Transforming growth factor beta2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis.

Vitamin A-deficient (VAD) quail embryos lack the vitamin A-active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFbeta2 is involved in RA-regulated cardiovascular development. In VAD embryos TGFbeta2 mRNA and protein expression are greatly elevated. The expression of TGFbeta receptor II is also elevated in VAD embryos but is normalized by treatment with TGFbeta2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFbeta2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFbeta2 protein to normal quail embryos mimics the excessive TGFbeta2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFbeta2 has a major role in the retinoic acid-regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFbeta2 signals during vertebrate heart formation.

Gallera method of chick embryo culture in vitro supports better growth compared with original New method.

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.

Hypoxia induces cardiac malformations via A1 adenosine receptor activation in chicken embryos.

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 A(1) adenosine receptors (A(1)ARs). 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 A(1)AR 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: A(1)ARs were found to be expressed in chicken embryos during early embryogenesis. Treatment of Hamburger and Hamilton stage 4 embryos with the A(1)AR 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 A(1)AR signaling was inhibited by the A(1)AR antagonist 1,3 dipropyl-8-cyclopentylxanthine or by siRNA-targeting A(1)ARs. 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 A(1)AR signaling.

Lithium Chloride and Trypan Blue Induce Abnormal Morphogenesis by Suppressing Cell Population Growth: (LiCl/trypan blue/chick teratogenesis/cell population growth inhibition).

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.

Cell Population Growth and Area Expansion in Early Chick Embryos During Normal and Abnormal Morphogenesis in vitro: (trypan blue, chick embryo/teratogenesis/cell population growth/blastoderm expansion).

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.