Assessment of the expression of genes involved in Notch-signalin in Spen- and Connexin43-ablated zebrafish embryos

Abstract

Our study on DCM-candidate gene spen in zebrafish suggests that loss-of Spen leads to development of heart failure and cardiac arrhythmia by downregulation of Connexin43 (Cx43). Since Spen is known to be a regulator of the Notch-signaling pathway via direct interaction with RBPJ, we assessed the regulation of Notch-pathway genes in spen- and cx43-morphants. Additionally, expressional levels of spen and cx43 were assessed in RBPJ-morphants. We found that there is no distinct and significant up- or down regulation of the analyzed Notch targets hey, her3.1, her6, her8.2, and myca in both, MO-spen and MO-cx43 injected embryos. The potential loss of Notch repression in spen morphants does not seem to be sufficient to decisively alter the expression of Notch target genes. Interestingly, the expression of notch1a, the membrane bound notch receptor, was significantly increased in both morphants. Intriguingly, in Notch signaling deficient embryos, which was achieved by rbpja/b-MO injection, we observed a downregulation of spen and cx43. Since RBPJ is the main switch of the Notch signaling pathway, one could assume that the Notch signal transduction cascade might somehow be involved in the regulation of cx43. Further deciphering of the molecular interplay of Cx43 and Spen is part of our future research. Methods: Morpholino and mRNA Injection Procedures: For all Morpholino-modified antisense oligonucleotide injection procedures, the TüAB wild-type strain was used. Morpholino-modified antisense oligonucleotides (MO; Gene Tools, LLC) were directed against the splice donor site of exon 1/intron 1 (5’ CTCAGCAAATCGTCACTTACCGTTT 3’) of zebrafish spen on chromosome 23 (MO-spensplice), the translational start site (5’ TCCCAACGCACTCCAGTCACCCATC 3’) of zebrafish connexin43 on chromosome 20 (MO-cx43ATG). For RBPJ knockdown a morpholine targeting the open reading frame was used (5' CAAACTTCCCTGTCACAACAGGCGC 3'). Reverse Transcriptase (RT)-PCR and quantitative real-time PCR: RNA isolation was performed by using Qiazol Lysis Reagent (Qiagen) according to the manufacturer’s instructions. cDNA was generated from RNA of 72 hpf MO-spensplice- and MO-Ctrl injected embryos using oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen). RT-PCR was performed according to standard protocols with cx43-, spen-, hey1-, her3.1-, her6-, her8.2- and myca- specific primers. Quantitative real-time PCR was carried out according to standard protocols with the SYBR-Green method (Bio- Rad) and an Eppendorf Realplex-2 cycler or a Roche LightCylcer480