# data[].attributes.body
First, we would get as many snake genomes as possible and look for the same gene repeated multiple times. If there is only one copy of the gene, gene duplication can be ruled out.

# data[].attributes.body
By comparing Shh gene expression during the development of snakes and lizards we could rule out regulatory evolution as a method for snakes losing their legs.

# data[].attributes.body
We would look at the sonic hedgehog gene in extant lizards and snakes and see if they are genetically similar using online DNA sequences. Since protein coding mutations are often widespread and fatal, we expect to see almost no mutation and thus proof that the change is not caused by protein evolution.

# data[].attributes.body
Sequence the genomes of organisms and compare for changes in the amino acid sequences

GWAS analysis

# data[].attributes.body
You can sequence the genome of a snake and other animals and see where the sequences differ. In animals with limbs, if the proteins encoded are the same, then protein evolution is likely not the cause for loss of legs.

# data[].attributes.body
You could look at genetic information for the Shh protein and compare the sequences to other closely related animals that have legs.

# data[].attributes.body
Download the section of the genome for the candidate gene for a snake and a lizard. Use GWAS to compare and see if there are significant differences in the DNA sequence. If there are, it would indicate that protein evolution is the mechanism for limb loss in snakes.

# data[].attributes.body
To rule out protein evolution, you can compare the protein with other species. Also, you can show that the protein is not evolving over time by comparing to past generations or showing high conservation across the species. We would conduct all of these measures on the snake species

# data[].attributes.body
To rule out regulatory evolution (gene expression) in Shh: , Researchers would need to show that Shh has the same spatial and temporal expression pattern, and similar expression levels, in both species.

# data[].attributes.body
Compare when and where SHH is expressed in snake embryos versus limbed animals. If SHH is still expressed normally in the limb region, then leg loss is probably not due to regulatory evolution at SHH.

# data[].attributes.body
Compare how many Shh gene are in a snake with other legged animals. If the number is the same then gene duplication can be ruled out.

# data[].attributes.body
We can test this by using PCR, to check if the gene we want is still present in the organism. Since the first gene always has the protein, and the other duplicated genes mutate.

# data[].attributes.body
Protein evolution - We would download the genomes of snakes and closely related species that have not lost legs and compare their genomes for changes in regions that would code for proteins that assist in leg growth. If there is no meaningful change, then protein evolution can be ruled out.

# data[].attributes.body
looking for which copies are most present with the genes associated with loss of legs. This can be seen between sensory and motor nerves being expressed more which then associates with the function of the shh gene.

# data[].attributes.body
We can look at the genome of lizards and see where the Sonic hedgehog is located and see where it aligns with the snake genome. If there is a homolog in the snake genome, then it was unlikely to be do to protein evolution. As an additional experiment, you replace the snake gene with the lizard gene corresponding for Sonic hedgehog and see if the snake develops limbs. If it grows legs, it's an example a protein evolution or something may be wrong with the coding part of the snake genome (obviously).

# data[].attributes.body
Download genomes and identify the non-coding regions in the snake genome as they compare to closely related species that still have legs. Using CRISPR, knock out the regulatory regions upstream of the gene of interest (Shh) in snakes. If the phenotype is regularly expressed after knockout, it was not due to regulatory evolution.

# data[].attributes.body
I would start looking at MFCs1/ZRS because that is the regulatory element related to limb buds

# data[].attributes.body
Floor plate of the spinal code, as this is were the previous shh regulatory elements tended to be. But also MFCs1/ZRS because that’s where limb development starts.

# data[].attributes.body
We would look at MFC/ZRS as its closest related to limb buds. Or Lmbr1

# data[].attributes.body
We would look at the MFCs1/ZRS gene because that correlates with the limb buds

# data[].attributes.body
We would start by looking at the floor plate and then causal diencephalon

# data[].attributes.body
We would start by looking at the MFCs1/ZRS which encodes for limb buds, because Shh targets limb development.

# data[].attributes.body
We would look at MFCs1 and ZRS because these are the regulatory elements for the limb buds

# data[].attributes.body
"The one that says 'limb bud': MFCs1/ZRS." -Chris

For the experiment, you start by replacing the snake regulatory sequence with an animal that develops legs, like lizards.

# data[].attributes.body
I would start looking at Limb bud associated genes such as in MFCs1 and ZRS

# data[].attributes.body
We would start looking at the SFPE1 gene. Because it appears right before Shh which means it could be a regulatory element of Shh.

# data[].attributes.body
We would start looking at SPEF1 and SPEF2 as they are regulatory elements for Shh and aid in the development of the floor plate of the spinal cord which is closer to limb development than the other

# data[].attributes.body
We would look at MFCs1/ZRS as this regulates limb buds

# data[].attributes.body
We would look at MFCs1 and ZRS

# data[].attributes.body
MFC/ZRS because closest realted to limb buds

# data[].attributes.body
We would start looking at the MFC/ZRS region because that is associated with the limb buds

# data[].attributes.body
We’d look at MFC/ZRS because it relates to the limb buds

# data[].attributes.body
It would be harder for viruses to be resistant against our immune system, whereas bacteria can evolve to be resistant to antibiotics