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Poult Sci 2007. 86:2384-2389. doi:10.3382/ps.2007-00152
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY: Research Note

The Expression Patterns of Hypoxia-Inducing Factor Subunit {alpha}-1, Heme Oxygenase, Hypoxia Upregulated Protein 1, and Cardiac Troponin T During Development of the Chicken Heart

S. Druyan*, A. Cahaner{dagger} and C. M. Ashwell*,1

* Department of Poultry Science, North Carolina State University, Raleigh, 276965; and {dagger} The Hebrew University, Faculty of Agriculture, Rehovot, Israel, 76100

1 Corresponding author: chris_ashwell{at}ncsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxygen is one of the critical determinants of appropriate embryonic and fetal development, including cardiogenesis. When the demand of tissues for oxygen exceeds oxygen supply, hypoxic conditions develop. In the developing embryo, hypoxia is associated with increased fetal mortality, cerebrovascular anomalies, cardiovascular dysfunction, and altered angiogenesis. Tissue hypoxia may elicit a broad range of responses, many of which are dependent upon hypoxia-inducible transcription factors. Three genes that are stimulated by hypoxia—hypoxia-inducing factor subunit {alpha}-1, heme oxygenase, hypoxia upregulated protein 1, and cardiac troponin T, which is responsible for binding tropomyosin to regulate calcium binding and contractility of heart muscle—were examined in the embryonic heart of the chicken to determine if expression patterns were altered throughout development. On embryonic day (E) 7, all 3 hypoxic-induced genes were expressed at their highest levels, followed by a decrease from E7 to E19 followed by an increase between internal (E19) and external pipping (E20). The cardiac troponin T exhibited a similar expression level for E7 and E15 with a similar significant increase at E19 and E20. During these periods of development, significant changes in the primary gas exchange organs occur. Based on our observation of upregulation of these hypoxia response genes, it appears that tissue hypoxia is likely a normal component of embryonic development in the chicken based on the upregulation of hypoxia response genes.

Key Words: hypoxia • gene expression • embryonic development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of the avian embryo is supported by 3 gas exchange organ systems: the yolk sac, chorioallantoic membrane, and the lungs. The vascular portion of the yolk sac is the principal gas exchange organ during early development (Baumann and Meuer, 1992). At approximately embryonic day (E) 8, the allantoic sac develops and fuses with the chorion to create the chorioallantoic membrane (CAM; Wangensteen and Rahn, 1970–1971). This highly vascular structure, in conjunction with the porosity of the eggshell, permits diffusion of oxygen and carbon dioxide between the environment and the blood, thus replacing the yolk sac as the primary source of oxygen uptake (Tullett and Deeming, 1982). Toward the end of incubation at approximately E19, the embryo pierces the air sac membrane with its beak internal pipping (IP), and after a period, which in the chicken embryo is approximately 24 h (Dawes, 1981; Burton and Tullett, 1985), it begins to rupture the eggshell external pipping (EP). In the chick embryo, breathing-like movements, and therefore the initiation of pulmonary air convection, begin at the onset of IP (Vince and Tolhurst, 1975; Dawes, 1981).

During this final phase of in ovo development, air convection occurs in conjunction with 2 gas exchange organs operating together, the CAM and lungs. Fifteen hours before hatching, there is a decline in the allantois gas exchange and regression of the CAM (Visschedijk, 1968a,b,c), and the embryo shifts to dependence on oxygen provided by the lungs (Menna and Mortola, 2002). This mismatch between the increasing metabolic rate of the embryo and the reduction in the CAM gas transfer capabilities is considered a key parameter in the timing of the hatching process (Pettit and Whittow, 1982). As the embryo develops, the restrictions imposed by the fixed gas conductance of the shell and the limited diffusion capacity of the CAM result in increasing diffusion gradients for O2 and CO2 (Pettit and Whittow, 1982). Eventually, the capacity for diffusion of adequate O2 and exchange of CO2 without inducing hypoxia and acidosis is exceeded. It has been demonstrated that the time of EP in the chicken is accelerated by decreased oxygen content as well as increased CO2 content in the air space (Visschedijk, 1968a,b,c).

