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ABSTRACT We used the chicken, Gallus domesticus, to investigate limb development in vertebrates.

We observed unincubated eggs, the egg shell, and embryos that had been incubated for 96, 72, 48, and 33 hours to identify important structures and understand the timing of the appearance of these structures during normal development. We stained the embryos and created whole mounts to preserve our embryos and identify additional structures we were unable to see on the unstained embryo. We also attempted to manipulate limb development by removing the AER and grafting beads of Fgf2 as well as implanting Shh beads into the anterior marginal zone of intact embryos in an effort to repeat Riddle’s 1993 experiment. The observations were valuable and we concluded that the number of somites and size of limb buds increases with the longer incubation time. We also concluded that the whole mount staining was an effective technique to visualize additional structures in the embryo. The limb bud manipulation experiments were not as successful since all of our embryos except one disintegrated in the KOH, but we were able to conclude from that embryo that Fgf2 implantation does rescue the wildtype phenotype as well as make important hypotheses about ways to improve the experiment and be more successful.

INTRODUCTION The chicken, Gallus domesticus, was used to study early development and limb formation. The chick is an excellent model organism since it is easy to obtain and the embryos are rather large compared to other model organisms. In addition, the chick is an example of vertebrate development and can be readily compared to the development of

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other vertebrates. We investigated the chick to learn the methods of staging using the Hamburger-Hamilton Staging Series, understand more about vertebrate anatomy, and practice and perform various manipulations. The chicken egg is similar to other birds and reptiles. The egg is an amniote egg which contains its own supply of food and water which allows the animal to travel far from a body of water to lay its eggs (Gilbert, 2006). There are four sacs within the amniote egg: the yolk sac, the amnion, the allantois, and the chorion; these sacs store the nutritional stores, contain the fluids surrounding the embryo, store the waste materials, and interact with the outside environment respectively (Gilbert, 2006). The chicken egg is considered a telolecithal egg because it has a large yolk mass that is located at one pole (Houston, 2008). The egg shell is a unique structure with dual functions; it is hard to protect the delicate embryo from dehydration and other environmental stresses, yet it allows oxygen to diffuse through its surface (Gilbert, 2006). The shell has three layers, the cuticular membrane on the outside, spongy layer in the middle, and the mammilliary layer on the inside (LM, Week 13). Just inside the shell is a thick white shell membrane whose function is to provide support, prevent water loss, and inhibit microbial infection (LM, Week 13). Shell deposition occurs as the egg rotates through the oviduct (Houston, 2008). During this rotation the yolk is shifted and the blastoderm slides off at a 45º angle; the higher region of the blastoderm then becomes the posterior end of the embryo, where the primitive streak begins (Gilbert, 2006). Another consequence of this rotation is the placement of the lighter yolk cytoplasm under this posterior blastoderm; the lighter yolk

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Gastrulation begins when the epiblast cells ingress and enter the primitive streak. 2006). 2006). We also examined a piece of the shell to identify its three layers. axis formation in the chicken is determined by gravity during the egg’s rotation through the oviduct (Gilbert. 2006).is thought to contain maternal determinants (Houston. analogous in function to the frog Organizer (Gilbert. albumin (thin and thick). and chalazae. these ingressing cells form the mesoderm and endoderm (Houston. and spinal cord (Houston. Tissues form progressively from anterior-to-posterior along the streak and the notochord and somites form anterior to the node (Houston. only the epiblast forms the embryo while the hypoblast functions in signaling to specify the epiblast cell migration (Gilbert. 2008). After egg laying. telencephalon + diencephalon. rhombencephalon. We examined an unincubated egg to identify structures. later vertical and equatorial cleavages occur to further divide the blastoderm into a multilayered tissue (Gilbert. additional divisions of the blastoderm occur to form the hypoblast and epiblast. 2008). 2008). 2006). Once the node forms the streak regresses. 2008). metencephalon + myelencyephalon. blastodisc. Every time we cracked the eggs—incubated or unincubated— 3 . The primitive streak is capped anteriorly by Hensen’s Node. 2006). Chicken cleavage is discoidal and begins at the animal pole of the embryo. 2008). and cells differentiate based on their location in the streak (Houston. mesencephalon. The nervous system is organized anterior-toposterior in the following order: prosencephalon. The streak progresses by a process called convergent extension where the lengthening of the streak coincides with the reduction of its width (Gilbert. Thus. particularly the yolk.

