A Study of Furosemide Inhibition On Skeletogenesis in Sea Urchin Embryos

• I. Introduction
• II. Materials and Methods
• III. Results and Discussion
• IV. Conclusion
• V. References
• VI. Acknowledgments
• Appendix A
• Appendix B: Results of Exposure to Various Furosemide Concentrations
• Appendix C: Results of Propidium Iodide Vital Staining
• Appendix D: Results of the Recovery of Skeletogenesis After Limited Furosemide Treatment
• Appendix E: Results of Immunostaining
• Appendix F: Results of Skeletal Growth Assay

Previous research has conclusively determined the detrimental effects caused by furosemide on sea urchin skeletogenesis. As many aspects of this system remained unresearched, the purpose of this project was to complete further investigations into its functioning. Several experiments were conducted, the first of which involved the determination of the effects of exposure to furosemide concentrations on skeletal growth and spicule calcification. This investigation confirmed a decrease in embryonic skeletal calcium concentration as well as a decrease in spicule length. A second investigation was conducted in order to discover the extent to which furosemide-treated embryos could recover if the furosemide was to be removed after varying lengths of exposure time. This showed the urchin embryo's ability to regain skeletal calcification after removal of furosemide. An immunostaining process revealed the PMCs location throughout the embryo to stray from the characteristic ring formation and instead be dispersed throughout the embryo when exposed to high furosemide concentrations. Finally, an investigation of the rapidity with which furosemide inhibits further skeletogenesis revealed negligible skeletal growth after addition of furosemide, allowing the conclusion that furosemide works almost instantaneously to inhibit skeletogenesis.