Oxygen tension is one of the critical determinants of appropriate embryonic and fetal development, including cardiogenesis (Sugishita et al., 2004a,b). When the demand of the tissues for oxygen exceeds the oxygen supply, hypoxic conditions develop. In the developing embryo, hypoxia is associated with increased fetal mortality (Chan and Burggren, 2005) and is known to cause decreased birth weight, cerebrovascular anomalies, cardiovascular dysfunction, and altered angiogenesis (Sharma et al., 2006). Wikenheiser et al. (2006) found that the embryonic chicken heart becomes exceedingly hypoxic, specifically during ventricular septation.

Tissue hypoxia may elicit a broad range of responses, many of which are dependent upon hypoxia-inducible transcription factors (HIF; Guillemin and Krasnow, 1997). The HIF are heterodimeric transcription factors consisting of inducible (HIF{alpha}-1, -2, -3) and constitutive subunits. The activity of HIF is largely determined by the induction of the HIF-{alpha} subunit in response to hypoxia. The HIF target and regulate the expression of more then 60 genes involved in diverse processes that are crucial for the hypoxic response, such as angiogenesis, anaerobic glycolysis, and cell survival (Koumenis and Maxwell, 2006).

In this study, 4 genes—hypoxia-inducing factor subunit {alpha}-1 (HIF1), heme oxygenase (HO1), [expressed in response to a panoply of stimuli that are associated with oxidative stress and inflammation, including hypoxia (Dawn and Bolli, 2005)], hypoxia upregulated protein 1 (HYOU1), also known as ORP150 (induced by hypoxia and thought to have an important cytoprotective role in hypoxia), and cardiac troponin T (cTnT; responsible for binding tropomyosin to regulate calcium binding and contractility of heart muscle)—were examined in the embryonic heart of the chicken to determine if expression patterns were altered throughout development. Samples were collected at E7 (before CAM development), E15 (CAM developed), E19 (IP), and E20 (EP) to capture the periods in incubation in which there is transition from the main gas transport organs, yolk sac to CAM to lung, and the potential for hypoxia to occur.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection

Fertilized chicken eggs were obtained from a broiler line. The eggs were incubated at 37.8°C in a moist atmosphere and turned automatically every hour. Four times during incubation [E7, E15, E19 (IP), and E20 (EP)], 6 eggs were randomly selected, broken out, and the embryos killed by decapitation. The E19 stage was verified and staged by determining if IP had occurred, before tissue collection. The E20 stage was identified by embryos that had EP. The heart was immediately removed, immersed in RNA-Later (Applied Biosystems-Ambion, Foster City, CA) according to the protocol of the manufacturer, and stored at –80°C.

RNA Extraction

Ribonucleic acid was extracted from individual hearts at the various developmental stages using a single-step, modified acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Saachi, 1987). Briefly, 100 mg of heart tissue was homogenized in RLT Lysis buffer (Qiagen, Valencia, CA). The homogenate was then extracted with acidic (pH 4.2) phenol:chloroform:isoamyl alcohol (125:24:1). The resulting mixture was separated by centrifugation, yielding an upper aqueous phase that was then decanted and extracted with chloroform-isoamyl alcohol (49:1), yielding an upper phase containing total RNA. The RNA was precipitated by addition of an equal volume of 100% isopropanol for 2 h and subsequent centrifugation. The RNA pellet was washed with 75% ethanol and then resuspended in diethylpyrocarbonate-treated water. Amount and quality of RNA were determined by Nanodrop (NanoDrop Technologies, Wilmington, DE) spectrophotometry. Only RNA of sufficient purity, having an absorbance ratio A260/280 > 1.8, was considered for synthesis of cDNA.

