Abstract

The Hedgehog (Hh) signaling pathway is essential for patterning and proper embryological development. Improper regulation of the Hh pathway has been observed in various cancers. A potent inhibitor of the Hh pathway has previously been shown to induce cyclopia and abnormal cranial formation in developing sheep. Therefore, to further study the role of Hh signaling, we decided to test the effect of cyclopamine on chicks. Control eggs were windowed at stage 1 and stage 9 of development, while the experimental group additionally received a 5 µg injection of cyclopamine. Potentially due to over heating or infection, only one experimental subject survived. The chick appeared to have improper forebrain, limb, and cartilage formation. Furthermore, it is possible that a single eye was seen, although that remained unclear. In the future, the experiment should be repeated without windowing the experimental group and using a larger sample. Based on the single chick that survived, we believe our results confirmed the detrimental effects of abherrant Hh signaling on development.

Introduction

Cyclopamine is a plant-derived teratogen belonging to a group of steroidal alkaloids. Cyclopamine inhibits the hedgehog (Hh) pathway, which is important in the development of the central nervous system and the facial structures (1). Cyclopamine suppresses the embryonic brain from dividing into two lobes (holoprosencephaly) and causes the formation of a single eye (cyclopia). The U.S. Department of Agriculture has verified that administration of cyclopamine during gestation in sheep directly induces cephalic malformations in offspring, including cyclopia (1). During normal Hh signaling, when sonic hedgehog (Shh) binds to patched protein, patched-mediated inhibition of smoothened is released. After a cascade of intracellular events, Hh’s target genes are transcribed. Chen et al has shown that cyclopamine acts antagonistically in the Hh pathway by directly binding to smoothened, altering its conformation and thereby repressing its function (1). Interestingly, cyclopamine induces a change in conformation that is structurally similar to that induced by patched. Abnormal Hh pathway activation has been seen in several cancers, including basal cell carcinoma and medulloblastoma (1). These cancers typically involve a mutation in either patched or smoothened (1). Since cyclopamine does not have an effect on adult organisms, it could one day serve as a cancer treatment by inhibiting aberrant smoothened activity.

As previously mentioned, mutations in smoothened and patched can induce excess activity of the Hh response pathway. Taipale et. al showed that cyclopamine blocks abnormal cell growth in oncogenic mutations of smoothened and patched (2).  Furthermore, treatment of adenoma and carcinoma colorectal epithelial cells with cyclopamine has been shown to induce apoptotic cell death (3). This data suggests that cyclopamine may serve as a potential treatment for colon cancer.

Shh secreted from the notochord and floor plate is an important morphogen for ventral neural tube patterning. One of the first steps in this patterning involves Shh repression of Pax3, Pax6, and Pax7 throughout the neural plate. Cyclopamine acts as an inhibitor the Shh induced dorsoventral patterning of the neural tube and somites (4). Following treatment with cyclopamine, cells formed by Shh signaling in the ventral neural tube are absent or misplaced in the ventral midline (4). Furthermore, the dorsal cells that are normally repressed appear ventrally (4). Sclerotome induction is also inhibited, as is evidenced by the expression of Pax7 in cyclopamine-treated chick embryos (4). Because somites give rise to the limbs, vertebrae, and cartilage, improper formation of the somites causes the absence of the ribs and vertebrae.

Shh activity has also been shown to regulate Pax6 expression. Under normal Shh signaling, Pax6 expression is reduced in the ventral retina and the lens (5). Inhibiting Shh activity causes Pax6 to be expressed in the optic nerve, which is normally devoid of Pax6 (5). In contrast, inhibiting Shh caused an exclusion of Pax2 from the optic nerve (5). Therefore, Pax6 is critical for the specification of the retina, which is why Shh gene disruption results in cyclopia (5).

In our experiment, we will investigate the effect of cyclopamine administration on craniofacial development and eye formation in chicks. We also want to discover whether limb and cartilage formation will be affected due to improper somite formation seen with aberrant Shh signaling. We hypothesize that holoprosencephaly and cyclopia will be induced in cyclopamine-treated chicks and that the limbs and cartilage will not form. 

