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BACKGROUND: The current understanding of the effects of hypoxia on early embryogenesis is limited. Potential mediators of hypoxic effects include adenosine, which increases dramatically during hypoxic conditions and activates A1 adenosine receptors (A1ARs). METHODS: To examine the influences of hypoxia and adenosine signaling on cardiac development, chicken embryos were studied. Real time RT-PCR assay was used to examine the A1AR gene expression during embryogenesis and after siRNA- mediated knock down. Cell proliferation was determined by counting cell nuclei and PhosphoHistone H3 positive cells. Apoptosis was determined by TUNEL assay. RESULTS: A1ARs were found to be expressed in chicken embryos during early embryogenesis. Treatment of Hamburger and Hamilton stage 4 embryos with the A1AR agonist N 6 -cyclopentyladenosine caused cardiac bifida and looping defects in 55% of embryos. Hamburger and Hamilton stage 4 embryos exposed to 10% oxygen for 6, 12, 18, and 24 h followed by recovery in room air until stage 11, exhibited cardia bifida and looping defects in 34, 45, 60, and 86% of embryos respectively. Hypoxia-induced abnormalities were reduced when A1AR signaling was inhibited by the A1AR antagonist 1,3 dipropyl-8-cyclopentylxanthine or by siRNA-targeting A1ARs. Hypoxia treatment did not increase apoptosis, but decreased embryonic cell proliferation. CONCLUSIONS: These data indicate that hypoxia adversely influences cardiac malformations during development, in part by A1AR signaling. Birth Defects Research (Part A) 82:121–130, 2008. 2008 Wiley-Liss, Inc.

Embryos can be exposed to environmental factors that induce hypoxia. Currently, our understanding of the effects of hypoxia on early mammalian development is modest. Potential mediators of hypoxia action include the nucleoside adenosine, which acts through A 1 adenosine receptors (A 1 ARs) and mediates adverse effects of hypoxia on the neonatal brain. We hypothesized that A 1 ARs may also play a role in mediating effects of hypoxia on the embryo. When pregnant dams were exposed to hypoxia (10% O 2 ) beginning at embryonic day (E) 7.5 or 8.5 and continued for 24–96 h, A 1 AR+/+ embryos manifested growth inhibition and a disproportionate reduction in heart size, including thinner ventricular walls. Yet, when dams were exposed to hypoxia, embryos lacking A 1 ARs (A 1 AR−/−) had much more severe growth retardation than A 1 AR+/+ or +/− embryos. When levels of hypoxia-inducible factor 1α (HIF1α) were examined, A 1 AR−/− embryos had less stabilized HIF1α protein than A 1 AR+/− littermates. Normal patterns of cardiac gene expression were also disturbed in A 1 AR−/− embryos exposed to hypoxia. These results show that short periods of hypoxia during early embryogenesis can result in intrauterine growth retardation. We identify adenosine and A 1 ARs as playing an essential role in protecting the embryo from hypoxia.

PR-39, a proline-arginine-rich angiogenic response peptide, has been implicated in myocardial ischemic reperfusion injury. The present study examined the cardioprotective abilities of PR39 gene therapy. Male C57Bl/J6 mice were randomized to intramyocardial injecton of 10(9) p.f.u. adenovirus encoding PR39 (PR39), FGFR1 dominant negative signaling construct (FGFR1-dn), empty vector (EV), or PR39 adenovirus plus 4 microg of plasmid endcoding a HIF1alpha dominant negative construct (PR39 + HIF1alpha-dn). Seven days later, hearts were subjected to 20 min of ischemia (I) and 2 h. reperfusion (R) ex vivo and aortic and coronary flow, left ventricular developed pressure (LVDP), and LVdp/dt were measured. Myocardial infarct (MI) size and cardiomyocyte apoptosis were measured by TTC staining and TUNEL, respectively. PR39 expression was robust up to 14 days after gene transfer and was absent after EV and FGFR1-dn. Hemodynamics showed no differences at baseline, and heart rate remained unchanged in all groups throughout the experiment. After I-R, hemodynamics remained unchanged in PR39 hearts, but deteriorated significantly in the other groups, except for aortic flow, which remained significantly higher in FGFR1-dn than in EV and PR39 + HIF1alpha-dn (p < 0.05), although it was lower than in PR39 (p < 0.05). MI was 8.7 +/- 0.9 % in PR39, 23.8 +/- 1.1% in FGFR1-dn, 29.9 +/- 2.2% in EV, and 30.8 +/- 2.7 % in PR39 + HIF1alpha-dn (PR39 vs. other groups: p < 0.05; FGFR1-dn vs. EV and PR39 + HIF1alpha-dn: p < 0.05). In PR39, HIF-1alpha protein was higher than in FGFR1-dn and EV. Importantly, cotransfection of HIF1alpha-dn with PR39 completely abolished cardioprotection by PR39. Cardioprotection by PR39 is likely conveyed by protective metabolic and survival responses through HIF1-alpha stabilization and not by angiogenesis, because baseline coronary flow was the same in all groups. Abrogation of FGFR1 signaling conveyed an intermediate degree of cardioprotection.