Quantitative Real-Time PCR

Total RNA from individual hearts was reverse-transcribed to produce cDNA in a 20-µL volume containing 1 µg of extracted RNA. Reverse transcription was carried out using an iScript kit according to the protocol of the manufacturer (BioRad, Hercules, CA). The reaction was incubated at 25°C for 5 min followed by 30 min at 42°C and 5 min at 85°C. Individual cDNA were diluted 1:20 before amplification. The mRNA expression levels of HIF1, HO1, HYOU1, and cTnT were analyzed by quantitative real-time PCR using the BioRad iQ instrument and with the iQ-SYBR Green Supermix kit using the protocols of the manufacturer.

Gene-specific primers were designed using Beacon Designer software (Premier Biosoft International, Palo Alto, CA) for SYBR Green detection according to the published cDNA sequences for each of the studied genes (Table 1Go).


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  		Table 1. Primers used for quantitative real-time PCR analysis of embryonic heart gene expression
 
Thermocycling parameters were as follows: 94°C for 5 min; 50 cycles of 94°C for 30 s, appropriate annealing temperature (Table 1Go) for 30 s, 72°C for 30 s; 72°C for 8 min. Fluorescence measurements were collected at every cycle during the extension step (72°C). Each gene was amplified independently in triplicate within a single instrument run. Standard curves were also generated to determine the efficiency of amplification by pooling undiluted cDNA from the heart samples across all ages and diluting the pooled cDNA to dilutions of 1:5, 1:25, 1:125, and 1:625. Cycle threshold (Ct) values were calculated for each sample automatically by the iQ software corresponding to the cycle in which amplification rate is maximal. Gene expression was normalized for RNA loading using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA (18S) as internal controls.

The n-fold change was calculated relative to that on E7 using the {Delta}{Delta}Ct method of Pfaffl (2001) including the efficiencies for both the experimental gene and GAPDH or 18S (internal controls).

Statistical Analysis

Data for the Ct ratio from 6 random repeats (sample gene Ct: sample GAPDH Ct and sample gene Ct: sample 18S Ct) during embryonic development were subjected to 1-way ANOVA according to the following model:


Formula 1([1])

with embryonic age (E7, E15, E19, or E20) as the main fixed effects.

The embryonic age was significant, and therefore, means of ages were compared using the Tukey test.

All statistical analyses were conducted using JMP software (SAS Institute, 2005).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In this study, we evaluated the expression of 4 genes. Three of these genes are involved in the response to tissue hypoxia and 1 gene, cTnT, is involved in cardiac development and contractibility during embryonic heart development (E7, E15, E19 IP, and E20 EP). Gene expression levels were determined by analyzing the resulting Ct values for each sample, normalizing to the level of GAPDH and 18S expression for the same RNA sample. The Ct ratios (gene Ct: GAPDH Ct or gene Ct: 18S Ct) for each of the genes in each of the developmental stages are shown in Table 2Go. The lower the Ct value, the lower the expression level; thus, the higher the Ct ratio, the lower the normalized gene expression level. The effect of embryonic age on gene expression was determined by using 1-way ANOVA, whereas differences in Ct ratio between different embryonic ages were determined to be significantly different using the Tukey test (Table 2Go).


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  		Table 2. Gene expression profiles in heart from embryonic day (E) 7 to external pipping (E20)
 
The embryonic age had a significant effect on the expression of all 4 genes (Table 2Go). Although the hypoxia-influenced genes HIF1, HYOU1, and HO1 all had a similar pattern of expression, the expression pattern of cTnT was different. In all 3 hypoxic genes, there was a linear increase in Ct value (decrease in fold change expression) between E7 to E19 with a significant difference in Ct ratio between E7 to E19. From E19 to E20, there was a significant decrease in Ct ratio (increase in fold change, Table 2Go). The cTnT exhibited a similar Ct ratio for E7 and E15 (1.10, 1.08 normalized to GAPDH and 1.98, 1.90 normalized to 18S respectively), with a similar significant decrease in Ct ratio on E19 and E20 (Table 2Go). There was no difference in the expression patterns of the 4 genes, which were identical regardless of which housekeeping gene the data were normalized to, either GAPDH or 18S.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As expected, cTnT expression was detected at all time points, in agreement with Antin and et al. (2002), who detected cTnT expression as early as stage 4 in embryonic development. The increase in expression level from E7 to E20 is correlated with the growth and maturation of the embryonic heart from a simple tube to a 4-chambered organ that is responsible for blood flow in the growing embryo. These results were as expected for the periods of incubation sampled and serve as a methodological marker of normal heart development.

