title: 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 (TGFβ2), 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 TGFβ2 and on several developmental genes linked to TGFβ 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 TGFβ2 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 TGFβ2 as well as the expression of several TGFβ2-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 TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.
authors.affiliation: Laboratory of Biodosimetry and Bioanalytical Methods, Department of Biology, University of Latvia, Riga, Latvia. inese.cakstina@lu.lv.
authors.forename: Inese
authors.initials: I
authors.lastname: Cakstina
authors.forename: Una
authors.initials: U
authors.lastname: Riekstina
authors.forename: Martins
authors.initials: M
authors.lastname: Boroduskis
authors.forename: Ilva
authors.initials: I
authors.lastname: Nakurte
authors.forename: Janis
authors.initials: J
authors.lastname: Ancans
authors.forename: Maija H
authors.initials: MH
authors.lastname: Zile
authors.forename: Indrikis
authors.initials: I
authors.lastname: Muiznieks
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000235:genetics; D050991:GATA4 Transcription Factor / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006321:Heart / Q000187:drug effects* / Q000196:embryology; D018398:Homeodomain Proteins / Q000235:genetics; D009024:Morphogenesis / Q000187:drug effects* / Q000235:genetics; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046509:Tissue Culture Techniques; D014157:Transcription Factors / Q000235:genetics; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D014714:Vertebrates / Q000196:embryology / Q000235:genetics; D014801:Vitamin A / Q000494:pharmacology*; D014815:Vitamins / Q000494:pharmacology
chemical_list: D030161:Avian Proteins; D005353:Fibronectins; D050991:GATA4 Transcription Factor; D018398:Homeodomain Proteins; D014157:Transcription Factors; D053781:Transforming Growth Factor beta2; D042461:Vascular Endothelial Growth Factor A; D014815:Vitamins; D014801:Vitamin A; D014212:Tretinoin