Once the chicken egg is laid it must be able to nourish the developing embryo until hatching. presence. a crucial component to the chicken’s ability to hatch (LM. 72.we cracked them at the blunt end. the eggs can be incubated for varying lengths of time to observe embryos at different time points (LM. Prior to our analysis we staged the embryos using the Hamburger-Hamilton Staging Series. have larger limb buds and more distinguishable nervous system features. or breaking the shell (LM. Week 13). eggs were incubated with their blunt side up. Week 13). Week 13). This formula defines parameters for the stages of the chick embryo based on the size. Week 13). Egg viability decreases as incubation time increases. but eggs can be incubated for less than 14 days at 80% humidity and 18. Finally. and 33 hours to identify structures. This end has a pocket between the shell and shell membrane called the air space. We observed embryos that had been incubated for 96. and that the whole mount staining would enable us to identify additional structures that we could not see in the unstained embryo. we hypothesized that the number of somites would increase with the longer incubation times. 48. In addition. the space fills with carbon dioxide and stimulates the chick to begin pipping. and location of characteristic structures. The chick is a popular organism to study for limb development and organogenesis because their limbs can be identified and easily manipulated without damaging the rest of 4 . when it exhales. we hypothesized that the curvature and limb bud size decrease with less incubation time. We hypothesized that the 96 hour embryo would be more curled up than any of the other time points. In order to ensure that our embryos were oriented near the air space. The chick first breaks the air space and inhales to fill its lungs. which takes about 21 days (LM.3ºC (LM. Week 13).

The ZPA’s activity is defined by the molecule sonic hedgehog (Shh). particularly the Apical Ectodermal Ridge (AER) and Zone of Polarizing Activity (ZPA) (Houston. 2006). Riddle et al transplanted Shh into the anterior margin of the chick limb 5 . the AER promotes limb growth by maintaining underlying mesenchymal cells in a continuously proliferative state (Gilbert. The AER performs its function through the action of Fibroblast Growth Factors (FGFs) which ultimately form a positive feedback loop between the underlying mesenchyme and the AER. The Zone of Polarizing Activity (ZPA) is a region of mesoderm at the posterior margin between the limb bud and body wall (Gilbert. When tissue from this region was transplanted to the anterior margin of the limb bud digits formed in a mirror image pattern (LM. We also implanted beads soaked with Fgf2 into the tip of limb buds after we removed the AER. 2006). Week 14). 2008). The AER runs along the anterior-posterior axis of the distal edge of each limb bud. 2006).the embryo (LM. We hypothesized that the Fgf2 would rescue the limb development and those embryos with the Fgf2 implant would have normal limb growth. 2006). the AER in turn secretes Fgf8 which stimulates the underlying mesenchyme to go through mitosis and produce Fgf10 (Gilbert. The limb bud contains regions that have important signaling functions. separates the dorsal and ventral surfaces. We hypothesized that embryos that had their AERs removed would fail to produce distil limb structures. When the AER is experimentally removed during limb development any undeveloped limb structures fail to form. We removed the AER to examine its role in the developing limb bud. the underlying mesenchyme produces Fgf10 which induces AER formation. Week 14). and is an important requirement for the outgrowth of the limb buds (Gilbert.

In order to assess the phenotypes of the implantation experiments we stained the embryos with Alcian blue.bud and obtained the same mirror image phenotype as the ZPA transplantation experiment (Gilbert. We then obtained eggs that had been incubated for 48 and 33 hours. We located the blunt end for future use and then cracked the egg into a Petri dish to examine the internal structures. We hypothesized that we would obtain the same duplication of digits in a mirror image. 2006). a cartilage stain. We repeated Riddle’s experiment and implanted a bead of Shh into the anterior margin of the limb bud. Next we obtained eggs that had been incubated for 96 hours and 72 hours. MATERIALS AND METHODS Observations We obtained an unincubated egg and observed its features beginning with the outside of the shell. Once the eggs were cracked we removed the pieces to create a hole at the top of each egg and peeled back the inner shell membranes to expose the embryos. We observed the embryos under the dissecting scope and labeled the structures that were visible. We obtained an insulin syringe of India ink in a 1:10 dilution in Ringer’s and gently injected it into the egg by puncturing the vitelline membrane and 6 . We oriented the eggs with the blunt end facing up and cracked them using the blunt end of our forceps. Since these embryos were much smaller we injected India ink below them to create more contrast and better view the structures. We noted these and then examined a piece of the egg shell under the dissecting scope and noted the different layers we could identify.