Our results showed that all 3 hypoxia-induced genes were expressed at E7. The relative expression of these genes, as compared with later time points, suggests the embryos were experiencing tissue hypoxia at E7. Our results are in agreement with those of Nanka et al. (2006), who demonstrated that embryonic quail tissues experience hypoxia under normal conditions on E4 and E6. They found a correlation between the tissue hypoxia and increased expression of HIF1 and vascular endothelial growth factor (VEGF) on those days. Vascular endothelial growth factor is a significant signaling protein that regulates vasculogenesis and angiogenesis. Our study also demonstrated that the expression of GAPDH was not significantly altered throughout development in the chick heart, and this gene can be used as an internal standard similarly to 18S ribosomal RNA. There has been some evidence that GAPDH expression in the heart responds to induced extreme hypoxia (Zhong and Simons, 1999; Yamaji et al., 2003), but we found this not to be the case for apparent normal tissue hypoxia during developmental transitions in the chick embryo.

During the first week of embryonic development, the vascularized portion of the yolk sac is the principal gas exchange organ (Baumann and Meuer, 1992). Oxygen during that time is carried by a primitive red blood cell (RBC) that originates from primary yolk sac erythropoiesis (Brown and Ingram, 1974; Moorman et al., 1987). These primitive RBC lack a stem cell compartment, and their final number is determined by the number of cells that initially committed to the erythroid pathway. Unlike RBC of adult origins, primitive RBC enter the circulation as immature erythroblasts and complete their differentiation inside the circulation. Although these primitive cells have embryonic hemoglobins that have been found to have a higher oxygen affinity than the adult hemoglobins, their main function is to create and maintain an adequate oxygen pressure gradient inside the embryo (Baumann and Meuer, 1992). The undifferentiated nature of the primitive RBC, their limited number, the low hemoglobin concentration of the blood, and the metabolic demands of the developing embryo may lead to tissue hypoxia. Tissue hypoxia is likely the reason why all 3 genes are expressed at E7 in the chick embryo. Tissue hypoxia is also likely the trigger for HIF1 induction, which can stimulate vascularization and angiogenesis of the CAM.

Our results show that there was a significant decrease in expression of the 3 hypoxia-inducible genes at E15 and a further decrease at E19. These reductions in gene expression may be a result of the CAM taking over as the major site for gas exchange beginning at the second week of incubation and supplying adequate oxygen to the embryo (Wangensteen and Rahn, 1970–1971). As incubation and embryo development progress into the third week, increased oxygen consumption occurs. With the restrictions imposed by the fixed conductance of the shell and the limited diffusion capacity of the CAM, a hypoxic condition should evolve, and hypoxia-regulated gene expression will be elevated. However expression may not be elevated during the second half of incubation, due to the continuous increase of blood oxygen affinity in the embryo (Bartels et al., 1966). The basis for this constant adaptation is provided by sequential changes in the total number of circulating blood cells (Tazawa, 1980), their type, hemoglobin type (Chapman and Tobin, 1979), and changes in the concentration of RBC metabolites that affect the affinity of hemoglobin for oxygen (Baumann et al., 1983).

From the second week of development, RBC with embryonic origins enter the vascular system. These cells synthesize adult hemoglobins A and D (D has a higher oxygen affinity) initially at a 1:1 ratio, which then shifts to the adult 3:1 ratio later in development (Baumann and Meuer, 1992). Furthermore, there is a rapid increase in the affinity of hemoglobin for oxygen during development. This is coupled with a decline in RBC nucleotide (adenosine triphosphate, uridine triphosphate, cytidine triphosphate) concentrations as the concentration of 2,3-bis-phosphoglycerate, a weak regulator of oxygen affinity, increases (Dragon and Baumann, 2003). As a result of this complex series of changes, the oxygen capacity of the blood changes from approximately 8 cm3/100 mL at E10 to about 13 cm3/100 mL at E18 with blood oxygen saturation around 90% (Tazawa, 1980). This adaptation to oxygen demand, the elevation in oxygen-carrying capacity, and the higher oxygen affinity of the heme may explain the significant decrease in expression of all 3 genes from E7 to E19 (IP). It appears that during this part of development, the embryo is less susceptible to hypoxia, and the CAM can supply most of the demand of the tissues for oxygen.

