Effects of retinoic acid on the development of cranial
facial bones of chicken embryos
Marissa Scalish
Figure 1: In vitro
cultured embryos
Figure 2: Windowed 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.
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.
Methods
25
20
B
10
15
Dosed, in ovo
10
Control, in ovo
Control, window
5
8
Dosed, in ovo
6
Control, in ovo
4
Control,
window
2
0
A
E
I
Craniofacial Measurement
0
G
K
H
Craniofacial Measurement
20
18
16
14
12
10
8
6
4
2
0
C
Dosed, in ovo
Control, in ovo
Control, window
B
C
D
J
Craniofacial Measurement
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.
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
A
12
Length (mm)
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 Ringer’s 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 Ringer’s 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
Advisor: Dr. Katy Lustofin
Length (mm)
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.
Length (mm)
Introduction
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. Women’s
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-cisretinoic acid on hindbrain and craniofacial
morphogenesis in long-tailed macaques (Macaca
fascicularis). Journal of Medical Primatology. 27:210219.
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.