the embryo was in the hole in the center of the ring. and we staged them using the Hamburger-Hamilton staging series. 48. We placed these embryos into a dish of Ringer’s.injecting the ink just below the embryo. For the 96 and 72 hour embryos we carefully removed them from the egg by grabbing a blood vessel with the forceps and cutting away the extraembryonic membranes. then we carefully placed this ring onto the vitelline membrane of the embryo inside the shell. We created a “lifesaver ring” by cutting a piece of filter paper into a doughnut shape. Once we had finished looking at the embryos we collected them for whole mount staining. observed them to identify additional structures. We placed the embryos in a Petri dish of Ringer’s solution and observed them again under the dissecting scope to identify additional structures. We used the forceps to lift both the filter paper and underlying vitelline membrane and quickly cut the extraembryonic membrane with scissors to remove the embryo. A few minutes later we 7 . The embryo was transferred to a fresh dish and a few drops of Carnoy’s Fix (3:1 100% ethanol: glacial acetic acid) was placed on the embryo to flatten the extraembryonic membranes. and 33 hours). Fixing and Whole Mount Staining The fixation was performed individually for each embryo. The 48 and 33 hour embryos were much smaller and had to be removed using a different technique. We collected and stained two embryos at each time point (96. Once we could clearly see the embryo we identified and labeled structures using the dissecting microscope. 72. and staged them using the Hamburger-Hamilton staging series. After a few minutes we added additional fixative to flood the tissue. We slid the embryo spoon under the embryo and detached it from the remaining extraembryonic membranes.

We transferred the embryos from the Histoclear onto the slides. Next we washed the embryos twice in 95% ethanol for 10 minutes each. The beads were incubated in the proteins for 30 minutes prior to implantation. We then added PBS to one dish. and placed it on the shaker for the remainder of the lab period. At the end of the lab period we replaced the Carnoy’s with 70% ethanol and incubated it until the next lab period. We placed a coverslip over the embryos and let them dry overnight before viewing them on the dissecting microscope. removed any remaining Histoclear. We left the embryos in the Mayer’s Carmalum for 24 hours until the embryos were a deep red. and covered them in Permount. We let the embryos soak for 10 minutes and replaced the water with Mayer’s Carmalum staining solution. and twice in Histoclear for 10 minutes each. At that time we replaced the 70% ethanol with fresh 70% ethanol for 10 minutes. The next week we removed the 70% ethanol and replaced it with distilled water.transferred the embryo to a small glass vial. and Shh (dissolved in PBS 1mg/ml) to the third dish. twice in 100% ethanol for ten minutes each. 8 . The stain was replaced with 70% ethanol and stored until the following week. AER Removal and Implantation of Growth Factor Beads Prior to any treatment we created growth factor beads by placing a drop of beads into three Petri dishes and removing any remaining liquid. recombinant Fgf2 (dissolved in PBS at 100µg/ml) to another dish. We made raised coverslip slides for all of our embryos by breaking coverslips and layering them with clear nailpolish to build up a slide with a depression that was large enough to hold the embryo. filled it with fixative.

but we left the AER intact for these embryos. We staged two embryos. placed a drop of Ringer’s on the embryo to keep it hydrated. In addition to these treatments we created a control embryo which was at stage 18 by not performing any treatment. We implanted the Fgf2 beads into the end of the limb bud by making a slit in the tissue with the tungsten needle and carefully inserting the bead with the forceps. removed the shell membrane only. 9 . We located the limb buds—either the wing or leg bud depending on what was easiest to locate—and used the tungsten needle to gently scrape off the AER of one limb. and staged the embryos. We opened the eggs at the blunt end. We staged two embryos at stage 17 and we implanted control beads soaked in PBS at the tip of the leg buds. and inserted Shh beads into the anterior margin of the leg bud and wing bud respectively.While the beads were incubating we obtained an egg that had incubated for 72 hours and began practicing AER removal. We also created a control by inserting a PBS bead into the anterior margin of the wing bud in an embryo at stage 18. We implanted the Shh beads at the anterior margin of the limb buds using the same technique of creating a slit and inserting the bead using the forceps. For the removal procedures we made sure to wash our tools and the egg shell with 70% ethanol before opening each egg to maintain a sterile environment. stage 18 and 19. We staged one embryo at stage 18 and implanted an Fgf2 bead at the tip of the leg bud and another embryo at stage 19 that we implanted an Fgf2 bead in the tip of the wing bud. We removed the AER of four embryos and these were used for the FGF treatments.

