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Although sea urchins and frogs have both served as useful model organisms for studying vertebrate development, in attempt to gain further insight into the mechanisms of higher vertebrate development, developmental biologists have looked to utilizing chicken embryos (Keller et al., 2009). Possessing characteristically similar organ development to that seen in mammals alongside overall quick development itself, chicken embryos have been immensely helpful in the experimental study of organogenesis and for these reasons developmental biologists have been able to extensively use chickens as a model organism in laboratory studies (Keller et al., 2009).
Unlike sea urchins and frogs, chickens undergo internal fertilization and development through a process known as incubation (Keller et al., 2009). Having already been subjected to the first stages of cleavage even before being laid, chick embryos continue to progress through incubation by developing a primitive streak from which both the head and backbone begin to form (24-33hrs in) (Keller et al., 2009). As development continues along the anterior/posterior axis (with anterior development being more advanced than posterior development) the eyes, blood vessels, and a beating, circulating heart become visible (48hrs in) (Keller, 2010). By the end of the third day of incubation (72+hrs in) alongside the clearly defined somites, limb buds for the wings and legs also become discernable (Keller, 2010). Approximately three weeks (or 21 days) into incubation the developing chick (now consisting of all the organs needed to sustain life) garners enough strength to peck its way out of its own shell and hatches (Keller et al., 2009).
Before beginning any series of experiments involving egg windowing, each of the eggs were initially oriented to lay on their sides (balanced internally by the chalazae) so as to allow the yolk and embryo to migrate to the surface (Keller, 2010). This not only made locating the embryo much simpler but hens themselves innately periodically rotate their eggs to prevent the embryo from sticking to the surface of the shell due to the massive amount of yolk (Keller, 2010).
The developing chick embryos were treated with Howard-Ringer's solution (which consists of penicillin and streptomycin and thus essentially acts as an antibiotic) to prevent the sensitive, susceptible embryos from acquiring any infections and dying when being exposed during experimentation (Keller et al., 2009). Egg windowing was ultimately carried out to act as an alternative method in complementing our observations of normal chick embryo development through the prepared slides (Keller et al., 2009). Thus correspondingly you should therefore also expect to see and observe normal chick embryo development when windowing an egg.
Over the course of chick embryo development and incubation, cells destined to become limbs/wings (limb bud cells) migrate and begin accumulating in their respected areas (Keller, 2010). Four or five days into incubation those same limb bud cells begin to generate limb/wing buds and it's at this very point that they also first become distinctly visible (Keller et al., 2009). However it isn't until after programmed cell death concludes in each of the developing chick's limb/wing buds that fully functional, digit comprising limbs/wings become inconspicuously apparent and thereupon come into existence (Keller et al., 2009). Limb bud cells are additionally known to possess properties of regeneration as a direct result of being largely totipotent and undifferentiated (Keller, 2010). Therefore this operation was performed to determine whether or not a chick embryo has the capability of regenerating a new limb if its original limb bud is removed surgically very early in development and since limb bud cells are characterized as being totipotent, you would expect the embryo to successfully regenerate a new limb even if the original bud was to be removed.
Candling serves as a method for locating large diverging blood vessels positioned far from the developing embryo (Keller, 2010). It is these blood vessels that ultimately in turn serve as sites of tissue graft (or in this case limb/wing bud) placement in the host embryo (Keller, 2010). Similarly, it is also why a host embryo aged differently (11-12 days) from the donor embryo (4-5 days) was used, to ensure that the host would have well -developed blood vessels capable of serving as sites of placement (Keller, 2010). However before being implanted, the particular chosen blood vessel was gently scratched to initiate a repair response and the growth of even more blood vessels to aid in the acceptance and incorporation of the newly transplanted limb/wing bud (Keller, 2010). Once implanted on a particular major blood vessel having been scratched, it wouldn't be unreasonable to expect to see an entirely new limb developing in that area over time as a consequence of the totipotent (regenerative) limb bud cells.
Programmed Cell Death
Without removing the vitelline membrane it would be nearly impossible for the stain to even reach the embryo let alone the dead cells, which is why its removal is necessary (Keller, 2010). Likewise, without the dye it would be nearly impossible to distinguish between which cells were still living and which had died, which is why treatment with the dye is crucial (Keller, 2010). In essence both the removal of the vitelline membrane and staining with the dye are needed in order to observe programmed cell death.
While you might expect a more highly developed embryo/organism to initiate apoptosis to combat mutated or abnormal cells, chick embryos at this stage (or early in development) trigger programmed cell death in response to further their development in the formation of digits and limbs (Keller, 2010). It is only in such cases that an embryo would actually want its healthy cells to die. Although killing cells to further development is important, the timing of initiating programmed cell death is more if not just as important. If the embryo were to commence cell death earlier or later than it intended to, the ratio of the number of digits to each bud in either situation would end up being abnormal and thus you would end up with too few or too many digits (Keller, 2010). Since the dead cells are what particularly take up the stain, it would therefore be fairly reasonable to expect the most prominent staining to occur in regions containing dead cells and specifically in this case between the digits of the limbs throughout their formation.
