1. Early embryonic development of the nervous system is relatively similar in most vertebrates. In most amphibians, including our model species Xenopus laevis, primary neurulation is responsible for formation of the neural tube. The process begins with the ectoderm differentiating into cells that will form into the epidermis, the neural tube, and the neural crest. After the neural plate has formed, its edges move upward to create the neural folds. The neural folds continue moving toward one another until the two sides merge, forming the neural tube. INTRODUCTION Through in situ hybridization the gene expression patterns of tnrc4, Xl.8933, and Xl.2595 on whole embryos were determined through relevant neural stages. These embryos were then cryosectioned to determine where these genes are expressed in relation to neural plate formation. Preliminary research indicates that all three genes are expressed in areas that suggest they are relevant to the formation of the neural tube. The genes tnrc4 and Xl.25952 will likely impact neurulation near the midline, while Xl.8933 will have more lateral effects. Evaluation of the effects of inhibiting translation through MOs will determine the projects direction from this point. Characterization of tnrc4, Xl.25952, and Xl.8933 in Early Neural Development Sydney Reichhardt and Christa Merzdorf Department of Cell Biology and Neuroscience, Montana State University April 2011 2011 Hughes Scholars Undergraduate Research Program ACKNOWLEDGMENTS DISCUSSION CONCLUSIONS RESULTS FUTURE WORK I want to thank the Merzdorf laboratory for its support and guidance through my research. I would also like to extend thanks to Martha Sellers and the Hughes Undergraduate Biology Program. Montana State University’s Hughes Scholars Program is supported by the Howard Hughes Medical Institute. Transcription factors, which are responsible for regulating gene expression in all organisms, consist of DNA binding domains that connect specific DNA sequences adjacent to the genes being regulated. Zic1 is a transcription factor that is important during early neural development. The genes tnrc4, Xl.8933, and Xl.25952 are all up-regulated by the transcription factor Zic1. Studying their expression patterns will lead to a better understanding of their roles and the role of Zic1 in early neural development. My research is to: 1) determine the expression patterns of tnrc4, Xl.8933, and Xl.25952 in different stages of development relevant to neurulation (i.e. stages 15-21); 2) explore the effects of over expressing and inhibiting expression of these genes on neural development; and 3) conclude the roles of these genes during embryonic development and relate this information to other species, such as humans and mice. I am working on analyzing the effects of the morpholino oligonucleotide (MO) injections. If significant morphological changes are observed in whole-mount embryos after inhibiting gene expression with the MO injections, then specific controls will be used to explore these alterations. Specifically, we will test whether the effects of the MOs can be rescued. This will be attempted with a plasmid that contains the tnrc4 or Xl.25952 gene, where, in the region that is hybridized by the MOs, the third nucleotides of codons are changed to result in the same protein, but do not allow the MOs to bind. Thus, these constructs can be co-injected with the MOs and the embryos will be tested whether the morphological defects are rescued. The genes tnrc4, Xl.8933, and Xl.25952 were discovered during a screening for genes that were up-regulated by the transcription factor Zic1. In other organisms, tnrc4 has been shown to participate in processing mRNAs. It is unknown at this point, what the Xl.8933 and Xl.25952 genes are and to what genes they may be homologous in humans or mice. Future work on this project will include cloning the Xl.8933 and Xl.25952 genes in an attempt to find genes that they may be equivalent to in other species. Then the roles of tnrc4, Xl.8933, and Xl.25952 during neurulation will be studied further. METHODS Figure 2. The major steps involved in the process of in situ hybridization that concludes with the specific areas of gene expression. (Copyright © 2000, Sinauer Associates) Figure 1. Neural tube formation in Xenopus laevis. (Copyright © 2000, Sinauer Associates) Cryogenic Sectioning Once stained, embryos are prepared for cryosection. The embryos are cryoprotected in an embedding medium (O.C.T. compound) that prevents crystal formation in and around the