Loss and Re-emergence of Legs in Snakes by Modular Evolution of Sonic hedgehog and HOXD Enhancers

Abstract

Limb reduction and loss are hallmarks of snake evolution. Although advanced snakes are 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 [1-5], suggesting that the mechanisms of limb development were not completely lost in snakes. Here we 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 Sonic hedgehog (SHH). Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. Our results suggest that degenerate evolution of the SHH limb enhancer played a role in reduction of hindlimbs during snake evolution. By contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in the 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 footplate, raising the possibility that re-emergence of hindlimbs during snake evolution did not require de novo re-evolution of lost structures but instead could have resulted from persistence of embryonic legs. VIDEO ABSTRACT.

Saunders's framework for understanding limb development as a platform for investigating limb evolution

“…Earlier work suggested that the limb fails to grow because of a degeneration of the AER resulting in the lack of Fgf and Shh production breaking the ectodermal-mesenchymal Fgf positive feedback loop (Cohn and Tickle, 1999). Recently, however, it was been discovered that pythons do initially induce Shh and Fgf8 in early hindlimb buds, only transiently and Shh expression is reduced due to degeneration of the ZRS (Kvon et al, 2016; Leal and Cohn, 2016). Further analysis revealed that a conserved Ets1 binding site in the ZRS is lost in all examined snake genomes and restoration of this site is sufficient to rescue the full activity of the ZRS (Kvon et al, 2016).…”

“…However, in snakes, this enhancer is only active in the genital tubercle (Infante et al, 2015). Interestingly, the cis-regulatory modules that control the early and late phase Hox expression in tetrapod limbs is conserved in pythons suggesting that they retain the ability to properly pattern the proximodistal axis of their diminutive limbs (Leal and Cohn, 2016). …”

<b><i>Hox</i></b> Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis

“…Consistently, Kvon et al [2016] demonstrated that through nucleotide-specific changes in the zone of polarizing activity regulatory sequence (ZRS, which is a limb-specific enhancer of Sonic hedgehog) in snakes the ZRS enhancer has progressively lost its function during snake evolution, leading to the disappearance of the limbs. In contrast, according to Leal and Cohn [2016], Hoxd limb enhancers are conserved in the python embryo. Expression of Hoxd13 spreads throughout the distal region of the python hind limb bud, overlapping with Hoxa13 expression in a pattern that resembles digitforming domains of Hoxa13 and Hoxd13 in limbed tetrapods [Montavon et al, 2011].…”

“…Expression of Hoxd13 spreads throughout the distal region of the python hind limb bud, overlapping with Hoxa13 expression in a pattern that resembles digitforming domains of Hoxa13 and Hoxd13 in limbed tetrapods [Montavon et al, 2011]. Therefore, because Hoxd expression in digits and external genitals are under shared regulatory control, it is possible that Hoxd distal enhancers were retained in snakes due to their essential role in external genital development [Lonfat et al, 2014;Leal and Cohn, 2016].…”

“…In python embryos, no apical ectodermal ridge could be found, and gene expression was not detected in limb ectoderm. More recently, Leal and Cohn [2016] proposed that hind limb development stops in python embryos as a result of mutations that eliminate essential transcription factor binding sites in the limb-specific enhancer of Sonic hedgehog. Consistently, Kvon et al [2016] demonstrated that through nucleotide-specific changes in the zone of polarizing activity regulatory sequence (ZRS, which is a limb-specific enhancer of Sonic hedgehog) in snakes the ZRS enhancer has progressively lost its function during snake evolution, leading to the disappearance of the limbs.…”