At that time we added fresh KOH and incubated the embryos overnight. The following day we washed them twice briefly in acid alcohol and incubated them overnight in 100% ethanol. and the yolk that surrounded the blastodisc was in a repeating pattern of light and dark regions.We left all of the embryos in their shells for these treatments and added a few drops of antibiotic following the manipulations. and placed them into tubes with 95% ethanol until the following lab period. We returned the following day to mount the embryos and view and analyze them.5% KOH and kept the embryos in the KOH until they sunk. We observed the embryos and identified the visible structures. During the next lab period we replaced the 95% ethanol with Alcian blue. Immediately surrounding the mass of yolk was a region of lightly colored thin albumin surrounded by the darker thick albumin. When we mounted the embryos it was very difficult to clearly see the structures so we only mounted two and left the other six in the tubes. RESULTS Observations We identified several structures in the unincubated egg.75mg/ml in acid alcohol (75% ethanol/25% acetic acid)] and incubated the embryos overnight. Figure 1a. The blastodisc was a light colored circle in the center of the mass of yolk. a cartilage stain. The next day we replaced the ethanol with 0. 10 . [0. decapitated the embryos. The chalazae were visible as thin threads extending out of the yolk. We covered the shells with parafilm and incubated them at 38ºC for two days. After two days we removed the eggs from the incubator.

Figure 1b. From this orientation we were also able to see a wing bud near the ventricle. and once the embryo was in a Petri dish it was much more curved. which is then surrounded by the darker thick albumin. The other two layers were slightly darker although they were both shades of white. The embryo was crescent-shaped and had a web of red blood vessels extending from the ventricle to the yolk. The shell (b) was viewed at 3X magnification on the dissecting scope and has three visible membranes: the cuticular membrane.5X magnification. optic cup. Just below the three layers is the thin shell membrane. The unincubated egg viewed with the naked eye (a). 11 . The blastodisc is the light circle in the center of the yolk. and spinal cord were visible as dark structures while the rest of the embryo was very lightly pigmented. The spongy layer was very bright white and did appear to have a spongy texture. The yolk is visible in the lower left region of the figure as a round structure with a repeating pattern of light and dark yolk. After removing the embryo from the shell it was much easier to identify structures. The somites were clearly visible along the dorsal surface and they extended from the base of the head to the tail. Nervous system structures of the telencephalon.We examined the shell under the dissecting microscope at 3X magnification. We could easily identify the cuticular membrane. layer. The embryo was viewed under the dissecting microscope at 1. The chalazae are visible as thin threads extending from the side of the yolk. and mammilliary layer. We first viewed the embryo that was incubated for 96 hours inside the egg after we had removed the shell at the blunt end. spongy layer. and mammilliary layer. The ventricle. The spongy layer is much brighter white and has a spongy texture. Figure 2a. spongy b Figure 1. Figure 2b. The heart was visibly beating and we were able to see the movement of blood through the vessels. Surrounding the yolk is the thin albumin. The shell membrane was also white and visible beneath these three layers. diencephalon.

