This document summarizes a proposed study to compare gene expression related to wing development in bats and birds. The study has two aims: 1) to compare expression of the Bmp3 gene related to digit elongation by knocking it out in bat and chicken embryos using CRISPR/Cas9, and 2) to compare expression of the Sonic hedgehog (Shh) gene related to light bone development by knocking it out in bat and chicken embryos. For both aims, gene-edited embryos would be observed to see if wing or bone development is altered compared to control embryos. Results could provide insights into genetic mechanisms underlying convergent evolution of flight in bats and birds.

1. Convergent Evolution of
Wing Morphologies in
Birds and Bats
By: Kevin Varty

2. Convergent Evolution
• The independent evolution of analogous
structures or features.
• Similar in function but different in
structure.
• Features not present in the last common
ancestor of individuals in question.
• Caused by environmental pressures.
• Selection for the traits of the individuals
best suited for their environment.

3. Convergence on the molecular level
SEBA’S SHORT-TAILED BAT BOTTLE-NOSED DOLPHIN

4. Opposable Thumbs
THE GIANT PANDA CHIMPS/APES/HUMANS

5. Evolution of Wings

6. What causes these similar
morphologies?
• Previous studies have shown similar genetic mechanisms are responsible for the
morphologies of wings in each group of animals.
• Digit elongation
• BMP Pathway genes (BMP3) (Welten et al. 2005; Sears et al. 2006; Sears,
2007; Wang et al. 2010)
• Small hollow or porous bones
• Sonic hedgehog (Shh) (Sears, 2007)

7. Seba’s Short-Tailed Bat (Carollia perspicillata)
• Found in deciduous and evergreen
forests near stagnant water.
• Predominately feeds on fruit,
occasionally flowers and insects.
• Foraging occurs near ground
level.
• Most common bat species found in
zoos today.
• Relatively easy to breed.
• Commonly used in studies (Cretekos,
et al. 2005; Sears, et al. 2006)

8. The Domestic Chicken (Gallus gallus domesticus)
• A subspecies of the red junglefowl.
• Outnumbers any other species of
bird or domestic animal.
• A huge source of food for people all
over the world.
• Found in almost all environments
where people can be found.
• Has been used in previous studies
(Zuo et al. 2016)

9. Aim 1
Compare gene expression in the extended forelimbs of bats and birds.
• Studies have shown Bmp3 to be a possible gene responsible (Welten et
al. 2005; Sears, et al. 2006; Sears, 2007; Wang, et al. 2010)
Methodology
• Obtain 10 embryos from each species
• Use laser capture microdissection (LCM) to take cells from the wings of
each embryo once the wings have been defined.
• Use CRISPR/Cas9 with a guide RNA sequence to knock out the Bmp3
gene in captured cells.
• Inject altered cells back into wings of embryos.
• Allow test subjects to be born and reach adulthood.
• Compare wingspans to established average ranges.
• 45-65cm for chickens and 21-25cm for bats

10. CRISPR/Cas9
1:05- 2:47

11. Aim 1
Expected Outcomes
• The wingspans of test subjects will fall below established averages.
• Based on previous studies (Wang, et al. 2010).
Possible Complicaitons
• Death of test subjects.
• Up-regulation of other BMP pathway genes
• Gene knockdown versus gene knockout.

12. Aim 2
Compare gene expression in the development of light bones in bats and in birds.
• Studies have shown Sonic hedgehog (Shh) to play a role in the reduction of length,
width, and density of the ulna in bat forelimbs (Sears, 2007)
Methodology
• Obtain 20 embryos from each species.
• Use LCM to capture cells from the ulna of 10 embryos from each species.
• Use CRISPR/Cas9 and a guide RNA sequence to knockout the Shh gene.
• Inject altered cells back into the 10 embryos of each species.
• Allow test subjects to be born and reach adulthood.
• Compare ulna size 20 experimental embryos versus 20 control embryos using dual
energy X-ray absorptiometry.

13. Aim 2
Expected Outcomes
• Ulna size of the 20 test embryos will be greater than the 20 control embryos in
length, width, and density.
• Based on previous studies (Sears, 2007)
Possible Complications
• Death of test subjects.
• Up-regulation of other genes.
• Gene knockdown versus gene knockout.

14. Broader Impacts
• Increased understanding of the genetic mechanisms behind
convergence.
• Increased understanding of limb development
• Shaping the minds of little children.

15. Questions?