Understanding the Development of the Respiratory and Cardiovascular Systems Using Avian Models

Abstract: 

The avian embryo has a long history of use as a model system for the study of morphological and physiological development.  Properties of avian eggs that make them good developmental models include the large range of functional maturity at hatching and the ability to easily manipulate the environment in which the egg is incubating.  The chicken embryo develops over a 20 day incubation period.  During the first 19 days of incubation, the embryo relies on the chorioallantoic membrane for gas exchange.  The embryo begins the hatching process on day 19 which involves internal pipping, external pipping, and finally hatching.  Our lab uses the chicken embryo to study the development of the cardiovascular system, specifically the ductus arteriosi.  The ductus arteriosi are a pair of blood vessels in the avian embryo which allow blood to bypass the non-ventilated lungs.  Upon initiation of lung ventilation these blood vessels must close.  Closure appears to be stimulated by increased oxygen in the blood due to increased lung ventilation during external pipping.  The properties of the avian model system will allow us to explore questions regarding the effect of environmental stress during development on the ductus arteriosus that cannot be addressed with typical mammalian models.

Table of Contents: 

    Introduction

    Biologists currently use a number of organisms as model systems. These systems include the mouse (Mus muscullus), the fruit fly (Drosophila), the nematode (Caenorhabditis elegans), the zebrafish (Danio rerio), and the chicken (Gallus gallus). Biologists use model systems in their studies because they allow investigators with common interests to focus on one species that possesses wanted characteristics. My lab takes advantage of a number of properties of the chicken embryo that makes it a good model system to study developing respiratory and cardiovascular systems. Here I outline the characteristics of the chicken embryo that make it an excellent model system. I then describe the respiratory and cardiovascular physiology of the developing chicken embryo and compare it with a typical mammalian model, the lamb fetus developing in utero. I finish by examining one specific aspect of the developing cardiovascular system studied in my laboratory, the ductus arteriosus.

    Why Study Development Using the Avian Egg

    When picking a species to study, comparative physiologists tend to follow the August Krogh principle. This principle states that “For every physiological question, there is an animal that is ideally suited for providing the answer. ”The avian egg and embryo possess a number of characteristics that make them excellent model systems for developmental studies.

    The first characteristic is its longevity as a model system in the scientific community. While most of us are familiar with the mammalian system and might expect it to be the longest studied system for development, this is not the case. The chicken embryo actually has the longest continuous history as an experimental model for developmental biology studies (Stern, 2004). There is evidence that the Egyptians studied the development of the avian embryo. Aristotle (384-322 BC) used the developing chicken embryo to study changes in morphology during development. In the 1600’s, the chicken embryo was used to study the development of the beating heart as well as the arteries and veins through which blood flows. In the 1800’s the chicken was used to study the fate of the different germ layers of the developing embryo. Throughout the 1900’s the chicken continued to serve as a model for developmental studies. Thus, there is a very strong background on the developing chicken embryo upon which future studies can build.

    A second advantage of the avian embryo is the wide range of developmental modes found in different species at hatching. Birds develop and hatch along a continuum of functional maturity. At one end of the spectrum, the chicken hatches fully feathered, able to walk about and search for food. It has the capacity to metabolically regulate body temperature (endothermic). This level of functional maturity makes them precocial hatchlings. At the other end of the developmental spectrum are altricial birds such as song birds. Hatchlings of altricial species hatch featherless, are unable to defend their body temperature (ectothermic), and must rely on their parents for food. They remain in the nest for the first few weeks of post-hatch life as they mature into endothermic juveniles. Thus, if one is interested in the development of metabolism and thermoregulation, the avian hatchling is an excellent model.

    One of the major advantages the developing avian egg/embryo model has over the mammalian fetus is the environment in which the organism develops. Most mammalian fetuses develop within the protection of the mother’s uterus. Developmental physiologists are often interested in determining the effect of developing in a hypoxic or low oxygen environment. In a mammal, this involves placing a pregnant female in a low oxygen environment which in turn would hopefully reduce the oxygen available to the fetus in utero. This is problematic, however, because the pregnant female utilizes a number of mechanisms to compensate for the decrease in environmental oxygen. By increasing ventilation rate or hemoglobin production the mother can partially offset the decrease in environmental oxygen. Thus, the fetus is actually exposed to higher oxygen levels than wanted by the investigator. In contrast, the avian embryo develops in a hard shelled egg with little input except for temperature from the mother after the egg has been laid. Once an egg is laid, it can be collected and incubated without any maternal influences. By placing an egg in a low oxygen environment, we know the exact level of oxygen exposure. Thus, when interested in environmental effects on development, the avian egg is an excellent system to use.