Background Fetal cells exfoliate in the uterine cavity during early pregnancy and are a potential source of material for NIPD. Aims This study was designed to test the hypothesis that fetal cells obtained from the uterine cervix during the first trimester of pregnancy could be utilized for prenatal diagnosis of chromosomal aneuploidy. Study design Fetal cells retrieved from the distal endocervical canal during the first trimester of pregnancy were hybridized with chromosome 21 specific FISH probes and analyzed with an automated fluorescence microscope. Subjects and outcome measures Cells with 3 copies of chromosome 21 were detected in 5 out of 5 trisomy 21 pregnancies. Results The number of trisomic cells detected ranged from 1 to 27 with a median value of 5. Conclusions FISH-based scanning can identify trisomy 21 pregnancies by analysis of routine cervical brushings. The approach offers the potential for non-invasive prenatal diagnosis as early as 5 weeks gestation.

The vitamin A deficient (VAD) quail embryo lacks active retinoids, fails to express normally GATA-4, and develops a nonlooping heart tube morphogenetic defect that is a model for congenital cardiomyopathy. VAD quail embryos, or chick embryos depleted specifically for GATA factors, show in addition abnormal foregut development, characterized by apoptosis of the endoderm cells associated with presumptive myocardium during the process of heart tube formation. Exogenous retinoic acid or transplantation of normal chick embryo anterior endoderm is sufficient to rescue apoptosis as well as GATA-4 expression and results in normal development and heart tube morphogenesis. Normal posterior endoderm also contains retinoids but is unable to rescue the VAD defect. Our results indicate that a retinoid-dependent transcriptional program, mediated at least in part by GATA factors, is critical in presumptive foregut endoderm for normal heart tube morphogenesis.

In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur earlier than previously thought and appear to be closely linked. The onset of skeletal myogenic program follows that of the cardiogenic program with the appearance of skeletal MLC2 at stage 8. We also observed that mRNA for the MEF-2 family of proteins appears as early as stage 2 and that for CMD-1, the chicken counterpart of MyoD, appears at stage 5. The temporal separation of activation of cardiac and skeletal MLC2 genes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consistent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological characteristics in the early embryo. The preferential appearance of BBF-1 activity in precardiac moesderm, relative to that of MEF-2, indicates that these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role of BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac differentiative state.

Full primitive streak stage chick embryos were cultured in vitro for 20 hrs and monitored every 4 hr for morphology, cell number and blastoderm area. In normal embryos, the cell population growth is exponential and correlates directly with Increasing morphological rank. The chick blastoderm area expands in two waves, one immediately after gastrulation and another after 16 hr in culture, while cell population growth is predominant between 4–16 hr. Trypan blue and LiCI inhibit cell population growth, epiboly and shaping of organ primordia. Both teratogens induce a similar spectrum of abnormalities although the severity of abnormal development is greater with LiCl for the given dose. In most abnormal embryos the cell population size and blastoderm area are inhibited most, which is detectable already after 12 hr of culture. We have established that the cell population growth, morphogenesis and area expansion constitute a parametric hierarchy with the cell population growth as the most independent parameter in regulating normal morphogenesis.