Methods

Twenty-four fertilized chicken eggs were obtained from Phil’s Farm Fresh Eggs. Eggs were kept in 13°C refrigerator until experiment began. 1 milligram of cyclopamine was obtained from Sigma-Aldrich. Cyclopamine was prepared by first mixing hydroxypropyl-β-cyclodextrin (HBC) in 1 milliliter phosphate buffered saline (PBS). A 45% HBC solution was made in order for cyclopamine to fully dissolve. Cyclopamine was suspended in the 45% HBC solution and was allowed to mix at room temperature for 24 hours. Following mixing, cyclopamine was stored at 4°C for several days.

Eight eggs were taken from 13°C refrigerator and windowed in order to expose the embryo at stage 1 of development. Approximately 3 milliliters of egg white was removed by using a sterile syringe to allow for easier manipulation of the embryo. Once the embryo was located, 5 µg of cyclopamine was added by simply placing 5 µl of solution on top of the vitelline membrane using a pipette. A small amount of Ringer’s solution containing antibiotics was added to prevent infection. Treated eggs were then closed using Scotch® tape and placed inside the 25°C incubator for 13 days along with eight control eggs which were not windowed. After 48 hours of development, eggs were checked for infection and viability. After developing for 13 days, chicks were removed from the egg and placed in trichloroacetic acid (TCA) for Alcian Blue staining as was discussed in class (6).

Eight more eggs were windowed 29 hours later in order for chicks to be at stage 9 of development. These eggs were also treated with cyclopamine by the same procedure as previously discussed; however, a small incision was made in the vitelline membrane and 5µg of cyclopamine was administered through this opening to reach the embryo. These eggs were also placed in the 25°C incubator until they reached day 13 of development. On day 13, the chicks were removed from the eggs and placed in TCA for Alcian Blue staining (6).

Results

Survival Following Treatment

All 24 chicks were checked for viability 48 hours after treatment with cyclopamine. At 48 hours, all chicks seemed to be developing normally and showed signs of vascularization. At day 13, all 24 eggs were harvested and surviving embryos were stained with Alcian Blue. Of the control eggs not treated with cyclopamine, 5 of 8 embryos were alive at day 13. These chicks appeared to be normally-developed 13-day-old chicks (Figure 1). The approximate length of control embryos was measured to be 3.5 cm (Figure 1)

Of the embryos treated with cyclopamine at stage 0 of development, only one of eight embryos survived. The seven embryos that died showed signs of drying out and therefore were unable to develop. This embryo was severely underdeveloped and only measured to be 1.2 cm (Figure 2,3). The size of this embryo was significantly smaller than the control embryo (Figure 2). No craniofacial structures were observable (Figure 3). Of the embryos treated with cyclopamine at stage 9 of development, no embryos were found to be viable at day 13. All eight embryos showed signs of drying out and therefore were not able to develop.

Alcian Blue Stain and Presence of Cyclops

All surviving embryos underwent staining with Alcian Blue. Alcian Blue stain was successful in staining the eyes, craniofacial, and cartilaginous structures of the 5 control chicks were stained dark blue along with cartilaginous structures of the spine and limbs (Figure 4). The embryo that was treated at stage 0 of development also underwent Alcian Blue staining. This embryo did not stain well, and stain was only present in the head and one of the limbs (Figure 5). There was no stain present in any of the other limbs or in the spinal cord. The small amount of stain in the head of the stage 0 embryo suggests cyclopia. Underdevelopment of facial structures is observed compared to control development. Because of the severity of the underdevelopment of the stage 0 embryo, we were not able to observe the detail of craniofacial abnormalities. 

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Figure 1:  Control embryos stained with Alcian Blue.

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Figure 2: Stage 0 embryo treated with cyclopamine compared to control chicks. 

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Figure 3: Embryo treated with cyclopamine at stage 0.

Discussion

Since the success rate of our experimental group was very low, it is difficult to support our hypothesis with certainty. Nevertheless, from the single chick that survived, several theories can be drawn.The surviving cyclopamine-treated chick showed signs of severe underdevelopment typically seen in cyclopamine-treated embryos.

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Figure 4: Magnification of control chick eye and face

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Figure 5: Alcian Blue stain of embryo treated with cyclopamine.

