1. Lithium Chloride Induces Exogastrulation and Teratogenesis in Developing Sea Urchin (Lytechinus variegatus) Embryos: COULD β -CATENIN BE INVOLVED? Cody Blymire Department of Biological Sciences, York College of Pennsylvania, York, PA 17405 Project Summary: The sea urchin is a very popular organism for scientific investigations. For this research project, Lytechinus variegatus, was studied in regards to the toxic effects of LiCl in delaying embryo development, causing exogastrulation, and affecting β- catenin expression during the gastrulation stage. Gastrulation is an extremely important developmental stage in the sea urchin, and drastic changes in cell arrangements occur. LiCl inhibits this developmental stage by causing the urchin to not form a gut tube or archenteron (Figure 1B). This phenomenon implies an effect on the cell-cell interactions and arrangements necessary for gastrulation to occur properly. β-catenin is a multifunctional protein that is part of the WNT signaling pathway that assists in cell-cell adhesion and cell fate determination. β- catenin expression is reported to be most abundant during sea urchin gastrulation, and accumulation is most abundant around the developing archenteron. LiCl is a well known vegetalizing agent, and has been demonstrated to delay development in various organisms. LiCl was presented at various concentrations of 0 mM, 15 mM, 30 mM, and 60 mM, and the sea urchins embryos were placed in these concentrations at the two-cell stage and allowed to develop. Morphological as well as developmental observations were made. Results showed that LiCl inhibited the development of embryos in a dose dependent manner in that the higher concentration the more inhibition was observed. Morphological dysfunctions included embryo fragmentations as well as exogastrulation. Due to lack of β- catenin antibody availability an ELISA test will be proposed and it is hypothesized that as the concentration of LiCl increases, the expression of β-catenin would increase as well in a dose dependent manner. This would imply that β-catenin may play a vital role in sea urchin gastrulation. Introduction: Gastrulation is a very important developmental occurrence in sea urchins. During this stage, many cell-cell interactions and cell relocations occur. It is during this stage that the urchin embryo forms what is termed the archenteron or gut tube (Figure 2). When sea urchin embyros are exposed to LiCl they do not form an archenteron, rather they exogastrulate. Urchins exposed to LiCl also demonstrate enhanced expression of β-catenin mRNA. This β-catenin may play a major role in signaling cells to successfully develop the archenteron. This is the question that my proposal aims to address, could β-catenin protein expression be enhanced by LiCl exposure, thus potentially be involved in causing this exogastrulation occurrence. Literature Review: 1 McGrath et al. (1991):  They noted that embryos exposed to LiCl demonstrated both a delay in development and an increase in exogastrulation in a dose dependent manner. 2 Miller et al. (1995):  They researched that β-catenin modulates cell-cell adhesion during gastrulation in the sea urchin.  They found that treating sea urchin embryos with LiCl enhanced the level of β-catenin mRNA detected through Northern Blot analysis.  They also noted that embryos treated with LiCl displayed an increase in endoderm and mesoderm cells. 3 Miller and McClay (1997):  They determined that β-catenin is expressed at its highest level during the gastrulation stage. 4 Logan et al. (1999):  They noted that when the archenteron is forming, β-catenin expression is localized to cells forming the archenteron. 5 Kitazawa and Amemiya (2001) :  They studied the effects of LiCl on developing sea urchin embryos (Peronella japonica).  They found that the developing archenteron was severely hindered due to exposure to LiCl at various concentrations. 6 Weitzel et al. (2003):  They observed that over-expression of β-catenin hinders primary mesenchyme cell (PMC) production.  They deterrmined that β-catenin is a powerful activator in the PMC linage during the gastrulation stage. Research Design: Literature Cited: 1. McGrath, Catherine, Robert McIsaac, and Susan G. Ernst. 1991. Altered Cell Fate in LiCl-treated Sea Urchin Embryos. Developmental Biology. 147: 445-450. 2. Miller, Jeffrey, Scott E. Fraser, and David McClay. 1995. Dynamics of Thin Filopodia During Sea Urchin Gastrulation. Development. 121: 2501-2511. 3. Miller, Jeffrey and David R. McClay. 1997. Changes in the Pattern of Adhesion Junction- Associated B-catenin Accompany Morphogenesis in the Sea Urchin Embryo. Developmental Biology. 192: 310-322. 4. Logan, Catriona Y., Jeffrey R. Miller, Michael J. Ferkowicz and David R. McClay. 1999. Nuclear B-catenin is Required to Specify Vegetal Cell Fates in the Sea Urchin Embryo. Development. 126: 345-357. 5. Kitazawa, Chisato and Sharon Amemiya. 2001. Regulating Potential In Development of a Direct Developing Echinoid, Peronella japonica. Develop. Growth Differ. 43: 73-82 6. Weitzel, Heather E., Michele R. Illies, Christine A. Byrum, Rongui Xu, Athula H. Wkramanayake, and Charles A. Ettensohn. 2004. Differential Stability of B-catenin Along the Animal-Vegetal Axis of the Sea Urchin Embryo Mediated By Dishevelled. Development. 131: 2947-2956.. Acknowledgements: York College of Pennsylvania Dr. Ricker for her continuous advice and support, you are the best!! Dr. Smith Anticipated Results : Regular GastrulationExo-gastrulation Figure 1: Regular gastrulating sea urchin embryo(A) vs. an exo-gastrulating sea urchin embryo exposed to LiCl(B). Figure 2: The gastrulation process in urchin embryos relies on both PMC’s (primary mesenchyme cells) and SMC’s (secondary mesenchyyme cells). 2-4 cell Eggs Sperm ~2000 embryos per beaker of seawater ± LiCl 0 mM15 mM30 mM60 mM Continuous Exposure Through Gastrulation Stage Simulated Microtiter Wells 0 15 30 60 Total Protein Lysates From ~100 Embryos Added to Each Coated Well; 10 Replicates, Each in Duplicate ELISA TEST Plate Reader ß-catenin Increase LiCl, increase ß-catenin Hypothesis: As LiCl concentrations increase, the expression of β -catenin protein will increase in a dose dependent manner A B Figure 3: The anticipated results of the ELISA test in regards to B-catenin expression and LiCl concentrations Figure 4: The anticipated results of the ELISA spectrophotometer reading values plotted against the varying LiCl treatments. Figure 5: The expected standard curve results of varying B-catenin concentrations and their absorbance readings from an absorbance spectrophotometer. Gut Exogastrulation Expose and Fertilize Urchin Gametes Allow Embryos to Develop to the 2-4 Cell Stage Add Embryos to Each Corresponding Beaker Once Embryos Reach Gastrulation Stage, Obtain Protein Lysates Absorbance Spectrophotometer