Abstract
BackgroundDysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome.MethodsUsing an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group.ResultsThe attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2).ConclusionsBased on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner.
Highlights
Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 deficiencies, mimicking human Holt-Oram syndrome
Multiple growth-related genes were downregulated in tbx5 knockdown embryos We screened growth-related genes that were downregulated after knockdown of tbx5 using a zebrafish microarray
We investigated gene expressions in dysmorphogenesis of a zebrafish tbx5-deficient model in order to reveal the role of tbx5 in altering transcription using an oligonucleotidebased microarray analysis, as it is sensitive to a single injection of tbx5 morpholino in zebrafish embryos
Summary
Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome. T-box transcription factor 5 (TBX5) is essential for cardiogenesis and forelimb development during embryogenesis in vertebrates. Mutation or haploinsufficiency of tbx in humans is related to Holt-Oram syndrome (HOS), which features congenital heart defects and forelimb deformities [1,2]. Tbx has essential roles in regulating progression of the cell cycle [13], cell growth, and apoptosis [14]. Blocking cell-cycle progression by tbx depletion at the RNA level leads to a decrease in the cardiac cell number, an alteration in the timing of the cardiac differentiation program, defects in cardiac sarcomere formation, and cardiac programmed cell death [13,15]
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