A Single Nucleotide Substitution in a Hox/Pax-responsive Enhancer Is Responsible for the Evolution of the Snake Body Pattern

Contributed by: Jongmin Nam

          Hox genes control the body plan of animals along the anterior-posterior axis (Figure 1). Their expression patterns and protein function determine the identity of each body segment, and changes in their functions are often responsible for the variations in the number and structure of body segments in vertebrates. 

Figure 1. The spatial expression zones of the Hox genes along the antero-posterior axis of a fly (a) and a mouse (b). Note the correspondence with the chromosomal order of the genes. This is referred to as colinearity. (From Carroll, S. et al. 2005, From DNA to Diversity, Blackwell.)

            Among many vertebrates, snakes are fascinating models for studying body plan evolution: they have more vertebrae than other vertebrates and have ribs even in the posterior parts of their trunk, where most vertebrates have rib-less vertebrae. It has been known in mice that Hox6 is expressed in the anterior trunk and Hox10 in the posterior trunk (Figure 2). The former is an activator of rib formation, and the latter a suppressor. When mouse Hox10 was ectopically expressed in the anterior trunk, rib development was inhibited. Interestingly in snakes, Hox6 expression is extended to the posterior trunk and overlaps with Hox10 expression (Figure 2). Because Hox10 function is dominant over Hox6 function in the rib development in mice, it was hypothesized that snake Hox10 lost its function to repress rib formation.

            To test this hypothesis, Guerreriro and colleagues (1) examined a gene regulatory network involving Hox10 in a recent PNAS paper. The authors hypothesized that loss of gene expression or protein function of Hox10 in snakes might be responsible for the posterior extension of ribs. However, expression patterns of Hox10 between mice and snakes were similar, and the snake Hox10 protein could still repress rib development in transgenic mice, rejecting the hypothesis.

Figure 2. Loss of Hox10 rib-suppressing activity in the trunk of the snake. In mice suppression of Myf5/6 in the posterior part of the body is responsible for rib-less phenotype. However, in snakes Hox10 cannot suppress Myf5/6 resulting in rib-bearing vertebrae in the trunk. (From Woltering JM, 2012, Current Genomcis, 13(4))

            If it is not Hox10, then are the downstream target genes of Hox10 responsible for the phenotypic difference? The authors examined several known downstream target genes of Hox10. Interestingly, they found a single base-pair difference in a highly conserved enhancer region of the snake Myogenic factor 5 (Myf5), a transcription factor gene that controls skeletal muscle development. In mice, this enhancer is repressed by Hox10 in the posterior trunk and is activated by Hox6 and Pax3 in the anterior trunk. When the snake mutation was introduced into the mouse Myf5 enhancer, ectopic reporter expression in the posterior trunk was observed, which is consistent with the posterior extension of ribs in snakes.

            Is this change specific to the snake lineage? The authors' survey of the enhancer sequences from a variety of vertebrate species discovered independent occurrences of the same mutation in at least two other lineages, elephant/hyrax/manatee and tenrec. Because the former three species also have longer rib-cages than other mammals, it is possible that the Myf5 enhancer is a hotspot (2) for morphological evolution. Note that the title is a bit misleading, because the causal mutation is fixed in the snake lineage, and therefore is not a polymorphism.

            Although the findings look clear-cut, it was puzzling that the single base-pair mutation in the snake enhancer is within the binding site of both Hox6 (activator) and Hox10 (repressor). How did the snake enhancer lose only repression, but not both activation and repression? The answer was quite unexpected: although Hox6 can bind directly on the enhancer in mice, Hox6 can also indirectly interact with the enhancer via interaction with Pax3, whose binding is not affected by the mutation. Because Hox10 does not interact with Pax3, Hox10's interaction with the snake enhancer is significantly reduced resulting in ectopic expression of Myf5. This result explains why snake Myf5 enhancer lost its repression by Hox10, yet maintained activation by Hox6.

Figure 3. Model for rib growth vs. repression in mice and snakes. In mice, Hox6, an activator of Myf5, is expressed in the anterior trunk, and Hox10, a repressor of Myf5, in the posterior trunk. In snakes, Hox6 is expressed in both anterior and posterior trunks and Hox10 only in the poster trunk. However, neither Hox6 nor Hox10 can bind directly to the snake Myf5 enhancer due to a single-nucleotide change within the enhancer. Unexpectedly in snakes, Hox6 still can activate Myf5 with the help of Pax3, whose binding to the enhancer is independent of the mutation. The newly discovered mechanism of Hox6/Pax3 is also conserved in mice. (From Mansfield JH, 2013, PNAS)

            The present study highlights the power of studying evolution in the framework of gene regulatory networks: it not only provided genetic clues for morphological differences, but also deepened our understanding on the underlying molecular mechanisms. Is functional variation within conserved genes the main underlying causes of morphological evolution? We need more of these studies to answer this question.

References

1.         Guerreiro I, et al. (2013) Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proceedings of the National Academy of Sciences 110:10682-10686.

2.         Stern DL (2011) Evolution, development, & the predictable genome (Roberts and Co. Publishers).

Abstract of the original paper Patterning of the vertebrate skeleton requires the coordinated activity of Hox genes. In particular, Hox10 proteins are essential to set the transition from thoracic to lumbar vertebrae because of their rib-repressing activity. In snakes, however, the thoracic region extends well into Hox10-expressing areas of the embryo, suggesting that these proteins are unable to block rib formation. Here, we show that this is not a result of the loss of rib-repressing properties by the snake proteins, but rather to a single base pair change in a Hox/Paired box (Pax)-responsive enhancer, which prevents the binding of Hox proteins. This polymorphism is also found in Paenungulata, such as elephants and manatees, which have extended rib cages. In vivo, this modified enhancer failed to respond to Hox10 activity, supporting its role in the extension of rib cages. In contrast, the enhancer could still interact with Hoxb6 and Pax3 to promote rib formation. These results suggest that a polymorphism in the Hox/Pax-responsive enhancer may have played a role in the evolution of the vertebrate spine by differently modulating its response to rib-suppressing and rib-promoting Hox proteins.