Abstract

CpG binding protein (CGBP) is a transcriptional activator that binds unmethylated CpG motifs. CGBP is ubiquitously expressed, and deletion of the CGBP gene in mice results in an embryonic lethal phenotype (E3.5–E6) indicating that CGBP is required for embryonic mammalian development. CGBP-null murine embryonic stem (ES) cells are viable but unable to differentiate and have a 60% decrease in CpG methylation compared to wild type ES cells. Based upon these results, it is hypothesized that CGBP is required for normal stem cell function. RT-PCR was used to identify a homologue of CGBP in zebrafish which is ~70% identical to the mouse CGBP. It was determined that CGBP is expressed in the zebrafish as early as 2 hpf. Morpholino oligonucleotides were designed to the zebrafish CGBP and injected into 1–2 cell embryos. Approximately 80% of the zebrafish treated with the CGBP morpholino oligonucleotides had little or no circulating red blood cells and had abnormal yolk sac morphology at 48 hpf. More than half of the CGBP treated zebrafish also exhibited cardiac edema, and ~14% were dead at 24 hpf. A similar phenotype was observed with a second morpholino designed to an independent site of the zebrafish CGBP cDNA. Zebrafish treated with a control morpholino oligonucleotide at the 1–2 cell stage exhibited normal yolk sac morphology and normal red blood cell circulation at 48 hpf. The control treated zebrafish did not have cardiac edema, and only 2–3% of these fish were dead at 24 hpf. The specificity of phenotypes observed following treatment with the CGBP morpholino oligonucleotide was assessed by co-injection of mouse CGBP mRNA with the CGBP morpholino oligonucleotide at the 1–2 cell stage. Only 37% of these fish exhibited a decrease in circulating red blood cells compared to 80% of zebrafish treated with CGBP morpholino alone. While 14% of the CGBP morpholino treated zebrafish were dead at 24 hpf, only 4% of the co-injected zebrafish were dead at 24 hpf. This experiment demonstrates that the mouse CGBP is able to rescue the phenotypes induced by a reduction of the endogenous zebrafish CGBP. These data indicate that CGBP is essential for normal zebrafish hematopoiesis and establishes the importance of CGBP in post-gastrulation development.

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