Background Our understanding of the mechanisms that protect the developing embryo from intrauterine stress is limited. Recently, adenosine has been demonstrated to play a critical role in protecting the embryo against hypoxia via adenosine A1 receptors (A1ARs), which are expressed in the heart, nervous system, and other sites during development. However, the sites of A1AR action that mediate embryo protection are not known. To determine if the heart is a key site of adenosine-mediated embryo protection, A1ARs were selectively deleted in the embryonic heart using a Cre-LoxP system in which the alpha-myosin heavy chain promoter drives Cre-recombinase expression and excision of the A1AR gene from cardiomyocytes. Results With increasing exposure of maternal hypoxia (10% O2) from 48-96 hours beginning at embryonic day (E) 8.5, embryo viability decreased in the cardiac-A1AR deleted embryos. 48 hours of hypoxia reduced embryonic viability by 49% in embryos exposed from E10.5-12.5 but no effect on viability was observed in younger embryos exposed to hypoxia from E8.5-10.5. After 72 hours of hypoxia, 57.8% of the cardiac-A1AR deleted embryos were either dead or re-absorbed compared to 13.7% of control littermates and after 96 hours 81.6% of cardiac-A1AR deleted embryos were dead or re-absorbed. After 72 hours of hypoxia, cardiac size was reduced significantly more in the cardiac-A1AR deleted hearts compared to controls. Gene expression analysis revealed clusters of genes that are regulated by both hypoxia and A1AR expression. Conclusions These data identify the embryonic heart as the critical site where adenosine acts to protect the embryo against hypoxia. As such these studies identify a previously unrecognized mechanism of embryo protection.

The purpose of this study was to determine both the short-term effects on cardiac development and embryo growth and the long-term effects on cardiac function and body composition of in utero caffeine exposure. Pregnant mice (C57BL/6) were exposed to hypoxia (10% O2) or room air from embryonic days (E) 8.5–10.5, and treated with caffeine (20 mg/kg, i.p.) or vehicle (normal saline, 0.9% NaCl). This caffeine dose results in a circulating level that is equivalent to 2 cups of coffee in humans. Hypoxic exposure acutely reduced embryonic growth by 30%. Exposure to a single dose of caffeine inhibited cardiac ventricular development by 53% in hypoxia and 37% in room air. Caffeine exposure resulted in inhibition of hypoxia-induced HIF1α protein expression in embryos by 40%. When offspring from dams treated with a single dose of caffeine were studied in adulthood, we observed that caffeine treatment alone resulted in a decrease in cardiac function of 38%, as assessed by echocardiography. We also observed a 20% increase in body fat with male mice exposed to caffeine. Caffeine was dissolved in normal saline, so it was used as a control. Room air controls were used to compare to the hypoxic mice. Exposure to a single dose of caffeine during embryogenesis results in both short-term effects on cardiac development and long-term effects on cardiac function.—Wendler, C. C., Busovsky-McNeal, M., Ghatpande, S., Kalinowski, A., Russell, K. S., Rivkees, S. A. Embryonic caffeine exposure induces adverse effects in adulthood.

The exponential growth and cell population during the early embryogenesis of chick, cultured in vitro correlates with a linear increase in the blastoderm area. To understand the relationship between these parameters and normal morphogenesis, we have used a known teratogen, trypan blue, as a probe. A method is developed in which each new embryonic structure is assigned a rank value of 1 and the total number of ranks allows quantification of development and establishment of a numerical relationship between the size of the cell population, blastoderm area and the morphological development. The teratogen inhibits cell population growth, morphogenetic movements and shaping of organ primordia, but not the epiboly and differentiation of cells which have already invaginated and positioned during primitive streak formation. In contrast, the cell population growth, but not the blastoderm area-expansion, is correlated with the extent of abnormal development. A graphic analysis of the rank order, log cell number and blastoderm area reveals that these three parameters coordinately regulate morphogenesis. It is suggested that head fold formation is the key event regulating the progress of early morphogenesis.

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro. Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.

We used the vitamin A-deficient (VAD) quail model to investigate the retinoid-dependent mechanism that regulates heart tube development. We showed previously that decreased levels of Gata4 in cardiogenic mesoderm and endoderm correlate with the cardiomyopathy caused by VAD, but that this could be rescued by transplanting normal anterior endoderm. Bmp2 is a known cardiogenic factor that is expressed normally in lateral plate mesoderm and cardiac-associated pharyngeal endoderm. Here we show that (like Gata4) transcripts encoding Bmp2 and BMP-dependent signaling activity are decreased throughout the heart-forming region of the VAD embryo. Addition of Bmp2 protein or forced expression of Gata4 in cultured VAD embryos leads to a partial rescue of the cardiomyopathy, and addition of both Bmp2 and Gata4 has an additive positive effect. Our data are consistent with a requirement for retinoid signaling to maintain expression of Bmp2, which regulates Gata4, and in addition acts with Gata4 to regulate genes important for normal morphogenesis of the primitive heart tube.