For all 3 hypoxia-induced genes, we found a significant upregulation between IP E19 and EP E20. These elevations in expression levels were likely induced by hypoxia that evolves during the 24-h gap between IP and EP. After the embryo internally pips the air sac, the respiratory system transitions from passive gas change by the CAM to active convective gas exchange via the lungs in preparation for hatching (Ar et al., 1980). During this period, a gradual hypoxia and hypercapnia develop due to the decline in the allantois gas exchange. Oxygen exchange by the CAM becomes the primary issue, rather than CO2, because the CAM resistance to CO2 flow is about half that to O2 (Piiper et al., 1980). Any additional metabolic requirement by the embryo depends exclusively on what the lungs can provide, thus limiting the O2 supply (Ar and Rahn, 1985). Visschedijk (1968a,b) suggested that EP is stimulated by a decreased O2 content and increased CO2 content in the air sac.

The apparent hypoxic conditions that are occurring early in incubation at E7 appear to induce the expression of all 3 hypoxia response genes evaluated. Especially important is the increase in HIF1, which functions to regulate the expression of other genes, like angiotensin and VEGF, which play a crucial part in blood system development and the development of the CAM and other organs. Certain genes are upregulated by HIF1 in almost all cells that have been studied, including VEGF (Carmeliet et al., 1996; Ferrara et al., 1996; Tomanek et al., 1999). Wikenheiser et al. (2006) showed that hypoxic sites in the myocardium expressed HIF1, suggesting elevated VEGF synthesis. The appearance of relatively hypoxic myocardium coincides spatiotemporally with the sites where the major coronary vessels will form. This leads to the hypothesis that hypoxia and the HIF1-regulated transcriptional responses are important for the formation and organization of vessels in the early embryo (Ryan et al., 1998). Our results support this hypothesis for coronary vessel development by the appearance of higher HIF1 expression at E7 when increased angiogenesis is needed during the morphological change from the tube-based primitive heart structure to the multichambered mature morphology. This process may be timed precisely as part of the developmental mechanism using hypoxia as a trigger not only for cardiac development but also for stimulating the vascular development of the CAM and its role as the primary gas exchange organ. Recent findings also suggest that HIF1 has a role in normal coronary development, whereas mice deficient in HIF1 had hypovascularity of the heart, among other abnormalities (Huang et al., 2004).

Our data along with others support the importance of hypoxia in normal embryo development and the potential for altering developmental processes through the manipulation of conditions, specifically the levels of oxygen at stages earlier in incubation than previously considered.

Received for publication April 12, 2007. Accepted for publication July 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antin, P. B., M. A. Bates, W. Zhang, R. J. Garriock, and A. A. Yatskievych. 2002. Precocious expression of cardiac troponin T in early chick embryos is independent of bone orphogenetic protein signaling. Dev. Dyn. 225:135–141.[CrossRef][Web of Science][Medline]

Ar, A., and H. Rahn. 1985. Pores in the avian eggshells: Gas conductance, gas exchange and embryonic growth rate. Respir. Physiol. 61:1–20.[CrossRef][Web of Science][Medline]

Ar, A., A. H. J. Visschedijk, H. Rahn, and J. Piiper. 1980. Carbon dioxide in the chick embryo towards end of development: Effects of He and SF6 in breathing mixture. Respir. Physiol. 40:293–307.[CrossRef][Web of Science][Medline]

Bartels, H., G. Hiller, and W. Reinhardt. 1966. Oxygen affinity of chicken blood before and after hatching. Respir. Physiol. 1:345–356.[CrossRef][Web of Science][Medline]

Baumann, R., and H. J. Meuer. 1992. Blood oxygen transport in the early avian embryo. Physiol. Rev. 72:941–965.[Free Full Text]

Baumann, R., S. Padeken, E. A. Haller, and T. Brilmayer. 1983. Effects of hypoxia on oxygen affinity, hemoglobin embryos pattern, and blood volume of early chicken. Am. J. Physiol. Regul. Integr. Comp. Physiol. 244:733–741.