the allantois was visible as a light grey sack-like structure extending from the ventral side of the embryo. Figure 2c. and myelencephalon were visible. The embryo was still more elongated than the 96 hour embryo. The heart was beating very quickly and the blood vessels were visibly extending from the ventricle into the yolk. they were much smaller and looked like small 12 . but they were not as distinct in this embryo as they were in the 96 hour embryo. Both wing and leg buds were visible.7X magnification. several blood vessels were still visible as well as the beating heart—it was beating much slower now. but there was a slight curve at the posterior end and a larger bend at the anterior end. but they did not extend as far into the tail as the somites in the 96 hour embryo. Based on the Hamburger-Hamilton Staging Series we determined this embryo was at Stage 23. metencephalon. The 72 hour embryo was viewed in its shell at 0. Inside the shell the ventricle and spinal cord were clearly visible. Figure 2d. however.5X magnification on the dissecting microscope. and myelencephalon were visible in the head of the embryo beginning near the eye and extending to the base of the head. The optic cup was easier to distinguish as a ring and the lens was visible inside. It appeared to be quite smaller than the 96 hour embryo and was much more elongated with just a slight bend near the anterior region. The wing and leg buds were clearly visible and the leg buds appeared to be slightly longer than the wing buds.mesencephalon. and the tail bud was evident in this embryo. Additionally. Once we removed the embryo from the egg some structures were much more apparent. Somites were visible in this embryo extending along the dorsal surface. Nervous system structures of the diencephalon. and we viewed it at 1. Although the embryo was no longer inside the egg. mesencephalon.

diencephalon. Surrounding these dark lines was tissue of a very light pigmentation that defined the edges of the embryo. although they were shorter and did not meet at the anterior end as they did in the older embryo.5X magnification under the 13 . The embryo was straight and elongated. mesencephalon. so we just identified structures once it was removed. The two lines that were visible in the 48 hour embryo were also visible. Figure 2e. The head fold. There were two dark lines extending from the anterior to the posterior end which defined the telencephalon. and the ventricle was visible and slowly beating. This embryo was very small and we viewed it at 2. This embryo was staged at stage 12 on the Hamburger-Hamilton Staging Series. although there were fewer somites than either the 96 or 72 hour embryos. The same nervous system components were still visible. metencephalon. lateral body fold. and lateral plate mesoderm were all visible once the embryo was removed from the shell. The 48 hour embryo was much smaller and more difficult to see inside the egg. At the posterior end was the open neural plate and primitive streak.5X magnification. We staged this embryo at stage 19 based on the Hamburger-Hamilton Staging Series.5X magnification. The spinal cord was visible just ventral to the somites.mounds of tissue at this point. After removing the embryo from the shell we could see a more definite shape of tissue. and viewed it under the dissecting microscope at 2. We used India Ink to better visualize the embryo and viewed it at 2. and optic cup boundaries. Figure 2g. We could not see the lens or the optic cup at this point. but we could also see somites at the posterior end of the embryo. Figure 2f. The 33 hour embryo was so small and lightly pigmented that we could not see it in the egg to identify any structures.

and ventricle were clearly visible along with limb buds. The embryos were viewed under the dissecting scope. wing buds. lateral body fold. This embryo was staged at stage 9 on the Hamburger-Hamilton Staging Series. nervous system structures were apparent as defined by two thick dark lines extending from the anterior to the posterior end. and it had dark staining of the somites.5X.7X magnification on the dissecting scope. somites and nervous system structures were apparent. The tail was slightly bent as was the anterior end. The 72 hour embryo was more elongated and smaller (c). at 1. and the allantois was distinct.7X inside the shell. a b a c d e f g Figure 2. nervous system structures were visible as labeled. Outside of the shell. The limb buds were large and easy to see. The heart was beating and the optic cup. the amnion and allantois were clearly visible. but the anterior neuropore and the open neural plate were obvious. A couple of somites were barely visible. Also. The 33 hour embryo was much smaller and could only be visualized outside of the shell (g). We viewed the 96 hour embryo at 0. the embryo was more rounded. Out of the shell. and we viewed it at 0. Whole Mount Staining The whole mount staining made some structures of the embryo darker and easier to see. Figure 3a. In the shell. The heart was beating and the ventricle and spinal cord were clear. spinal cord. the embryo had visible somites and nervous system structures (d). The 96 hour embryo was viewed at 0. The nervous system structures were easier to distinguish. Once removed from the shell we viewed it at 1. and leg buds. and lateral plate mesoderm were evident. Visible nervous system structures included the diencephalon. and somites were much more distinct (b). The anterior neuropore and open neural plate were distinguishing features. This embryo was at stage 19. the mesencephalon was a round structure and the diencephalon and 14 . telencephalon.5X both inside and outside of the shell. This embryo was at stage 12. The 48 hour embryo was much smaller and straight (e). viewed at 2. ventricle.5X. and mesencephalon. (f) the same nervous system structures were visible as well as somites in the posterior end.7X inside the shell (a).dissecting microscope. The head fold. This embryo was at stage 9. The embryo was at stage 23.

