Further studies into our understanding as to why snakes have no legs

Decrease in size or length of the arm/leg (animal’s limb), or complete loss of limbs, has been a great fascination to some of us in developmental biology. The best experimental model for studying limb reduction and loss is to examine snake evolution. Ever since altered homeobox (HOX) gene expression was shown [Nature 1999; 399: 474] to block the transcription factor “sonic hedgehog (SHH) signaling to form limbs in the python, further studies of these signaling pathways have been carried out. Although ‘the true snake’ is completely limbless, ‘basal’ and ‘intermediate snakes’ retain pelvic girdles and small rudiments of the femur. Moreover, legs may have re-emerged in extinct snake lineages, suggesting that the mechanisms of limb development were not completely lost in snakes.

In the Curr Biol paper [attached], authors report that hindlimb development arrests in python embryos as a result of mutations that abolish essential transcription factor-binding sites in the limb-specific enhancer of the gene encoding SHH. Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. These results suggest that degenerate evolution of the SHH limb enhancer plays a role in reduction of hindlimbs during snake evolution. In contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in hindlimb buds progresses to the distal phase, forming an autopodial (digit) domain. Python hindlimb buds then develop transitory pre-chondrogenic condensations of the tibia, fibula, and even the footplate (!) –– raising the possibility that re-emergence of hindlimbs during snake evolution might not have required de novo “re-evolution of lost structures” but instead could have resulted from persistence of embryonic legs.

In the Cell paper [attached], authors identified snake-specific DNA sequence changes within an otherwise highly conserved SHH long-range limb enhancer. Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates –– including fish, but not in snakes. Genomic substitution of the mouse Shh enhancer with its human or fish ortholog resulted in normal limb development. On the other hand, replacement with snake SHH orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor-binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. These fascinating data demonstrate that changes in a regulatory sequence can be associated with a major body plan transition. This report highlights the role of gene enhancers in morphological evolution.

Cell 2o16; 167: 633–642   and   Curr Biol 2o16 26, 2966–2973

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