The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos

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.

Anti-apoptotic effects of IGF-I on mortality and dysmorphogenesis in tbx5-deficient zebrafish embryos

“…This study showed that exogenous IGF-I induced positive effects on embryogenesis in wild-type zebrafish embryos (Fig. 3C and I ), whereas a previous study reported that exogenous GH had little effect [ 21 ]. Additionally, it has been reported that the embryogenic effect of GH probably occurs via an IGF-I-independent mechanism [ 25 ].…”

“…tbx5 is a regulator of apoptosis and cell growth, and tbx5 deficiency can induce cell apoptosis and alter cell cycling [ 22 – 24 ]. Although the role of IGF-I is not well-characterized in zebrafish embryogenesis, previous studies have found that tbx5 morphants have down-regulated gh and GH-related genes, including igf1 [ 21 ]. In this study, we found that exogenous recombinant human IGF-I administration ameliorated dysmorphogenesis, increased akt2 and erk1 expression , normalized bcl2 and bad expression, suppressed cell apoptosis, and improved survival rates in tbx5 morphants.…”

“…We have previously observed that exogenous human growth hormone (GH) treatment can enhance expression of downstream mediators in the GH and IGF-I pathways and partially restore dysmorphogenesis in tbx5 morphants [ 21 ]. Therefore, this study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in tbx5 -deficient zebrafish embryos by microinjecting exogenous recombinant human IGF-I into tbx5 -deficient zebrafish embryos at an extremely early embryonic stage.…”

Pectoral Fin Anomalies in tbx5a Knockdown Zebrafish Embryos Related to the Cascade Effect of N-Cadherin and Extracellular Matrix Formation

“…We used the Zebrafish AB strain carrying a specific transgene (Tg, cmlc2::EGFP; cmlc2:H2AFZmCherry) cy3 in this study. The details of the maintenance and breeding of zebrafish and embryo collection have been previously published [21,22,23].…”