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Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A.

ABSTRACT: BACKGROUND: Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGF?2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGF?2 and on several developmental genes linked to TGF? signaling during heart morphogenesis. RESULTS: We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGF?2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGF?2 as well as the expression of several TGF?2-linked developmental genes is regulated by RA. CONCLUSIONS: Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGF?2 and other genes involved in vertebrate early cardiovascular morphogenesis.

SUBMITTER: Cakstina I

PROVIDER: S-EPMC3939001 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A.

Cakstina Inese I Riekstina Una U Boroduskis Martins M Nakurte Ilva I Ancans Janis J Zile Maija H MH Muiznieks Indrikis I

BMC developmental biology 20140219

<h4>Background</h4>Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), inv ...[more]

PMID: 24552295

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Project description:cDNA clones encoding Japanese quail chorioallantoic membrane and chicken kidney 1,25-dihydroxyvitamin D3 receptors were isolated and the total 448-amino acid (aa) sequence was deduced. The sequences of the chicken and quail receptors are identical. The nucleotide and deduced amino acid sequences of the avian receptors are similar but not identical to the reported rat or human receptor sequences. There is a 78% similarity in the nucleotide sequences and 98.5% and 87.5% similarities in the amino acid sequences of the DNA-binding and ligand-binding domains, respectively. Two avian receptor proteins (58 and 60 kDa) arise from a single mRNA transcript by alternate initiation of translation. The avian 1,25-dihydroxyvitamin D3 receptors were produced using a bacterial expression system. Form A receptor was expressed from a cloned cDNA that contains the first translation signal (ATG) and corresponds with the 60-kDa avian receptor protein, and form B receptor was initiated from the third ATG on the same mRNA transcript to give rise to the 58-kDa protein. The cysteine-rich DNA-binding domain is almost conserved among human, rat, and avian receptors. The position of the nine cysteines was conserved in all three sequences. The avian receptor differs in the second zinc finger domain, where a methionine replaces a leucine, a serine replaces an asparagine, and a lysine replaces an arginine at aa 77, 83, and 87, respectively, of the avian sequence. The increased length of the avian receptor results from a 20-aa extension of the N-terminal region. RNA hybridization indicates there is a single mRNA species of approximately 2700 bp for both the chicken and quail receptors compared to 4400 bp for the rat transcript. Surprisingly, the translated avian sequence is larger (448 aa) than the 423-aa rat receptor protein. Therefore, our results confirm that despite the difference in molecular mass between different receptor proteins, there is a similarity in gene organization such that the DNA-binding and hormone-binding domains are positionally conserved from the C terminus.

Dataset Information

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A.

Publications

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A.

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