PROMPT 1: Dan, here is quick my book report-level summary of the paper:
The authors hypothesized that using snake-specific sequence changes, a genomic substitution of the mouse enhancer with its human or fish ortholog would result in normal limb development. Else, replacement with snake orthologs would cause severe limb reduction. This study focused on limb reduction in snakes using CRISPR/Cas9 genome editing. Results show an increase in the rate of sequence changes and progressive in vivo loss of function for a distant-acting limb enhancer in snakes, especially those that have lost skeletal limb structures.
PROMPT 2: Dan, the specific new hypothesis I am testing is:
I predict that the great egret, which has the longest necks in the class Aves, has mutations in their Mef2 cervical spine enhancer that makes them express more Mef2 in their mesoderm than other Aves-class members such as the hummingbirds, which have very short necks.
PROMPT 3: Dan, the specific experiments I would perform are:
Using CRISPR/Cas9 genome editing, I would remove the Mef2 cervical spine enhancer in the hummingbird and replace it with a Mef2 cervical spine enhancer from a great egret. I would conduct the in-situ hybridizations to look at Mef2 transcription in embryos using digoxigenin-labeled antisense riboprobes and then stain embryos with a bone-specific stain to look at the enlargement of the cervical spine. I would then rehydrate the embryos using reverse methanol series, followed by an extensive wash. The cervical spine enlargement would be noted using a Leica MZ16 microscope and Leica DFC420 digital camera.
PROMPT 4: Dan, the specific results I might see are:
I would expect to see an increase in expression of Mef2 in the mesoderm of the modified hummingbird and, ultimately, long neck. I would not the modified hummingbird struggle for survival compared to non-modified hummingbirds since a longer neck would increase its weight and disorient its motor functions due to decreased stability. The rest of the hummingbird’s anatomy and physiology would be the same.
PROMPT 5: Dan, my interpretation of the possible results would be:
The elongation of the modified hummingbird’s neck would mean that Mef2 regulation has been active, causing evolutionally shorter necks. It would also mean that DNA sequence is the main regulator in the evolution of vertebrates, which is one of the premises of Kvon et al. (2016). As such, it would be plausible to say small mutations that occur through evolution are the cause of the anatomical and physiological characteristics of living things.
Kvon, E., Kamneva, O., Melo, U., Barozzi, I., Osterwalder, M., & Mannion, B. et al. (2016). Progressive Loss of Function in a Limb Enhancer during Snake Evolution. Cell, 167(3), 633-642.e11. doi: 10.1016/j.cell.2016.09.028