embryos. Individual embryo blocks are prepared and frozen using the cryoprotected embryos. Once frozen, the embryo are sectioned 12  m thick slices using a microtome. The slices are evaluated using photography and microscopy. Figure 3. The figure shows the process of sectioning a stained embryo on the microtome after it has been embedded in O.C.T. compound. (Current Protocols in Cytometry) A. B.. The transcription factor Zic1 plays an important role in formation of the nervous system. Our laboratory has identified a number of genes, including tnrc4, Xl.8933, and Xl.25952, whose expression is directly regulated by Zic1. These three genes had not been previously studied during early neural development. Through this research, we are learning about the roles of these new genes and Zic1 during early neural development. The expression patterns of the genes tnrc4, Xl.8933, and Xl.25952 were studied to determine their role in neural plate and neural tube formation. The in situ hybridization staining suggested that all three genes are expressed in the neural plate and in the neural tube. Areas with a dark purple or brown precipitate are the regions where a specific gene is expressed at that point in development. DISCUSSION Our results suggest that tnrc4 and Xl.25952 may be expressed in regions that are closer together in the neural plate, while Xl. 8933 may be expressed further away from the midline of the embryos. Based on the cross sections prepared, Xl.25952 and tnrc4 are likely expressed in the neural plate region. Cross sections of Xl.8933 indicate that this gene is expressed more laterally and more widely spread. Evaluation of the expression of Xl.8933 was more challenging due to the overwhelming background observed in cross sections. Further experiments are being designed to clarify the expression of Xl.8933. Our goal was to compare staining present in the cryosections with the landmark structures of neural plate, notochord and somites. Preliminary observations suggest that the genes tnrc4 and Xl.25952 are expressed in the neural plate. The expression pattern of the gene Xl.8933 is more lateral to the neural plate and suggests that Xl.8933 may be expressed in the neural crest. Initial morpholino oligonucleotide injections have been done, but the results have not yet been sufficiently evaluated. In Situ Hybridization In situ hybridization is a specific staining technique used to determine the location of gene expression during embryonic development. The process incudes probe synthesis, fixing the embryos, probe incubation, antibody incubation, and an alkaline phosphate reaction. Anti-sense RNA probes that bind to specific mRNAs are used to over-express certain genes of interest to be stained. The embryos are prepared and fixed in formaldehyde and stored in ethanol in order to preserve them and eliminate the effect of RNases, which digest RNA. Prior to exposure to the antisense RNA probe, the embryos are blocked with a buffer containing random RNA to ensure that the antisense RNA probe only binds to its complementary mRNA. The RNA probes used for in situ hybridization have digoxigenins bound to the uracils. Before and during antibody incubation the non-specific binding sites are blocked using a protein solution so that when the anti-digoxigenin antibody is added, it does not bind to random protein sites. Lastly, the embryo is stained using a solution of NBT/BCIP substrate. The alkaline phosphate attached to the antibody reacts with the NBT/BCIP and causes precipitation that marks the location of gene expression. METHODS Morphollino Oligonucleotides Inhibition of gene expression is achieved by injecting one side of embryos at the eight-cell stage with morpholino oligonucleotides specifically designed to the tnrc4 and Xl.25952 genes. Morpholino oligonucleotides alter gene expression by blocking the region containing the AUG start codon of the mRNAs for the target genes tnrc4 and Xl.25952, which prevents translation. The morpholino oligonucleotides are co-injected with a green fluorescent protein (GFP) tracer to create a colored stain on the injected side of the embryo. The embryos are observed for morphological changes throughout the course of neural development using a fluorescent microscope. RESULTS Figure 4. Morpholino oligonucleotides block the translation of specific mRNA sequences when injected into Xenopus laevis embryos. (/www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMorpholino.shtml) tnrc4 Embryo 1 from 7/29/2010 in situ-whole mount and cross section (~stage 16)