Programmed cell death (PCD) is a biological process that shapes human development. Yet, cancer cells are insusceptible to this process leading to the proliferation of tumors. Research on PCD can produce cancer therapies which increase tumor susceptibility to PCD for tumor eradication.

The exact mechanisms of PCD are currently unknown. My research aims to uncover the role of the gene outsiders in the scheme of PCD in Drosophila melanogaster (fruit fly) embryos. During embryogenesis, Drosophila germ cells travel across the embryo to the gonads for proper development. Mutants with the outsiders gene respond less to PCD resulting in the correct number of germ cells in the gonads, but an excess outlying the peripherals.

To decipher the mechanisms involved in PCD, outsiders will be excised from the genome using the CRISPR-Cas9 genetic engineering technique. This knock-out phenotype will provide insight on the network of PCD for human health applications.

Long non-coding RNAs (lncRNAs) are important players in epigenetic regulation of gene expression during development and disease (Niland et al, 2012). A number of mechanisms have been proposed for lncRNA action, however, few functional studies of lncRNAs have been described. We are using Transcription Activator-Like Effector Nucleases (TALENs), engineered site-specific nucleases, to create targeted mutations in a novel zebrafish lncRNA. We previously mapped a highly penetrant retinal tumor model to transgene disruption of the zebrafish lncRNAis18 gene. The objective of this project is to isolate a second zebrafish lncRNAis18 allele that contains a deletion of part of the lncRNAis18 gene. Two TALEN pairs were designed to simultaneously target double-strand breaks to exons 2 and 5 of lncRNAis18. Injection of 25-40pg of the TALENs targeting individual exons into zebrafish embryos resulted in efficient mutagenesis of the target sites. To isolate the lncRNAis18 deletion allele we co-injected embryos with the TALEN pairs targeting both exons 2 and exon 5. We predicted co-injection of TALEN pairs targeting exons 2 and 5 of lncRNAis18 would create a 147kb deletion after loss of the intervening sequence and repair by the non-homologous enjoining pathway. PCR products spanning the fusion of exons 2 to 5 were amplified from somatic tissue in 9 out of 14 co-injected embryos. We verified the deletion allele by sequencing PCR products from 3 embryos. We have identified one founder that transmits the deletion allele to the F1 generation. F1 embryos are being raised to establish a new lncRNAis18del line. The lncRNAis18 deletion allele will provide a new genetic tool to study the function of lncRNAis18 in zebrafish development and cancer.

The retina is responsible for sensing light and transmitting the signal to the brain in the form of chemical and electrical signals. In our lab, we focus on the development of one set of neurons in the retina, the retinal ganglion cells. These cells receive visual information and send that information as a signal to the brain via their axons, which make up the optic nerve. Studying these cells is important for medical advancement treating diseases such as glaucoma, in which the death of these cells eventually leads to blindness. The goal of my research is to identify the genes most critical to development of healthy retinal ganglion cells by characterizing the mRNA expressed in these cells in the developing chick retina at different time points. Identifying these critical genes and the time points at which they are expressed could contribute to successful ganglion cell generation in vitro. The cells could then be used to replace unhealthy cells that are causing disease and blindness.

Recent studies estimate that there are at least 30 different types of retinal ganglion cell in the mouse eye. These cells are responsible for the connection between the eye and the brain and, therefore, have important functions in image formation. In addition, these are the cells that die in glaucoma, the second leading cause of blindness. Even though the 30 types have been characterized by their physiology, the genetics of each type is unknown. This includes those genes that distinguish one type from another. The goal of our project is to begin to characterize the differences between these cells on a genetic level. In the lab, there is a mouse that has been engineered to express a red fluorescent protein in 8 different types of ganglion cells. Previously, students isolated red cells from the mouse retinas and identified sets of mRNAs that were expressed in each cell. Through these mRNAs, predictions have been made as to which genes define which ganglion cells. However, since the number of cells analyzed by this method was small, larger scale validations need to be performed. Our objective was to take genes that were identified in this initial screen and analyze their expression in retinal ganglion cells in much more detail.

