Fig. 2. In situ hybridisation in Xenopus wild type and N1 line after induction of extra limbs. (A-C)lmx1 expression in the wild-type limb is restricted to the dorsal mesenchyme (A) but is present in the entire mesenchyme in the endogenous limb of the N1 line (B,C), indicating a dorsalisation of the limb; the extra limb shows normal lmx1 expression restricted to the dorsal mesenchyme (C). (D-F) en1 expression is restricted to the ventralectoderm in the wild type (D) but is absent in the N1 endogenous limbs (E), whereas extra limb shows normal expression of en1 (F). (G-I)Expression of fgf8 is restricted to the AER in wild-type limbs (G) but is expressed in a broader domain in N1 endogenous limbs while normally expressed in the AER of the extra limbs (H,I). Arrowheads indicate endogenous limbs; arrows indicate extra limbs.
Fig. S1. Western blot. The p-smad2/3 antibody recognizes two bands in tadpoles (wt, N1) and one band in embryos (DMSO, Alk 4/5/7 inh). Whereas the upper band decreases in the N1 tadpoles after noggin overexpression, the lower one increases. However, when embryos are treated with the Alk 4/5/7 inhibitor for 24 hours, it is the lower band that vanishes when compared with the DMSO embryo control. This indicates that the activity of the Tgfβ pathway increases rather than decreases in N1 tadpoles after noggin overexpression. Loading control: α-tubulin.
Fig. S2. Differential expression of RA modifier genes assessed by real-time PCR. Wt zebrafish embryos were treated with Bmp inhibitor or DMSO (control) at 6-7 hpf and RNA collected at the indicated hpf. Genes in the RA pathway are not differentially expressed (exception crabpII at 24 hpf), indicating that Bmp does not act upstream of the RA pathway and therefore must act in parallel. Standard deviation is shown. Note that expression is relative to the highest expression for every gene, which is arbitrarily set at one for each experiment and then accumulated.
