Presentation on theme: "Effects of retinoic acid on the development of cranial facial bones of chicken embryos Marissa Scalish Advisor: Dr. Katy Lustofin Introduction Accutane,"— Presentation transcript:
Effects of retinoic acid on the development of cranial facial bones of chicken embryos Marissa Scalish Advisor: Dr. Katy Lustofin Introduction Accutane, also known as 13-cis-retinoic acid, is a very effective acne medicine that has been on the market for over 24 years (Goodheart, 2006, 9). Retinoic acid is derived from Vitamin A, which is why this drug is considered a retinoid (Makori et al., 1998, 210-211). Vitamin A is known to be a vital component of embryonic development, tissue differentiation, and epithelial cell growth (Garcia-Fernadenz et al., 2005, 36). If Vitamin A concentrations become too high or too low, developmental defects can arise (Huang et al., 2005, 202). Five million people in the United States, more than half of whom are female, are currently taking Accutane (Meadows, 2001, 19). Accutane is known to cause major birth defects in embryos whose mothers are on Accutane. Defects include limb abnormalities, craniofacial defects, incomplete development of neural tube, heart defects, and digit deletion (Pullarkat et al., 1992, 3). As a result, it is recommended that Accutane is prescribed as a last resort treatment option (Goodheart, 2006, 9). Several features make the chicken embryo a good model organism for developmental experiments. They have similar development to humans because they are both vertebrates (Wolpert et al., 2007, 98). Chicken embryos incubate outside their mother, which allows for easy access to the embryo and better control of the dose that reaches the embryo. Chicken embryos are tolerant of experimental manipulation of the egg, including making a hole in the egg shell (Drake et al., 2006, 67), or taking the embryo completely out of its shell (Drake et al., 2006, 67). There were two hypotheses tested in this experiment. The first hypothesis was that the Accutane would cause craniofacial bone defects. The second hypothesis was that among the three injection methods, the in vitro would have the most severe defects because the injection was made through the yolk. Methods White Leghorn chicken eggs were obtained from the OSU Poultry Farm. Eggs were placed in the 1500 professional incubator at 37.5 ° C. Four different trials were conducted using 2 dozen eggs each trial. Eight eggs were used for each culturing methods. On day 3 of incubation, 4 eggs for each culturing method were injected with 50 μl Accutane in corn oil. The other 4 eggs were injected with 50 μl corn oil as a control. Three culturing methods were used: in ovo, in vitro, and windowing. All egg shells were disinfected with 70% EtOH to prevent contamination. The in ovo eggs had a hole made through the egg shell into the airspace. After the injection was administered, the hole was resealed with wax. The eggs were placed in the rotating incubator. The in vitro embryos were taken completely out of the egg shell and placed into an incubation chamber made of Saran wrap, dixie cups, rubber bands, and petri-dish lids (refer to Figure 1). The dose was injected through the yolk and 20 μl of Ringers solution was added as well to prevent the embryo from drying out while being exposed to the air. These embryos were placed in the non-rotating incubator. The windowed eggs had a hole cut through the side of the egg shell with scissors (refer to Figure 2). Once the dose was injected, 20 μl of Ringers solution was added and the hole was resealed with scotch tape. Eggs were placed in the non-rotating incubator. On day 14 of incubation, chicken embryos were sacrificed, decapitated and skinned. The heads were stained and cleared using the procedures by McLeod, 1980. Ten different craniofacial bone measurements were taken (refer to Figure 3) using a dissecting microscope with the Lumenera software and an electronic calibrator. The SPSS software was used to perform a MANOVA and T-test to see if craniofacial bone measurements showed significance between the dosed and control groups. Discussion This experiment attempted to test the effect retinoic acid would have on the craniofacial bone development of chicken embryos. It was hypothesized that the Accutane would cause craniofacial bone deformalities, especially in the in vitro cultured eggs. However, after completing four trials and the statistical analysis, the hypotheses were not supported. The only measurement that was very close to be being considered significant was head length with a p value of 0.084. Because there was such a high mortality rate among the different culture methods, this could be why the data was not statistically significant. Also, there is very little published about cranial facial defects found in chick embryos that have been exposed to retinoic acid. This could be because chickens are not the best model organism for Accutane testing. Since Accutane is a type of retinoid, perhaps chickens are not a good model to use when testing other retinoids as well. It would be beneficial to repeat this experiment with more trials to see if some of the craniofacial measurements would be significant since there would be a larger sample size. References Drake VJ, Koprowski SL, Lough JW, Smith SM. 2006. Gastrulating chick embryo as a model for evaluating teratogenicity: a comparison of three approaches. Birth Defect Research. 76:66-71. Garcia-Fernandez RA, Perez-Martinez C, Alvarez JE, Nararrete AJ, Garcia-Iglesias MJ. 2006. Mouse epidermal development: effects of retinoic acid exposure in utero. Veterinary Dermatology.17:36-44. Goodheart HP. 2006. Accutane for severe acne. Womens Health OB-GYN Edition. 2:9-14. Huang FJ, Hsuuw YD, Lan YD, Kang HY, Chang SY, Hsu YC, Huang KE. 2006. Adverse effects of retinoic acid on embryo development and the selective expression of retinoic acid receptors in mouse blastocysts. Human Reproduction. 21(1):202-209. Maclead, MJ.1980.Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology.22:299-301. Makori N, Peterson PE, Blankenship TN, Dillard-Telm L, Hummler H, Hendrickx AG. 1998. Effects of 13-cis- retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis). Journal of Medical Primatology. 27:210- 219. Meadows M. 2001. The power of Accutane. FDA Consumer. 35(2): 18-23. Pullarkat RK, Azar B.1992.Retinoic acid, embryonic development, and alcohol-induced birth defects. Alcohol Health & Research World.16(4):317. Su B, Debelak KA, Tessmer LL, Cartwright MM, Smith SM. 2001. Genetic influence on craniofacial outcome in an avian model of prenatal alcohol exposure. Alcoholism: Clinical and Experimental Research. (25)1: 60-69. Wolpert L, Jessell T, Meyerowitz E, Robertson E, Smith J. 2007. Principles of developmental biology (NY): Oxford University Press; 520p. Figure 1: In vitro cultured embryos Figure 2: Windowed eggs Results As seen in figure 4, there was no statistical difference between the dosed in ovo eggs and the control windowed eggs. The in vitro cultured eggs were not considered in the analysis because none of them survived any of the trials. P values ranged from 0.084-0.985. P values less than 0.05 were considered to significant. Measurements A, B, E, and G had a dosed measurement that was higher in length than the control windowed eggs. Measurements D, I, J, and K had data that showed the in ovo control to be the largest measurement. Measurements C and H had similar lengths between the dosed in ovo and control in ovo eggs. Key: A: head length B: head height C: ocular diameter D: maxillary length E: mandibular length G: oral gap H: nasal width I: nasal length J: mandibular length (dorsal view) K: maxillary length (dorsal view) Figure 3: Craniofacial bone measurements. Figure 5: Average length of craniofacial features. Measurements were grouped into three graphs (A, B, C) based on similar sizes. Bars represent standard error. Acknowledgements I would like to thank Dr. Lustofin for her help with every aspect of my project. Dr. Brown for his support and feedback. The biology department for funding my project. Kristin Lutes for her help with the statistical analysis. Erin Nicola for her help with the electronic calibrator. My friends and family for all their support and encouragement through this capstone process.