Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework. Animals with a backbone can look remarkably different from one another, like fish and birds, for example. Nevertheless, these animals – which are also known as vertebrates – have many genes in common that shape their bodies during development. These genes include a family called the Hox genes, which control how an animal’s body parts develop from its head to its tail and are needed to shape the animal’s limbs. Hox genes are found clustered in groups within a vertebrate’s DNA, and large regions of DNA on either side of a Hox cluster can, in some cases, physically interact with the Hox genes to regulate their expression. So how do the same genes produce different body shapes? Different vertebrates regulate where and when their Hox genes are switched off and on in different ways. As such, it is likely that differences in gene regulation, rather than in the genes themselves, lead embryos to develop into the distinct shapes seen across the animal kingdom. Snakes – for example – evolved from a lizard-like ancestor into elongated limbless animals as they have adapted to a burrowing lifestyle. However, it was not known if changes in how Hox genes are regulated have played a role in shaping the distinct body plan of snakes. Guerreiro et al. have now compared how Hox genes are regulated in snakes, mice and other vertebrates, focusing on corn snakes and one particular cluster of Hox genes called the HoxD cluster. The comparison revealed that these Hox genes are regulated differently in developing snakes than in other vertebrate embryos. This is particularly the case for tissues that show the most differences when compared with other animals (such as the torso and genitals) or that are absent (such as the limbs). Although Hoxd genes are activated at different times and places in snakes than in other vertebrates, snake Hox genes appear to be regulated using the same general mechanisms as mouse Hox genes. Guerreiro et al. suggest that changes to Hoxd gene regulation have contributed to the evolution of the snake’s shape and have most likely influenced the body shapes of other vertebrates as well. However, the findings also suggest that these gene regulatory changes have been constrained by an existing regulatory mechanism that has been maintained throughout evolution. It remains for future work to address whether these changes in Hox gene regulation are a cause or a consequence of the snake’s extreme body shape, or indeed a combination of the two.