and ventricle were apparent after the staining.5X magnification. We viewed it at 2. Several nervous system structures were more easily distinguished with the staining including the telencephalon. The somites were not as easy to distinguish and appeared as a dark line along the dorsal side of the embryo. Both the 96 and 72 hour embryos had several structures on their ventral surface that all appeared to be on top of each other. The somites were much clearer and easy to distinguish. The 33 hour embryo also was damaged during processing. Also. The 48 hour embryo. diencephalon. Figure 3b. but the tissue was folded over the embryo so the whole thing was not visible in the whole mount. although smaller than the 96 hour embryo. The optic cup was a dark circular structure surrounding the lighter lens The 72 hour embryo.metencephalon were visible on either side of it. This embryo was viewed at 2. appears to have been damaged during the processing as the head is slightly torn apart. but it was impossible to determine which nervous system structures we could see. mesencephalon. metencephalon. it was difficult to distinguish them because it looked like they were piled on each other. These structures included the atrium. and somites. The somites and the dark lines that defined the nervous system were slightly visible. Although the staining seemed to have made these more apparent. The wing buds and leg buds were darkly stained and easy to see. the infundibulum. mesencephalon. and the lighter lens was visible inside. Figure 3c.7X magnification on the dissecting microscope. and metenchephalon were clearly distinguishable as individual structures. 15 . The telencephalon. The optic cup was visible. was viewed at 0. pharyngeal pouch. and aortic arch.5X magnification on the dissecting scope. Figure 3d.

The whole mount staining embryos as viewed under the dissecting microscope. ventricle. There were also two fully formed wings with additional blue staining where the bones would develop. Figure 4 shows the expected outcomes of the limb bud experiment. allantois. The embryo had definite blue staining of all the structures of the spinal column and limbs. Figure 4c. There appeared to be two fully formed legs with blue staining through their length. somites.7X magnification and the nervous system structures as well as the somites. but the nervous system structures. (c) was viewed at 2. The AER removal was expected to result in the embryo’s failure to develop distal limb structures. The 48 hour embryo. and limb buds were visible (a). It was difficult to distinguish any structures. The only one we could see was the embryo that was at Stage 17 when we performed the treatments and had Fgf2 implanted in the leg bud.a b c d Figure 3. Figure 4a. ventricle. lens. 16 . ventricle. and lateral plate mesoderm were all visible.5X magnification. both legs were symmetrical and identical. limb buds and spinal cord were all visible (b). but the somites and some nervous system tissue were visible. AER Removal and Growth Factor Implants When we returned to analyze our limb bud experiments nearly all of the embryos had disintegrated in the KOH and there was nothing to see. The Fgf2 implantation was expected to result in the rescue of the wildtype phenotype. Figure 4d. Figure 4b.5X magnification and seemed to have been damaged during processing as the head was torn apart. The 96 hour embryo was viewed at 0. This embryo was also viewed at 2. The Shh implantation was expected to give additional ectopic digits in a mirror image to the normal digits. amnion. The 72 hour embryo was also viewed at 0. The 33 hour embryo was very small (d) and also appeared to have been damaged as it looked like there was a fold of tissue covering the embryo.7X and nervous system structures as well as the lens.

4/28/08). 4/28/08). The expected results for the Shh grafting experiment were a duplication of the digits in a mirror image (c) (image modified from Dr. Houston’s Lecture notes. The expected results for the Fgf2 grafting experiment (b) are to have the phenotype rescued (image modified from Dr. The limb buds were visible and larger on the 96 hour embryo than the 72 hour embryo. The only limb bud experiment that could be analyzed was the Fgf2 implant into the leg bud (a).a b c Figure 4. The limb buds were not visible at all on the 48 or 33 hour embryos. especially concerning the development of the limbs. Houston’s Lecture notes. 4/28/08). The expected results for the removal of the AER (d) were a failure to form distal limb structures (image modified from Dr. and the younger embryos are more elongated than the older ones. We hypothesized that the 96 hour embryo would be more curled up than any of the other time points. Houston’s Lecture notes. and the limb buds would be larger. Our observations support our hypothesis. supporting our hypothesis. 17 . The observations were valuable and the whole mount staining was successful. d DISCUSSION Overall the chick experiments were helpful in visualizing development of a model organism. Growth factor bead implants. whose limb buds were small mounds of tissue. the 96 hour embryo was much more curled up than any of the other embryos. The phenotype was rescued as the embryo developed 2 legs with proximal and distal structures.