    Basic Physiology of the Developing Avian

    In order to introduce the basic respiratory and cardiovascular physiology of the developing bird I will focus on the chicken embryo. The chicken embryo follows a set pattern of development during the 20 days of in ovo incubation. From day 0 to day 19 of incubation the embryo relies on the chorioallantoic membrane (CAM) for exchanging oxygen and carbon dioxide with the environment. The CAM is a highly vascular tissue that develops just under the shell and the two shell membranes. The development of the CAM is complete by the 12th day of incubation at which point it covers the entire egg (Ackerman and Rahn, 1980). Gases enter and leave the chicken egg by diffusing through the 10,000 pores in the shell. Oxygen diffuses from the environment, across the shell, across the outer and inner shell membranes, and into the blood in the capillaries of the CAM. The blood then returns to the embryo and supplies the tissues with the oxygen necessary for development. The removal of carbon dioxide occurs by the same process, but in the opposite direction. Along with exchanging oxygen and carbon dioxide with the environment, for proper development the developing avian egg must lose between 11% and 15% of its initial mass. This mass loss occurs as water vapor diffuses across the egg’s shell from the fully saturated egg and into the less humid environment. As water is lost to the environment, an air cell or air space develops between the outer and inner shell membranes at the blunt end of the egg. The embryo will take advantage of this air cell during hatching.

    On day 19 of incubation, the embryo has developed to the point where it begins the multi-step process of hatching. During the first step of hatching, the embryo breaks through the CAM and inner shell membrane into the air cell with its beak. This process is known as internal pipping (IP; Figure 1). This marks the initiation of lung ventilation by the embryo. During the internal pipping stage of hatching, the embryo relies on both the CAM and the lungs to obtain oxygen from the environment (Menna and Mortola, 2002; Sbong and Dzialowski, 2007). Approximately 12 to 24 hours after internal pipping the embryo breaks the egg shell with its beak in a process known as external pipping (EP; Figure 1). At this point the amount of oxygen acquired by the lungs rapidly increases and the oxygen acquired by the CAM rapidly diminishes (Meena and Mortola, 2002). External pipping is accompanied by a rapid decrease in blood flow to the CAM and increased blood flow to the lungs (Rahn et al., 1985). The embryo then hatches within the next 8 to 12 hours and relies solely on its lungs for gas exchange (Figure 1). The entire process of hatching takes between 18 and 36 hours in the chicken, during which time there is a gradual increase in the reliance on the lungs.

    The timing of hatching in the chicken embryo contrasts with that of parturition in the mammal. During development in utero, the fetus relies on the placenta and associated circulation for oxygen acquisition and carbon dioxide removal. The CAM and placental circulation have very similar functions during development. At birth, the neonate rapidly loses the placenta and its associated oxygen acquisition and begins to rely solely on the lungs for gas exchange (Figure 1). Where this transition from embryonic gas exchange to neonate gas exchange takes place over 24 hours in the chicken hatchling, it occurs in a matter of seconds in the mammalian neonate (Figure 1).

    Even though the timing of hatching and parturition differ in the bird and mammal, many of the cardiovascular and respiratory parameters of the developing mammal fetus and bird embryo are similar (Table 1). At term, both the chicken embryo and lamb fetus have similar cardiac outputs, a measure of the amount of blood pumped by the heart. The rate of blood flow through the umbilical cord to the placenta in mammals and CAM artery to the CAM in birds are nearly identical. The same is true for the oxygen and carbon dioxide blood gas levels in both the chicken and lamb at this stage of development. Thus, even though there are differences in the timing of hatching and the source of oxygen and nutrients, the respiratory gases in the blood during development are very similar in birds and mammals. The increased time during which the chicken hatches after initiation of lung ventilation should be beneficial to developmental physiologists.

