Development: Sonic Snakes and Regulation of Limb Formation

RESEARCH HIGHLIGHTS

Nature Reviews Genetics | Published online 15 Nov 2016; doi:10.1038/nrg.2016.149

DEVELOPMENT Sonic snakes and regulation of limb formation

Evolutionary limb loss is a funda- expression in the zone of polarizing Leal and Cohn identified three mental and overt change in body activity in developing limbs, deletions at the 5ʹ end of the ZRS plan, but the underlying molecular this activity is progressively lost in that occur in snakes but not limbed causes have remained debated and ZRS sequences from basal through vertebrates; the middle deletion largely elusive. Two new studies to advanced snakes. Furthermore, is the same 17 bp deletion studied pinpoint enhancer alterations knock-in to replace a 1.3 kb core by Kvon et al. They showed using associated with limb loss during region of mouse ZRS with the equiv- engineered expression constructs in snake evolution. alent sequence from python (a basal cell culture that each deletion reduces Mechanisms of major morpholog- snake) resulted in mice with partially enhancer activity (although effects on ical transitions have been challenging truncated limbs, and replacement limb development in vivo were not to elucidate. The continued presence with the sequence from cobra (an explicitly tested). Although Leal and of known gene families for limb advanced snake) caused substantially Cohn note loss of an ETS binding formation in limbless organisms such more severe limb truncations. site caused by the 17 bp deletion, as snakes, as well as fossil indications For further mechanistic insight, their co-expression experiments and that limbless species might retain the Kvon et al. searched for transcrip- electromobility shift assays indicated capability to re-evolve limbs, suggests tion factor binding motifs that that each of the three identified ZRS that these major macroevolutionary were disrupted by snake-specific deletions disrupt binding by HOXD changes may be caused by subtle deletions, identifying ETS and homeodomain transcription factors, regulatory alterations. HOX transcription factors as key as a potential molecular explanation In their search for mechanistic candidates. Strikingly, one particular for curtailed SHH signalling and limb answers, Kvon et al. examined regu- ETS binding site was disrupted by a development in snakes. lation of the Sonic hedgehog (SHH) 17 bp deletion in all snakes, and using It will be interesting to dissect cell-fate gene. They focused on a CRISPR technology to re-introduce the relative importance of the evo- characterized limb SHH enhancer these 17 bp into the python ZRS core lutionary binding site disruptions — known as the zone of polarizing sequence of the knock-in mouse identified by Kvon et al. and Leal and activity regulatory sequence (ZRS) restored limb formation, thus high- Cohn, as these sites seem to function — which is known to be essential for lighting the importance of individual in a redundant manner. For example, proper limb development in several transcription factor binding sites in limb formation can be rescued by vertebrates. Taking advantage of the morphological changes. restoring a single 17 bp deletion, as recent availability of six draft snake In a related study, Leal and Cohn shown by Kvon et al. It remains to be genomes, they compared snake ZRS compared SHH signalling in the seen whether restoring this deletion sequences with those of limbed ver- rudimentary developing limb buds could over-ride ZRS enhancer defi- tebrates. The ZRS enhancer showed of pythons with those in the limbed ciency in the context of other binding progressive degeneration, relative to green anole lizard. They noted that site deletions, as well as the relative limbed vertebrates, in basal snakes SHH expression was not sustained in importance of ETS versus HOXD (which retain vestigial rudimentary pythons, resulting in a breakdown of binding events for limb loss and limbs) through to advanced snakes the feedback loop and limb develop- re-establishment. (which have lost all skeletal limb ment. Hence, they too sought regula- Finally, knowledge of the identified structures). This degeneration tory explanations for disrupted SHH regulatory mechanisms may help the did not occur in other limb expression in snakes. They found no clinical interpretation of non-coding enhancers examined. differences in the expression levels mutations in human patients with To prove that ZRS sequence of ZRS-binding transcription factors limb malformations. Darren J. Burgess

sayanjo65/iStock/Getty differences have functional con- between the snake and lizard but, sequences, the authors tested 16 similarly to Kvon et al., used compar- ORIGINAL ARTICLES Kvon, E. Z. et al. Progressive ZRS sequences from a range of ative genomics to identify progressive loss of function in a limb enhancer during snake evolution. Cell 167, 633–642 (2016) | Leal, F. & snakes and limbed vertebrates in degeneration of ZRS sequences Cohn, M. J. Loss and re-emergence of legs in an in vivo mouse enhancer reporter during snake evolution and demon- snakes by modular evolution of sonic hedgehog assay. Whereas ZRS enhancers from strated functional consequences in and HOXD enhancers. Curr. Biol. http://dx.doi.org/ 10.1016/j.cub.2016.09.020 (2016) limbed species drive robust reporter mouse reporter assays.