Normal heart development in a chick embryo begins with the formation of a heart tube (Keller, 2010). This tube eventually progresses through several folds, bulges, and turns until finally the two individual heart primordia fuse into a single (four chambered) beating heart (Keller, 2010). In order to induce cardia bifida (or the presence of two individual beating hearts), these two heart primordia have to be surgically prevented from fusing together (Keller, 2010). However, performing surgery with the chick embryo still inside the egg would not only prove to be difficult but a much more risky process as well, which is why the embryos were explanted instead (Keller, 2010). Within the egg the chick embryo is positioned dorsally, so to be able to reach the heart (and specifically the anterior intestinal portal) you would have to make an incision through the back which could potentially rupture/damage spinal/neural tissue and subsequently lead to the death of the embryo (Keller, 2010). Conversely, with the embryos explanted you can position them ventrally making the already intricate procedure easier to accomplish (Keller, 2010). Overall a total of two embryos were explanted so that one could serve as a control against the other embryo exhibiting cardia bifida.
Present along both sides of the neural tube in the developing chick, somites are pieces of mesoderm that can be used to obtain a more accurate, precise representation of the stage/age of an embryo as opposed to simply judging by the structures (organs, limbs) present, which explains their use (Keller, 2010).
Soon after the heart forms, the embryo also begins immediately producing red blood cells so that heart (and essentially the embryo's lifeline) can in effect start performing its function in circulating blood throughout the body (Keller, 2010). Thus in the explant where cardia bifidia was not induced (or the control) you could expect to see normal singular heart development, where as the opposite would be true for the other explant. In the expant where cardia bifida was induced you could expect to see two separate, individual functioning hearts to develop because the heart primordia were surgically prevented from fusing together.
Part B (Windowing)
Under ideal conditions, windowing an egg would allow you to clearly observe chick embryo development (in conjunction with organogenesis) as it progressed through each of its various stages. However by being vulnerable to infections/diseases and having an extreme sensitivity to changes in temperature chick embryo development is not always successful as witnessed in my results. Opting to observe normal chick embryo development by explanting the entire embryo along with all of its yolk instead of windowing, sadly proved to be just as unsuccessful if not more so, as after 48hrs (a point at which signs of development should have been evident) the embryo had remained undeveloped. The initial minuscule red speck of an embryo was largely still submerged in a sea of yellow yolk and although this could be attributed to a number of factors, infection and the actual process of explanting itself could be considered the most prominent suspects/causes.
Part C (Limb Regeneration)
Observations of limb regeneration itself were largely limited due to the fact that the specific chick embryo used to contribute its limb/wing bud cells (so that regeneration could be seen), died shortly after abstraction. However considering that early on in chick embryo development its limb/wing bud cells are still distinguished as being totipotent and thus undifferentiated, the occurrence of limb development should be inevitable regardless of whether or not its original limb/wing bud cells were to be removed. Therefore had the embryo survived and continued through normal development, chances that it would induce the regeneration of new "second" limb/wing (to take the place of the removed one) would be highly likely.
Part D (Tissue Grafting)
Depsite the donor embryo having died during the extraction of its limb/wing bud cells, the formation of a new limb however was successfully observed when those very bud cells were implanted in an older host embryo. In addition to there being notably far more blood vessels present in the region (as an outcome of scratching), the newly accepted limb/wing bud cells themselves had visibly started to take the shape of a limb (72hrs later). This not only verified their totipotent, undifferentiated nature but provided further evidence for the chick embryo's believed regenerative capabilities.
Part E (Programmed Cell Death)
As a more detailed means of trying to distinguish between stages of development in a chick limb and in an effort to achieve a better understanding of cellular movements, the dead cells of developing chick embryos are stained with colored dye. Although observations of a distinct stage (possibly 24/26) in the formation of digits was unsuccessful (since the embryo seemed premature), once administered with the dye Trypan blue the embryo initially became completely engulfed before the dye appeared to begin clumping up in specific regions, which could signify potential regions of dead cells.
Part F (Cardia Bifida)
As with all vertebrates, normal heart development (which involves fusion of the heart primordia) results in a single, fully functioning, beating heart. This was clearly expressed in the control, which also consisted of a single beating heart observed hard at work circulating the blood. However, inhibiting the fusion of these primordia results in the abnormality known as cardia bifida, where instead two individual, independently functioning hearts develop. This too was successfully observed; by surgically inducing cardia bifida (accomplished by severing the anterior intestinal portal just before fusion) the resulting chick embryo now consisted of two clear freely pumping hearts (as depicted in figure 1.).
To gain even more knowledge about the regenerative properties of chick embryos and their totipotent limb/wing bud cells, I would design an experiment that would specifically determine and test the boundaries of these capabilities by attempting to generate limbs in regions where you wouldn't normally find them (e.g. head, chest, back). Following a procedure similar to one performed in lab, limb/wing bud cells would be excised from a donor embryo but subsequently implanted on various regions located directly on the developing host embryo instead. Observations would then be made to see if limbs would actually form and then continue to form even in regions that could prove to be detrimentally fatal for the chick embryo upon completion of development.
Keller Laura, John Evans, Thomas Keller. 1999 "Embryonic Chick Development" Experimental Developmental Biology. Pg 21-30.
Keller, L 2010. Lecture Notes. Mar 29