    A Special Vessel: The Ductus Arteriosus

    The cardiovascular system of reptilian, avian, and mammalian embryos differs from that of the adult (Bergwerff, et al., 1999). The pulmonary artery of the adult carries deoxygenated blood from the heart to the lungs where it becomes oxygenated. Oxygenated blood returns to the heart through the pulmonary vein, where it is pumped by the left side of the heart out of the aorta and to the tissues of the body. As the lamb fetus and chicken embryo develops in utero and in ovo, the lungs are filled with fluid and are not ventilated with air. As noted above, the placenta and CAM are the sites for embryonic oxygen acquisition in mammals and birds, respectively. Therefore, the lungs only need enough blood to deliver oxygen necessary to support proper lung tissue development. The embryo has a special blood vessel, the ductus arteriosus, not found in the adult. The ductus arteriosus is a blood vessel which connects the embryonic pulmonary artery with the aorta. This provides a right-to-left shunt of blood away from the non-ventilated lungs and to the body and embryonic gas exchanger. This is called a right-to-left shunt because the blood from the right ventricle, which goes to the lungs in an adult, is shunted to the aorta and the body. Mammalian fetuses have a single ductus arteriosus and bird embryos have two ductus arteriosi. It is essential that this vessel remain open during in ovo or in utero development. Once hatching or birth begins, the lungs are ventilated and require an increase in blood flow. This is achieved by closing of the ductus at hatching or birth.

    There are a number of serious fetal and neonatal pathologies involving the human ductus arteriosus that require knowledge of the mechanism regulating the tone or tension of the vessel in order for proper treatment. Individuals may be born with a ductus arteriosus that does not close, requiring intervention to ensure closure. A persistent ductus after birth can result in necrotizing enterocolitis, intracranial hemorrhage, pulmonary edema/hemorrhage, bronchopulmonary dysplasia, or retinopathy (Clyman, 2006). This may occur in pre-term babies where the ductus is not fully developed and may not close properly at birth. Therefore, it is necessary that we have a good understanding of the mechanisms involved in maintaining ductus patency prior to birth and ductus closure at birth. This brings us to the major focus of the research conducted in my lab, determining factors that regulate ductus arteriosus tone using the avian embryo as our model system.

    The regulation of ductus arteriosus tone is a balance between contractile and relaxing factors. Prior to birth, the ductus must remain open. This is achieved in the mammalian system by the relaxing effect of prostaglandins (Clyman et al., 1980). Blocking prostaglandin production by the ductus results in constriction of the mammalian ductus. We have found that the avian ductus is insensitive to prostaglandin inhibitors suggesting that prostaglandins play little role in maintaining patency of the avian ductus.

    Closure of the ductus at birth or hatching requires a shift in the balance from the relaxing factors to the contractile factors. It is clear that oxygen is the major stimulus for the shift in this balance and the closure of both the mammalian and the avian ductus. As mammalian and chicken embryos approach full term, the sensitivity of the ductus arteriosus to oxygen increases. We have found that on day 19 of incubation, the chicken ductus responds to an increase in oxygen with a slight contraction. This contractile response matures during the hatching process. There is a doubling of the contraction strength generated by the ductus in response to oxygen from a day 19 embryo to an externally pipped embryo. The mammalian DA exhibits similar increased responsiveness to oxygen with gestation (Noel and Cassin, 1976; Coceani et al., 1979).

    The initiation of the functional closure of the ductus, occurring during external pipping, correlates with an increase in arterial and venous oxygen levels due to a greater reliance on the lungs for oxygen exchange (Tazawa et al., 1983). Pulmonary ventilation begins during internal pipping when the embryo begins to ventilate its lungs with the hypoxic gas of the air cell (110 mmHg; Tazawa, et al., 1983). With the initiation of lung ventilation, there is a slight increase in arterial and venous blood oxygen from 30 mmHg to 53 mmHg (Tazawa et al, 1983). This increase is a result of the combined use of the chorioallantoic membrane and the lungs for gas exchange. The oxygen induced contraction of the internally pipped and pre-pipped day 19 ductus is weak. Accordingly, the ductus is wide open and does not appear to be closing (Figure 2).

    It is not until external pipping that the ductus arteriosus begins to constrict in the hatching animal (Figure 2C & Figure 2D). Externally pipped embryos experience a further increase in blood oxygen levels (80 mmHg) resulting from an increase in the reliance on the lungs, which are now breathing normoxic gas (Tazawa, et al., 1983). The timing of the increase in blood oxygen levels at external pipping correlates with the increased response of the avian ductus to oxygen and a dramatic decrease in the diameter of the vessel opening (Figure 2C & Figure 2D). Rahn, et al. (1985) show that there is a gradual increase in blood flow to the lungs, and thus decrease in DA flow as the embryo hatches.

