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How Snakes Lost Their Legs: An Introduction to Evolutionary Development and Gene Regulation

Shana Lee McAlexander
Product Developer

Fossil, embryological, and genetic evidence support the view that ancestors of modern snakes had legs. Use the absence of legs in the snake body plan as an engaging case study to introduce students to the concepts of cell differentiation, mutation, and gene regulation in the context of evolutionary development.


Cell differentiation

To understand how the snake lineage may have lost its legs, we first need to understand how cells differentiate during embryonic development.

All the tissues in vertebrates are a result of the cellular divisions from the same fertilized egg. These cells differentiate to serve different forms and functions in the body. Cells receive signals that determine their fate, and genes control these signals. A gene is "expressed" when it is transcribed into mRNA, and that mRNA is translated into a protein. In order to develop correctly and survive, an organism must be able to express genes at specific times and under specific conditions.


Genetic variation by mutation

Vertebrates have a common ancestor, yet genetic mutations over millions of years have influenced the variation among them. Species that have similar DNA sequences evolved from a recent common ancestor. Organisms that are more distantly related have, over time, accumulated more differences in their DNA sequences via mutations.

Harmful mutations, which hinder an organism's fitness and its ability to survive and reproduce, are less likely to be passed on to the next generation. Mutations that generate advantages for an organism may be gleaned through natural selection, the driving force of evolution. Changes that occurred in the snake lineage likely provided an advantage for survival and reproduction.

It is believed that members of a group of genes that regulate the vertebrate body plan, called Hox genes, were the result of a gene duplication. A gene duplication event, a specific kind of insertion mutation, occurs when an entire section of DNA in the genome that codes for 1 protein is mistakenly copied twice. If all the regulatory sequences that control transcription and translation of the protein are present in the duplication, both regions will code for the same protein. When a second copy of the gene is available, 1 of the 2 DNA sequences can change—possibly to serve a new function—while the other copy maintains its original function.


Gene regulation and Hox genes

Hox genes regulate the differentiation of cells in the animal embryo. The expression of Hox genes along the long axis (head to tail) of an animal provides the basis for anterior–posterior tissue specification, which leads the head portion to become the head portion and the hind portion to become the hind portion. A mutation in a Hox gene, or its expression in a new time or place, can lead to entirely different organs developing in new parts of the body. Mutations in 1 region of DNA can alter gene expression in another because some genes, or portions of genes, serve to regulate (turn on, turn off, turn up, or turn down) levels of transcription.

Specific to vertebrates, the pattern of Hox expression determines the fate of the cells that will become the vertebrae. Certain combinations of Hox signals create thoracic vertebrae (having ribs), while others create cervical (neck) vertebrae or lumbar (lower) vertebrae.


Loss of forelimbs

Fossil findings of snakes with present hind limbs—but missing forelimbs—suggest that the snake lineage lost its ability to generate forelimbs first. Some extant snakes, such as vipers, are completely limbless, while more primitive extant snakes, such as boas and pythons, have pelvic girdles and rudimentary femurs. Researchers have studied the loss of forelimbs and the loss of hind limbs as separate evolutionary events.

In most vertebrates, the forelimb arises in the tissue based on a particular combination of Hox gene expression. Specifically, the forelimb develops immediately anterior (toward the head) to a domain of HoxC-6 expression. When both HoxC-6 and HoxC-8 are expressed, the vertebrae are regulated to become thoracic vertebrae (with ribs). In the case of nearly all snakes, the region of HoxC-6 and HoxC-8 expression extends along most of the body. Without the absence of HoxC-6 expression, no tissue becomes forelimb. Snakes' four-legged ancestors likely had an absence of HoxC-6 where their tissue was signaled to develop into forelimbs. Over many generations, the RNA expression of HoxC-6 extended toward the head, eliminating forelimb bud formation.


Loss of hind limbs

Some snakes have rudimentary hind limbs. Examining a proposed phylogenic tree from Tickle and Cohn's research in 1999, snake ancestors, the Mosasauridea, had forelimbs and hind limbs. Among the oldest known extinct snakes, Pachyrhachis had complete hind limbs but no forelimbs. Of the extant snakes, like the python and boa, the Boidae have rudimentary hind limbs only. The most modern snakes, like the viper, Colubroidea, are completely limbless.

Because expression of HoxC-6 occurs in the time and location of hind limb formation, there is strong evidence that the hind limb is controlled by another regulatory mechanism, but this mechanism is not completely understood. Even in those snakes with hind limbs, the tissue never fully forms into legs. Some signal—or absence of signal—does not allow this tissue to develop as it does in many other vertebrates. Studies have shown that the timing of expression of a gene, called sonic hedgehog, is critical to normal hind limb development.

Performing experiments in mice, researchers examined how changes in sonic hedgehog expression affect tissue originally destined to become hind limbs and found that correct regulation of the sonic hedgehog protein is important. If the sonic hedgehog regulatory region of DNA is mutated, limbs develop abnormally. If the regulatory sequence is deleted completely, the mice develop with no feet. The appearance of the abnormal limb of the mutant mouse closely resembles the tissue of the python's rudimentary hind limb. Because the python expresses functional sonic hedgehog RNA in other parts of its body corresponding to other vertebrates, but not in the hind limb region, the sonic hedgehog gene is believed to play a critical role in the loss of snakes' hind limbs.


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References

M. T. Cohn, C. Tickle. 1999. Developmental basis of limblessness and axial patterning in snakes. Nature 399: 474-479.

T. Sagai, M. Hosoya, Y. Mizushina, M. Tamura, and T. Shiroishi. 2005. Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Development 132: 797–803.

J. Zhu, E. Nakamura, M. Nguyen, X. Bao, H. Akiyama, and S. Mackem. 2008. Uncoupling sonic hedgehog control of pattern and expansion of the developing limb bud. Developmental Cell 14: 624–32.

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