Health Science Reviews

By Sibusisiwe Matewe

Numerous studies on evolution have allowed for scientists to establish a particular pattern that is followed by vertebrates in limb development, which involves enhancers – short DNA sequences increase transcription of one or more genes – seen consistently in reptiles for millions of years. However, at some point, snakes took a different path which led to the progressive loss of limbs and the familiar appearance we associate with snakes today. This occurred as a result of a particular limb enhancer of a gene called Sonic hedgehog (yes, like the popular video game character), known as the ZRS, whose function was lost in most snakes yet retained in other limbed animals. The question then becomes why and how has this occurred over time, and how does this phenomenon compare to the evolution of other vertebrates? The study by Kvon et al. (2016) set out to trace this molecular unravelling that led to limb loss in snakes, and to study the molecular and functional evolution of the enhancer responsible for this, both in various types of snakes as well as in other vertebrates. The end-goal of the researchers was to demonstrate how experiments can be used to recreate the gradual loss of function of a regulatory DNA sequence over the course of evolution, as well as to illustrate the importance of enhancers in morphological evolution.

To this end, the researchers made use of the following techniques and procedures: phylogenetic analysis and alignment of genomes of numerous vertebrate species including basal snakes and advanced snakes, transgenic mouse enhancer reporter assaying, CRISPR-Cas9 genome editing, and examining of putative Transcription Factor binding sites at the ZRS enhancer. By using these strategies, the scientists were able to address the following aims: 1. Explore the potential role of the ZRS enhancer in snake evolution 2. Establish whether variation in the snake ZRS changes the enhancer’s function in snakes’ bodies; 3. Compare development in limbed vertebrates and snakes; 4. Visually localise ZPA activity (the region which is regulated by the ZRS) in limb buds of various species; 5. Find any snake-specific nucleotide deletions in the ZRS and restore them, to examine the effects on limb development; 6. Determine the effects of deleting the mouse ZRS enhancer on limb development; 7. Determine the effects of transferring both the restored and unaltered snake ZRS enhancer into mice; 8. Identify potential  binding sites which may be implicated in the loss of ZRS enhancer function seen in snakes.

The authors of this paper were able to find several interesting results, including a distinction between basal snakes and advanced snakes in terms of the extent of limb loss, as well as a snake-specific 17 base-pair microdeletion in the ZRS that is responsible for the lack of limbs seen in snakes. Of note, it was found that restoring this microdeletion is sufficient to re-instate limb development, whereas replacement of the ZRS in limbed vertebrates with the unaltered snake ZRS causes extensive limb reduction. In addition, loss of important Transcription Factor binding sites including the E1 ETS site was identified as the likely cause of ZRS enhancer functional loss in snakes.

While the work of this paper allowed for a deepened understanding of the ZRS’s role in morphological evolution, and in particular, how its functional loss leads to reduced limb development in snakes, there are likely other enhancers and regulatory sequences that contribute to this, so this is not the end of the story. Nevertheless, this study has highlighted how phenotypic change can be driven by evolution involving the non-coding (i.e., regulatory) genome, and the likely causal relationship between enhancer evolution and morphological change. As evolutionary degeneration of the ZRS enhancer in snakes mirrors how mutations in the very same regulatory sequence can cause human congenital limb disorders, it could be worthwhile to make use of the models used in this paper to study how various ZRS mutations can result in human developmental diseases.

Reference:

Kvon, E.Z., Kamneva, O.K., Melo, U.S., Barozzi, I., Osterwalder, M., Mannion, B.J., Tissières, V., Pickle, C.S., Plajzer-Frick, I., Lee, E.A. and Kato, M., 2016. Progressive loss of function in a limb enhancer during snake evolution. Cell, 167(3), pp.633-642. DOI: https://doi.org/10.1016/j.cell.2016.09.028.