Zebrafish (Danio rerio) serve as a very useful model organism because they have a fast generation time, clear embryos and a well mapped genome. In this research project, the students in the Developmental Biology lab course and the students in the Freshmen Research Initiative have used these characteristics to conduct a screening of the zebrafish genome in order to identify genes that are required for development. The CRISPR/Cas9 system (a protein that creates double strand breaks at specific sites in the genome that are then repaired by the cellular machinery) was recently specialized for the use in zebrafish. However, there are usually mistakes made when repairing the break. By using this system we can create mutations at specific sites in the genome and even delete entire sections. We can then observe if the mutation has created any notable phenotypes in the developing embryo. That information can give us insight into what the genetic requirements are for development or how those mechanisms can go wrong in diseases such as cancer.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. Once these nerve cells die, the patient’s muscles waste away, resulting in paralysis and eventually death. Two primary forms of ALS exist: Familial and Sporadic. Familial constitutes 5-10% of cases and is defined by ALS being present in one or more cases in a family’s lineage. Sporadic makes up 90-95% of ALS cases and is essentially when no family history exists with ALS but an individual has ALS. Mutations in SOD1 have been the most studied in regards to ALS. However there are many other genes linked to ALS that have not been studied. VCP is a gene that has been linked to several different diseases including familial versions of ALS. The protein has been linked to many different cellular processes including protein degradation and programmed cell death. To gain a better understanding into the development and eventual death of motor neurons, we are using both TAL-effector nuclease (TALEN) mediated mutagenesis and a VCP CRSPR to create zebrafish that are mutant for VCP. These mutant fish will hopefully allow us to create a new model of motor neuron degeneration or ALS.

Amyotrophic lateral sclerosis (ALS) is a well-known neurodegenerative disease caused by motor neuron death within the spinal cord and brain. Soon after the nerve cells die, the patient’s muscle cells degenerate resulting in paralysis and eventually death. Another debilitating human disease is primary open-angle glaucoma (POAG). POAG is an ocular disease triggered by the rise in internal eye pressure which damages the optic nerve, reducing image signals to the brain. Most cases of ALS are sporadic and the direct causes for the increase of internal eye pressure are questionable, meaning that a clear genetic and molecular understanding of the mechanisms leading to the diseases is not well understood. The gene optineurin (OPTN) has been identified and implicated as a contributor to the mechanisms leading to the onset of both of these diseases. To gain a better understanding of the cellular functions of OPTN, we are using TAL-effector nuclease (TALEN) facilitated mutagenesis. The TALEN specific for the OPTN gene in zebrafish has been generated, injected, and has produced mutations. The mutations are being characterized for their consequences on zebrafish eyes and motor neurons to hopefully allow us to create new zebrafish models for ALS and glaucoma.

Amyotrophic lateral sclerosis (or Lou Gehrig's disease) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Once these nerve cells die, the patient's muscle cells degenerate, resulting in paralysis and eventually death. Most cases of ALS are sporadic, meaning that a clear molecular and genetic understanding of the mechanisms by which the motor neurons die is lacking. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one gene that has been implicated in ALS, as well as several other neurodegenerative diseases. To gain a better understanding into the function of UCHL1 in non-diseased neurons, we are using TAL-effector nucleases (TALEN) to create UCHL1 mutant zebrafish. Specifically, I have generated these TALENs and begun to inject them to generate the mutant fish. Once created, these fish will provide insights into the normal function of UCHL1 and, hopefully, allow us to create a new zebrafish model of nerve cell degeneration.

Endothelial tubulogenesis is the formation of functional blood vessels. The purpose of this research is to understand how actin drives this process. To observe the function of actin, certain transgenic gene lines will be characterized. These transgenic gene lines are LifeAct, Moesin, Moesin-Actin-Binding Domain, and Alpha-Catenin. These genes will allow us to see how actin contributes to vascular formation. This formation will be visualized using fluorescent proteins RFP and GFP. These proteins will act as markers on each transgenic gene line to monitor the process of endothelial tubulogenesis in zebrafish using confocal microscopy. The zebrafish will express fluorescents in these gene lines using the GAL4-UAS system. This system is expressed in the vascular system, and binds to the UAS sequence to activate fluorescents for the gene of interest. Based on our research, actin acts as structural units to form vasculature. Actin is a functional protein that forms microfilaments that can be used in this process. This is based on where actin was expressed in zebrafish during endothelial tubulogenesis. Actin was expressed from major vessels, and expressed more in areas with continual vasculature formation. This shows that actin is an important factor in endothelial tubulogenesis.

Amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease, is a neurodegenerative disease caused by the death of motor neurons in the central nervous system. The death of these nerve cells leads to the degeneration of the patient’s muscle cells, resulting in paralysis and eventually death. Because the majority of ALS cases are sporadic, there is not a clear understanding of the molecular and genetic mechanisms that lead to the death of the motor neurons. Superoxide dismutase (SOD1) is one gene that has been implicated in ALS, but there are also a large number of genes that are linked to ALS but have not been studied in depth. Sigma non-opioid intracellular receptor 1 (SIGMAR1) is one gene that has been found to have a connection to different forms of ALS, including juvenile ALS, but the relationship between the two is not understood. To gain a better understanding into SIGMAR1’s role in the development and death of motor neurons, we are using Tal-effector nuclease (TALEN) mediated mutagenesis to create SIGMAR1 mutant zebrafish. Once created, these fish will hopefully provide insights into the function of SIGMAR1 and allow us to create a new model of nerve cell degeneration.