We observed that chordin, an inhibitor of bmp signaling, can antagonize the formation of bone fusion induced by shh, suggesting an interaction between shh and bmp signaling.

In the developing limb bud, shh signaling is thought to be regulated by feedback loops with other proteins such as fibroblast growth factors ( 32, 33).

Halpern, Carnegie Institute of Washington, Baltimore), a 2.5-kb fragment encoding the zebrafish chordin cDNA ( 27), was inserted into the pCS2+ expression vector containing the CMV promoter. pCMV5 bmp2b, a modified version of pCMV5 vector, was made to create convenient restriction sites ( EcoRI and KpnI sites) to place the bmp2b cDNA under the control of the CMV promoter. Position 632 of the shh sequence corresponds to the cleavage site of the shh precursor. It was cloned into the pCMV5 vector at the HindIII and BglII sites. pCMV5 shh-NH 2, a 632-bp, N-terminal fragment of the shh cDNA ( 26), was obtained by PCR by using the following primers: forward, 5′-ATTAAGCTTGCGGCAAAATGCGG-3′ reverse, 5′-GAAGATCTCCCCCAGATTTCGCAGC-3′. PCMV5EGFP, the enhanced GFP gene (EGFP) was excised from plasmid pEGFP-C1 (CLONTECH) and ligated into the multiple cloning site of pCMV5 (CMV, cytomegalovirus) (Department of Molecular and General Biochemistry, University of Texas, Southwestern Medical Center). No ectopic bone deposition occurs in the mesenchyme interior of the lepidotrichia (L) as marked with asterisks. Arrows in G and I indicate the region of ectopic bone matrix deposition. ( E, G, I) Cryosections (16 μm) in the region of normal bifurcation ( E) or fusion ( G, I). ( D, F, H) Whole-mount control and fused rays proximal part of the fin is to the left. Constructs were injected in the region indicated by white arrows ( F, H). ( D– I) Normal bifurcating ( D, E) and N-shh ( F, G) or bmp2b ( H, L) injected fused rays stained with Alcian blue. Injections were performed at 2 dpa (high cut shown by the dotted line). ( C) Ectopic expression of EGFP 24 h after injection of the pcmv5EGFP reporter construct in the tissue separating the branches of lepidotrichia (L) of two adjacent rays. ( B) Sequence of shh expression patterns after a low cut, leading to ray bifurcations: ( B1) 2 dpa, an unbifurcated ray expresses one shh domain in the distal blastema ( B2) by 4–5 dpa shh is expressed in two domains, preceding the formation of a bifurcation ( B3) two newly formed sister rays each exhibit one shh expression domain ( B4) the process repeats itself to form a second round of bifurcations (not to scale). The diagram illustrates the position of amputations, relative to the bifurcation of the lepidotrichia “low cut” and “high cut” are performed one to two segments proximal and four to six segments distal to the origin of bifurcation, respectively. Dermal bone forms two segmented, concave and opposing hemirays. These results implicate shh and bmp2b signaling in the proliferation and/or differentiation of specialized bone-secreting cells in the blastema and suggest shh expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.įigure 1 ( A) Schematic representation of the skeleton of a ray in the zebrafish fin. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of shh expression and a rapid abolition of ptc1 expression. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. Ectopic expression of shh or bmp2 in the blastema-induced excess bone deposition and altered patterning of the regenerate. We established an in vivo method of gene transfection to express ectopically genes in the blastema of regenerating fins. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes.

Altered reciprocal inhibition