However. since our control had also disintegrated. we could not distinguish these structures. These were structures that we were unable to see in the unstained embryo. Our results offer some support of this hypothesis. supporting our hypothesis. we were only able to analyze the phenotype of one of our limb bud experiments as the rest of the embryos disintegrated in the KOH. however. comparable to the 96 hour embryo. and the whole mount staining did. Although we did not count an exact number. we cannot definitively state that the AER was effectively removed. the 72 hour embryo also had a very developed and defined nervous system. If the AER were not removed.We also hypothesized that the nervous system would be more distinguishable in the 96 hour embryo. This embryo had 2 legs that were equal in length and developed structures. The only embryo we could analyze was the embryo with the Fgf2 implant in the leg bud. allow us to better visualize structures on the embryos. and the technique was pointless. The nervous system structures were more apparent and much more developed in the 96 hour embryo than in the 48 or 33 hour embryos. Unfortunately. the phenotype we observed would have been a result of the 18 . however. it was evident by observation that the number of somites each embryo had did increase with age. so we could support our hypothesis that Fgf2 rescues the phenotype of the embryos allowing them to produce distal limb structures after the removal of the AER. so the staining was beneficial in that it allowed us to see structures we would otherwise be unable to see. In the future it may be beneficial to visualize the stained embryos before and after mounting to identify structures that could potentially get squished when the embryos are mounted. One draw back to the whole mount staining was that we had some structures end up overlapping others once the embryos were mounted. After mounting them. in fact.

Fallon performed a similar experiment to our Fgf2 implants and was able to show that the implantation of the Fgf beads rescued the wildtype phenotype (LM. but analyzing the results when the KOH is added instead of one day later. in fact. past experiments suggest that our hypotheses are. When we changed the KOH the embryos were visible and the blue dye had clearly stained the cartilage of the developing embryos. nor could we confirm or reject our hypothesis that Shh implants will result in ectopic digits in a mirror image. 2006). however. Also.normal function of the AER. In retrospect we should have visualized the embryos the same day we added the KOH so we could have more data to analyze the limb bud phenotypes. Although our results do not allow us to address the validity of our hypotheses. Additional experiments could include repeating the limb bud experiments and Alcian blue staining. the KOH may have been too strong for the embryos. Riddle performed the Shh grafting experiment and was able to show mirror image digit duplication (Gilbert. Although we followed the lab manual. it would be interesting to implant the Fgf2 bead on the top or bottom of the limb bud instead of at the tip and see if the phenotype differs since the protein is in a 19 . The disintegration could have been due to the KOH. We could not confirm or reject our hypothesis that AER removal results in the embryo’s failure to form distal limb structures. not the implanted Fgf2. or 24 hours may have been too long to wait before analyzing the results. These experiments allowed us to understand some of the molecular mechanisms involved in limb development in vertebrates. accurate. all of the embryos had disintegrated except one. Also. when we returned after allowing the embryos to incubate in the KOH overnight. Week 14).

Hamilton H. 2006. Developmental Dynamics 195:231-272.F. Understanding human mechanisms for limb growth could have some important medical consequences. Lecture Notes: April 15 and 22. by familiarizing ourselves with the natural process of limb development. Houston. S. Hamburger V. 20 . A series of normal stages in the development of the chick embryo (1992). REFERENCES Developmental Biology 002:135 Lab Manual. Developmental Biology 8th Edition.different location. 2008. By learning the mechanism behind limb development in a vertebrate model organism. MD. we can make hypotheses about similar mechanisms in humans. D. Sunderland. we become closer to developing a process to regenerate human limbs after amputation. Spring 2008 Gilbert. Sinauer Associates Inc.