Question

Snakes have lost their limbs over the course of evolution. Recent data show a reduction in Shhexpression in their limb fields due to loss of a portion of the limb enhancer, noting that other enhancers remain intact.

First, indicate whether this is an example of heterotopy, heterochrony, heterometry, or heterotypy, defining your chosen term. Second, describe how you might use the mouse embryo as a model system to test the hypothesis that this reduction in Shh expression in the limb field is responsible for the loss of limbs in snakes. Be sure to provide a brief description of the relevant technique(s), and to include at least one control

Answer 1

The information described above talks about the snakes losing their limbs over the course of evolution. This is because of the effect of the absence of the enhancer element that governs the expression of the Shh or the Sonic HedgeHog gene, responsible for limb development. Understand it in this way: the enhancers are a stretch of DNA that could interact with the proteins called activators or transcription factors and this whole setup then further activate a specific gene (by activation I mean they induce its transcription) and thus its expression (in our case the gene is Shh). In the evolutionary process if there is a loss of a particular enhancer (in the particular group of cells present at a specific site in the developing embryo) that ultimately leads to the decrease in the activation of a gene(say Shh, which is important for limb development) is termed as heterometry. In the case of a heterometry, there is no mutation that leads to a change in the expression of a gene but the amount of expression of particular gene changes. Here the enhancer is called ZRS that governs limb specific expression of Shh.

Now say, to prove this hypothesis, that ZRS enhancer is responsible for the amount of expression of the Shh gene we can design the following experiment. Remember a Hypothesis is a scientific question that is asked to address a gap in the knowledge or in the literature. The gap is filled by answering that research question and thus generating enough conclusive data. Here, to implicate the role of ZRS in the activity of Shh we can delete the enhancer element altogether and then observe the expression pattern of the Shh gene in the embryonic mice model.

In order to do that we will be needing a mice embryo model which will have to be subjected to deletion of the target enhancer element, ZRS. So the mice model will be subjected to CRISPR/Cas9 induced knockout of the enhancer. CRISPR/Cas9 is a recent and widely utilized tool for making genetic alterations that are efficient and target specific. CRISPR/Cas9 system incorporates the use of a gRNA that is target-specific (shows complementarity, and thus Watson & Crick base-pairing is seen). Once the CRISPR/Cas-9 associated gRNA is bound to the target sequence it triggers the activity of the Cas-9, an endonuclease that will lead to the cleavage of a target sequence.

Coming to the point of inducing the knockout or deletion of the ZRS, this could be achieved by giving the required concentration of Cas9 and sgRNA through injection to the FVB mouse embryos (at a single-celled stage). The knockout of the ZRS can be validated via PCR or Polymerase chain reaction. Now to compare the effects of these genetic manipulations made in the experimental group we will also have to include a Control group, i.e. those mice embryos that are not altered with the CRISPT/Cas9 and thus have no election of the ZRS. Such a group will be known as the wild type group. Now remember that the mammals are diploid and thus have homologous chromosomes, this means if the ZRS is knocked out from one chromosome then there is another chromosome left to compensate the absence of the earlier knocked-out fragment. So CRISPR/Cas 9 will have to be used to knock out the enhancer sequence on the homolog chromosomes as well. This could be done by injecting the Cas9 and the sgRNA in the eggs of the initial mice subjected to the first cas9 induced deletion. Only the founder line with null ZRS detection (through PCR) will be utilized to study the effect of the ZRS knockout. The PCR specific to ZRS will tell us that whether or not the CRISPR/Cas9 tool was successful in deleting the ZRS element, as the presence of the ZRS will be indicative of the failure of the Cas9 mediated knockout. However wild type will show amplification of the ZRS sequence.
What should we expect to see is a truncated or abnormal limb development in the CRISPR/Cas9 induced ZRS knockout mice but a fully normally developed limbs in the Wild type control group of mice. Thus this will answer the research question or the hypothesis made earlier that during the course of evolution the loss of the enhancer activity ultimately leads to the decrease in the activity of an important development-specific gene such as Shh that led to limb loss in reptiles such as snakes.