The first known tetrapods to emerge already display a specifically arranged limb, composed of proximal endoskeletal elements (humerus, radius and ulna) plus distal elements (wrist bones and digits). While limbs with digits have been regarded as an evolutionary novelty, part of their skeletal organization was already in place in fish fins, as the closest relatives to tetrapods, sarcopterygian fishes, possess three proximal bones and a distal endoskeletal region composed of segmented radials. Among modern lungfish, the genus Neoceratodus has retained distal radials whereas Lepidosirenids (Protopterus e Lepidosiren) have lost them. Genetic or pharmacological inactivation of Sonic Hedgehog signaling (SHH) in developing limbs and fins leads to loss of distal endoskeleton, digits or distal radials, respectively. Shh expression during appendage development is controlled by a highly conserved enhancer element termed ZRS, present in all vertebrates including snakes. Here, we investigate the role of SHH signaling during lungfish fin regeneration by: (i) cloning and in silico analysis of ZRS from lungfish species of the three living genera; (ii) Semiquantitative PCR for Shh, Ptch1 and Gli1 and (iii) pharmacological modulation of SHH signaling during lungfish fin regeneration. Comparative analysis of vertebrate ZRS sequences revealed a 17 bp deletion of a key ETS transcription factor binding site in Lepidosirenid lungfish but not in Neoceratodus. Furthermore, as seen in salamander limb regeneration, SHH signaling is activated and is necessary for lungfish fin regeneration. Last, we show that SHH activation increases the number of distal radials in lungfish fin. Overall, our data suggests that SHH signaling, a key pathway underlying digit development in tetrapods, also controls distal radial development in lungfish fins, providing support for the homology of digits and distal radials.
|
