Calcium is an ubiquitous and critical signaling molecule that mediates a wide range of cellular processes. Cytosolic calcium is maintained at concentrations that are orders of magnitude below both the extracellular space and intracellular organelles. Even small, sustained increases in cytosolic calcium can have harmful effects on the cell. As such, calcium homeostasis mediated in large part by the endoplasmic reticulum (ER) and mitochondria that serve to sequester calcium enable normal functioning of the cell. To examine cellular mechanisms that mediate calcium homeostasis, we are using mechanosensory hair cells of the zebrafish lateral line. Mechanosensory hair cells can be stimulated via a mechanical stimulus or drug-mediated calcium release from intracellular stores. The hair cells' abilities to easily uptake drugs and respond to physical stimuli make them an ideal tool to examine calcium homeostasis. Preliminary studies using transgenic zebrafish in which the calcium indicator GCaMP3 is targeted to the cytosol vs. the mitochondrial matrix demonstrates that stimulation of hair cells causes an immediate increase in cytosolic calcium in contrast to a gradual increase in mitochondria calcium. The question is whether the calcium in the ER is changing in response to the changes in mitochondrial and cytosolic calcium. As a first step to investigating the role of ER in calcium homeostasis, we will use gateway cloning to generate transgenic zebrafish lines in which calcium indicators are targeted to the ER. We predict that stimulation of mechanosensory hair cells will cause changes in ER calcium levels. These studies may provide valuable insight in how cells maintain health by buffering intracellular calcium levels in response to activity. 

3 million Americans and 65 million people worldwide have been diagnosed with epilepsy. In two-thirds of these patients, the causes of epilepsy, and therefore the therapeutic targets, are unknown. Recently, de novo mutations in the EEF1A2 gene, which encodes for a protein translation regulator, were found in several individuals with pediatric epilepsy and intellectual disability. In order to study how a mutation in this gene causes the neurological defects, we generated mutant eef1a2 zebrafish using the CRISPR/Cas9 method. Zebrafish models to study diseases have gained momentum due to their genetic similarity to humans, large amount of offspring in each generation, easy genetic manipulation and amenability to large-scale drug screens. Our initial in situ hybridization studies showed that eef1a2 has a limited expression pattern in the developing zebrafish that includes neurons involved in movement and escape behavior. To understand whether the mutation causes observable behavioral dysfunction or seizures, we tracked wild type (WT) zebrafish and heterozygous and homozygous mutants in various behavioral assays and genotyped tracked fish using Sanger sequencing. We are expecting that the mutants to have defect in movement and escape response. In a future study, the electrophysiology of the forebrain will be recorded to determine whether eef1a2 mutants reveal abnormal electrical discharge characteristic of a seizure. These studies with the zebrafish model will provide more insight about the EEF1A2 protein and its neurological function, and may lead to a better understanding of epilepsy etiology and the design of targeted therapeutic approaches.