Since cyclopamine interferes with Hh pathway signaling, Shh was inhibited from inducing the dorsoventral patterning of the somites and neural tube. Since Pax7 expression was expanded, somites did not differentiate properly. The major effects of cyclopamine-treated organisms are holoprosencephaly and cyclopia.   The surviving experimental chick shows little staining in the head and there was staining in only one of the limbs. Based on the physical characteristics of the chick, it appears that the forebrain did not divide properly and we believe that cyclopia was induced. The limited staining implies that proper cartilage and limb formation did not occur, which confirms the findings of other studies (4). Unfortunately, we did not have any viable stage 9 cyclopamine-treated chicks.  Therefore, it cannot be concluded that the rib cage and vertebrae did not form as would be seen by little staining.

The study needs to be repeated for our hypothesis to be supported. We believe that the primary cause of death was due to overheating. We should have closed the widowed eggs more securely by utilizing multiple pieces of tape instead of just a few pieces. Hot air from the incubator likely escaped into the windowed eggs, essentially cooking the eggs and preventing the embryos from developing. The control eggs were not windowed, explaining why most of them survived. Therefore, it is difficult to compare unwindowed eggs to windowed ones since windowing the eggs creates a more stressful environment for the developing chicks. Although we tried to be as sterile as possible, using the 70% ethanol to clean the outside of the eggs and also using the Bunsen burner to disinfect the equipment, windowing the eggs could have induced bacterial contamination. In the future, cyclopamine needs to be more carefully injected into the vitelline membrane. We were afraid to pierce the yolk through injection, but in either case, we would have to try injecting the cyclopamine or try to secure the windowed eggs more properly. Additionally, the 5 µg of cyclopamine that we used may have destroyed the eggs.

We modeled this concentration of cyclopamine from the study by Incardona et al.; however, for future experiments we would like to use a smaller dosage (4). Optimally, we would like to begin with 1 µg of cyclopamine and increase our way up to 4 µg by 1µg increments. That way, we could use several eggs for each concentration of cyclopamine and evaluate the impact of increasing cyclopamine concentration on the developing chicks. Overall, based on our single surviving stage 0 experimental embryo, we believe the chick displayed abnormal craniofacial development based on the chick’s appearance and very little staining. Our result is consistent with cyclopamine-induced malformations caused by interruption of Shh pathway.

Conclusion

Overall, evaluating the effect of cyclopamine on chick development was difficult considering the survival of only one experimental chick embryo. We did infer, however, that the chick may have presented with cyclopia and decreased cartilaginous tissue formation. In the future, more experimentation needs to be done with varying concentrations of cyclopamine and a larger number of chick embryos.

References

Chen, J. K., J. Taipale, M. K. Cooper, and P. A. Beachy. Inhibition of Hedgehog Signaling by Direct Binding of Cyclopamine to Smoothened. Genes & Development 16, no. 21 (Nov 1, 2002): 2743-2748.

Taipale, J., J. K. Chen, M. K. Cooper, B. Wang, R. K. Mann, L. Milenkovic, M. P. Scott, and P. A. Beachy. Effects of Oncogenic Mutations in Smoothened and Patched can be Reversed by Cyclopamine. Nature 406, no. 6799 (Aug 31, 2000): 1005-1009.

Qualtrough, D., Buda, A., Gaffield, W., Williams, A. C., & Paraskeva, C. (2004). Hedgehog signaling in colorectal tumour cells: Induction of apoptosis with cyclopamine treatment. International Journal of Cancer.Journal International Du Cancer, 110(6), 831-837.

Incardona, J. P., W. Gaffield, R. P. Kapur, and H. Roelink. The Teratogenic Veratrum Alkaloid Cyclopamine Inhibits Sonic Hedgehog Signal Transduction. Development (Cambridge, England) 125, no. 18 (Sep, 1998): 3553-3562.

Zhang, X. M. and X. J. Yang. Temporal and Spatial Effects of Sonic Hedgehog Signaling in Chick Eye Morphogenesis. Developmental Biology 233, no. 2 (May 15, 2001): 271-290.

Smith, Pliny. “Alcian Blue Stain, Lab Manual.” LFC. 2011.