Joy Williams and Dr. Emily Bates, Biochemistry Department
Birth defects arise because of both genetic and environmental factors. Children with genetic alteration of the inwardly-rectifying potassium ion channel Kir2.1 channel have heart arrhythmias, impaired cognitive function, and facial and limb abnormalities. Children that suffer from Fetal Alcohol Syndrome have cognitive and social development problems, heart defects, and facial abnormalities—including small eyes, small head, small upper jaw, and a small upper lip1. Kir2.1 is bound and blocked by alcohol2 and thus we predicted that it is the molecular target of Fetal Alcohol Syndrome. We designed experiments to determine how Kir2.1 is required during development and found that within 14 days of prenatal development, both facial and limb abnormalities are present in knockout mice embryos.
First, we raised a colony of mice with the Kir2.1 gene conditionally knocked out. We made crosses with these mice and waited 12, 13, and 14 days into the embryos’ gestation period. With each litter, we stopped gestational development by sacrificing the pregnant mother and harvesting her embryos. We took a small DNA sample from each embryo and performed PCR and gel electrophoresis to genotype them as either wild-type or knockout mice; the knockout gene is heterozygous. Using a cryostat machine, we sectioned samples of the embryos’ limbs and cranium onto microscope slides and stained them with 4’ 6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl tranasferase dUTP nick end labeling (TUNEL). Bromodeoxyuridine (BrdU) allowed us to identify the cells that were dividing to populate the limbs, and the DAPI stain allowed us to determine the percentage of cells that were using Kir2.1 while replicating, specifically in the notochord region; the notochord is a flexible axis to which muscle tissue attaches. The TUNEL stain allowed us to identify apoptosis, or cell death, in both the interdigital and craniofacial regions.
TUNEL staining indicated a larger amount of apoptosis in the palate of the knockout mouse than the wild-type mouse. DAPI staining positively identified the Kir2.1 channel as necessary for notochord development in the limb.
Trans-membrane channels are readily targeted pharmacologically and medicinally. Kir2.1 may be a good therapeutic target for treating Fetal Alcohol Syndrome. Possible treatments could include bone and tissue regeneration. We still have a few more steps to complete for this project. We need to TUNEL stain our limb sections. We also need to specifically activate Kir2.1 after 12, 13, and 14 days of gestational development, then record the recovered amount of bone, tissue, and rate of survival. We will compare this data to that of mice without active Kir2.1 channels.
Alcohol blocks BMP signaling, which is a developmental signaling pathway that depends on Kir2.13. Blocking this gene blocks the BMP signaling pathway and prevents tissue from developing in the craniofacial area, and does not allow apoptosis between digits. Kir2.1 is essential in structural development during gestation and may be a future therapeutic target for treating Fetal Alcohol Syndrome.
- A.D.A.M. Health Solutions, Ebix, Inc. et al. “Fetal alcohol syndrome” PubMed. July 2012.
- Aryal, P. et al. “A discrete alcohol pocket involved in GIRK channel activation” Natural Neuroscience. Aug, 2009.
- Dahal, G. R. et al. “K+ channel required for patterning” Development. 2012.