Brown, J. L., and V. M. Ingram. 1974. Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249:3960–3972.[Abstract/Free Full Text]

Burton, F. G., and S. G. Tullett. 1985. Respiration of avian embryos. Comp. Biochem. Physiol. 82A:735–744.

Carmeliet, P., V. Ferreira, G. Breier, S. Pollefeyt, L. Kieckens, M. Gertesenstein, M. Fahrig, A. Vandenhoeck, K. Harpal, C. Eberhardt, C. Declercq, J. Pawling, L. Moons, D. Collen, W. Risau, and A. Nagy. 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439.[CrossRef][Medline]

Chan, T., and W. Burggren. 2005. Hypoxic incubation creates differential morphological effects during specific developmental critical windows in the embryo of the chicken (Gallus gallus). Respir. Physiol. Neurobiol. 145:251–263.[CrossRef][Web of Science][Medline]

Chapman, B. S., and A. J. Tobin. 1979. Distribution of developmentally regulated hemoglobins in embryonic erythroid populations. Dev. Biol. 69:375–387.[CrossRef][Web of Science][Medline]

Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.[Web of Science][Medline]

Dawes, C. M. 1981. The effects of cooling the egg on the respiratory movements of the hatching fowls, Gallus G. domesticus, with a note on vocalization. Comp. Biochem. Physiol. 68A:399–404.

Dawn, B., and R. Bolli. 2005. HO-1 induction by HIF-1: A new mechanism for delayed cardioprotection? Am. J. Physiol. Heart Circ. Physiol. 289:H522–H524.[Free Full Text]

Dragon, S., and R. Baumann. 2003. Hypoxia, hormones, and red blood cell function in chick embryos. News Physiol. Sci. 18:77–82.[Abstract/Free Full Text]

Ferrara, N., K. Carvermoore, H. Chen, M. Dowd, L. Lu, K. S. Oshea, L. Powellbraxton, K. J. Hillan, and M. W. Moore. 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442.[CrossRef][Medline]

Guillemin, K., and M. A. Krasnow. 1997. The hypoxic response: Minireview Huffing and HIFing. Cell 89:9–12.[CrossRef][Web of Science][Medline]

Huang, Y., R. P. Hickey, J. L. Yeh, D. Liu, A. Dadak, L. H. Young, R. S. Johnson, and F. J. Giordano. 2004. Cardiac myocyte-specific HIF-1{alpha} deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J. 18:1138–1140.[Abstract/Free Full Text]

Koumenis, C., and P. H. Maxwell. 2006. Low oxygen stimulates the intellect. Symposium on hypoxia and development, physiology and disease. EMBO Rep. 7:679–684.[CrossRef][Web of Science][Medline]

Menna, M., and J. P. Mortola. 2002. Metabolic control of pulmonary ventilation in the developing chick embryo. Respir. Physiol. Neurobiol. 130:43–55.[CrossRef][Web of Science][Medline]

Moorman, A. F. M., M. P. A. Schalekamp, P. A. J. Deboer, W. J. C. Geerts, W. H. Lamers, and R. Charles. 1987. Immunohistochemical analysis of the distribution of histone H5 and hemoglobin during chicken development. Differentiation 34:161–167.[CrossRef][Web of Science][Medline]

Nanka, O., P. Valásek, M. Dvoráková, and M. Grim. 2006. Experimental hypoxia and embryonic angiogenesis. Dev. Dyn. 235:723–733.[CrossRef][Web of Science][Medline]

Pettit, T. N., and G. C. Whittow. 1982. The initiation of pulmonary respiration in a bird embryo: Tidal volume and frequency. Respir. Physiol. 48:209–221.[CrossRef][Web of Science][Medline]

Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e45.[Abstract/Free Full Text]

Piiper, J., H. Tazawa, A. Ar, and H. Rahn. 1980. Analysis of chorioallantoic gas exchange in the chick embryo. Respir. Physiol. 39:273–284.[CrossRef][Web of Science][Medline]

Ryan, H. E., J. Lo, and R. S. Johnson. 1998. HIF-1{alpha} is required for solid tumor formation and embryonic vascularization. EMBO J. 17:3005–3015.[CrossRef][Web of Science][Medline]

SAS Institute. 2005. JMP® Version 6 User’s Guide. SAS Inst. Inc., Cary, NC.

Sharma, S. K., J. L. Lucitti, C. Nordman, J. P. Tinney, K. Tobita, and B. B. Keller. 2006. Impact of hypoxia on early chick embryo growth and cardiovascular function. Pediatr. Res. 59:116–120.[CrossRef][Web of Science][Medline]

Sugishita, Y., D. W. Leifer, F. Agani, M. Watanabe, and S. A. Fisher. 2004a. Hypoxia-responsive signaling regulates the apoptosis-dependent remodeling of the embryonic avian cardiac outflow tract. Dev. Biol. 273:285–296.[CrossRef][Web of Science][Medline]

Sugishita, Y., M. Watanabe, and S. A. Fisher. 2004b. Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract. Dev. Biol. 267:294–308.[CrossRef][Web of Science][Medline]

Tazawa, H. 1980. Oxygen and CO, exchange and acid-base regulation in the avian embryo. Am. Zool. 20:395–404.[Web of Science]

Tomanek, R. J., A. Ratajska, G. T. Kitten, X. Yue, and A. Sandra. 1999. Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis. Dev. Dyn. 215:54–61.[CrossRef][Web of Science][Medline]

Tullett, S. G., and D. C. Deeming. 1982. The relationship between eggshell porosity and oxygen consumption of the embryo in the domestic fowl. Comp. Biochem. Physiol. 72A:529–533.[Medline]

Vince, M. A., and B. E. Tolhurst. 1975. The establishment of lung ventilation in the avian embryo: The rate at which lungs become aerated. Comp. Biochem. Physiol. 52A:331–337.[CrossRef][Medline]

Visschedijk, A. H. J. 1968a. The air space and embryonic respiration. 1. The pattern of gaseous exchange in the fertile egg during the closing stages of incubation. Br. Poult. Sci. 9:173–184.[CrossRef][Web of Science][Medline]

Visschedijk, A. H. J. 1968b. The air space and embryonic respiration. 2. The times of pipping and hatching as influenced by an artificially changed permeability of the shell over the air space. Br. Poult. Sci. 9:185–196.[CrossRef][Web of Science][Medline]

Visschedijk, A. H. J. 1968c. The air space and embryonic respiration. 3. The balance between oxygen and carbon dioxide in the air space of the incubating chicken egg and its role in stimulating pipping. Br. Poult. Sci. 9:197–210.[Web of Science][Medline]

Wangensteen, D., and H. Rahn. 1970–1971. Respiratory gas exchange by the avian embryo. Respir. Physiol. 11:31–45.[CrossRef]

Wikenheiser, J., Y. Q. Doughman, S. A. Fisher, and M. Watanabe. 2006. Differential levels of tissue hypoxia in the developing chicken heart. Dev. Dyn. 235:115–123.[CrossRef][Web of Science][Medline]

Yamaji, R., K. Fujita, S. Takahashi, H. Yoneda, K. Nagao, W. Masuda, M. Naito, T. Tsuruo, K. Miyatake, H. Inui, and Y. Nakano. 2003. Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: Involvement of Na+/Ca2+ exchanger. Biochim. Bio-phys. Acta 1593:269–276.[Medline]

Zhong, H., and J. W. Simons. 1999. Direct comparison of GAPDH, β-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem. Biophys. Res. Commun. 259:523–526.[CrossRef][Web of Science][Medline]





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