    In contrast with the chicken ductus, the mammalian ductus begins to close as soon as the neonate begins to ventilate its lungs. This difference in timing between DA closure in the chicken and the mammal may be due to the timing of the switch from the fetal gas exchanger to the neonatal gas exchanger. During fetal life in mammals, the ductus is exposed to similar low oxygen levels in the blood (28 mmHg; Smith, 1998). At birth, mammals switch from fetal gas exchange to pulmonary respiration within seconds, bringing about a rapid large increase in arterial oxygen levels. The late stage avian embryo is exposed to similar oxygen tensions, between 20-30 mmHg inside the egg (Tazawa et al., 1985). However, with the initiation of lung ventilation, the embryo respires hypoxic air cell gas with a Po2 of 110mmHg (Tazawa et al., 1983) and arterial oxygen levels rise only slightly. It is not until external pipping that the oxygen levels increase to the levels of the mammalian neonate. Therefore, the increase in arterial oxygen levels occurs much slower during avian hatching than during mammalian birth, resulting in slower closure of the avian ductus than the mammalian ductus. The switch from complete chorioallantoic respiration to complete pulmonary respiration is estimated to take between 22-30 hours in the chicken (Tazawa et al., 1983). This slower progression from the embryonic gas exchanger to the neonatal gas exchanger should provide us with greater resolution during closure and make the avian embryo an optimal system to study.

    Conclusions

    The avian egg and developing embryo have been used to study development for more than 2000 years. One of the major advantages of the avian egg over the mammalian fetus is the ability to isolate the developing embryo from the mother. The hard egg shell in which the avian embryo develops allows for easy manipulation of the incubation environment. My lab uses the avian embryo to study the mechanism regulating the closure of the ductus arteriosus during hatching. Because of the similarities between the avian and mammalian cardiovascular system, our findings can be directly applied to treat neonates in which the ductus is not functioning properly.

    References

    • Ackerman, R. A. and Rahn, H. 1980. In vivo O2 and water vapor permeability of the hen's eggshell during early development. Respir. Physiol. 45, 1-8.
    • Clyman, R.I. 2006. Mechanisms regulating the ductus arteriosus. Biol. Neonate. 89: 330-335.
    • Clyman, R.I., Mauray, F., Rudolph, A.M., and Heymann, M.A. 1980. Age-dependent sensitivity of the lamb ductus arteriosus to indomethacin and prostaglandins. J Pediatr 96: 94-98.
    • Coceani, F., White, E., Bodach, E. and Olley, P.M. 1979. Age-dependent changes in the response of the lamb ductus arteriosus to oxygen and ibuprofen. Can. J. Physiol. Pharmacol. 57, 825-831.
    • Menna, T.M. and Mortola, J.P. 2002. Metabolic control of pulmonary ventilation in the developing chick embryo. Respir. Physiol. 130, 43-55.
    • Noel, S. and Cassin, S. 1976. Maturation of contractile response of ductus arteriosus to oxygen and drugs. Am J Physiol. 231, 240-243.
    • Rahn, H., Matalon, S., and Sotherland, P.R. 1985. Circulatory changes and oxygen delivery in the chick embryo prior to hatching. In Cardiovascular Shunts (ed. K. Johansen and W. W. Burggren), pp. 199-211. Copenhagen: Munksgaard.
    • Sbong, S. and Dzialowski, E.M. 2007. Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos. Comp. Biochem. Physiol A. doi: 10.1016/j.cbpa.2007.03.013
    • Stern, C.D. 2005. The chick: a great model becomes even greater. Dev. Cell. 301, 9-17.
    • Tazawa, H., Visschedijk, A. H. J., Whitman, J. and Piper, J. 1983. Gas exchange, blood gases and acid-base status in the chick before, during and after hatching. Resp. Physiol. 53, 173-185.

    Table 1: Comparison of oxygen and carbon dioxide levels in the chicken embryo and fetal lamb (table compiled by Rahn et al., 1985).

      Units Sheep Chicken
    Cardiac Output mlkg-1 min-1 500 500
    Umbilical/CAM flow mlkg-1 min-1 200 250
    Po2 artery mmHg 23 24
    Pco2 artery mmHg 46 49
    pH artery   7.35 7.32
    Po2 vein mmHg 35 48
    Pco2 vein mmHg 42 40
    pH vein   7.40 7.45
    Fetal Tissue Po2 mmHg 10 10
     

    Figure 1: Changes in respiration in the developing human and bird.

    Figure 1: Changes in respiration in the developing human and bird

    Figure 2: Cross-sections of the ductus arteriosus

    Figure 2: Cross-sections of the ductus arteriosus