It is known from nutritional studies that vitamin A is an important factor for normal hematopoiesis, though it has been difficult to define its precise role. The vitamin A–deficient (VAD) quail embryo provides an effective ligand “knockout” model for investigating the function of retinoids during development. The VAD embryo develops with a significant reduction in erythroid cells, which has not been noted previously. Activation of the primitive erythroid program and early expression of the erythroid marker GATA-1 occurs, though GATA-1 levels eventually decline, consistent with the erythropoietic and hemoglobin deficits. However, from its early stages, the GATA-2 gene fails to be expressed normally in VAD embryos. The bone morphogenetic protein (BMP)–signaling pathway regulates GATA-2, and BMP4 expression becomes reduced in the caudal embryonic region of VAD embryos. Adding BMP4 to cultured VAD-derived explants rescues the production of erythroid cells, whereas normal embryos cultured in the presence of the BMP antagonist noggin are defective in primitive hematopoiesis. We find that cell clusters of primitive blood islands undergo an inappropriate program of apoptosis in the VAD embryo, which can explain the deficit in differentiated primitive blood cells. We propose that vitamin A–derived retinoids are required for normal yolk sac hematopoiesis and that an embryonic retinoid–BMP–GATA-2 signaling pathway controls progenitor cell survival relevant to primitive hematopoiesis.

In comparing the pathological state of cardiac hypertrophy with early embryonic growth and development of the primitive heart, important and informative aspects of mechanisms that underlie activation of the gene expression pattern become apparent. Interestingly, in both cases the muscle phenotypes share the expression of a ‘fetal’ gene expression program, raising the question whether the same genetic mechanism is being called upon by signals associated with the onsets of cardiogenesis and myocardial hypertrophy. A cell specific transcription factor, CLP-1, was recently identified in our laboratory that is likely to play a crucial role, in conjunction with other known regulatory factors, in early cardiac events leading to cardiogenic cell specification and differentiation. We have also identified a novel mechanism that involves activation of the Jak/Stat signaling pathway that is linked to the autocrine angiotensin-II loop associated with the hypertrophic response in cardiomyocytes. Since early cardiac cell development and the hypertrophic state involve the expression of the same battery of genes, one may speculate that common transcription factors may account for assembling a competent apparatus responsible for transcribing the genes. Our present studies are designed to investigate the potential role of these factors in control of both processes.

Publisher Summary This chapter discussed molecular adaptation of the transcriptional apparatus in cardiac hypertrophy and embryonic development. The heart occupies a central position in the ontogeny of all vertebrate organisms. As one of the first organs to arise during development, it becomes essential for development of the embryo and fetal life, and also to the viability of the vertebrate organism throughout its life. Virtually all aspects of development and organogenesis in vertebrates rely on the interaction and inductive influences operating among the three primary germ cell layers of mesoderm, ectoderm, and endoderm. The earliest event in development of the heart is the establishment of the cardiogenic cell lineage from one of these cell layers, in this case, the mesoderm. Mesodermal cells that acquire the cardiogenic cell fate go on to contribute to the cardiac primordia or primitive heart tube. The chapter also explains regulation of myosin light chain-2 gene activity in hypertrophy.

We describe the isolation and characterization of a cDNA clone, called cCLP-1, that is a candidate for the previously described early cardiac-specific transcription factor BBF-1. BBF-1 binds the MEF2 (or element B) binding site within the cardiac myosin light chain 2 (MLC2) gene promoter. We used the element B sequence as a probe to screen an expression library constructed from mRNA obtained from the presumptive heart-forming regions of stage 6 chicken embryos. This yielded the cCLP-1 cDNA clone. Gel-shift analysis of stage 6 embryonic chicken protein extracts suggests that a protein that is recognized by the anti-cCLP-1 antibody binds to the same element B binding site to which BBF-1 binds. cCLP-1 mRNA was detected early in chicken development, prior to cardiac fate assignment at stage 4. The expression pattern of cCLP-1, based on whole mountin situhybridization, coincides remarkably well with the established morphogenetic field of early heart formation. The nuclear localization of cCLP-1 is phosphorylation-dependent, suggesting that cCLP-1 may be a member of that class of transcription factors whose activity is regulated by cytoplasm to nucleus transport. Taken together, these data suggest that cCLP-1 may encode a novel transcription factor whose expression pattern is in agreement with that of the cardiogenic precursor cells of the early chicken embryo.