Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

Abstract

BackgroundThe Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for.Methodology/Principal FindingsThe zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development.Conclusions/SignificanceThe current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development.

Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

“…Our attempts to establish prp2 MO reagents met with some success, reproducing aspects of previous studies [27], [28]. However, these reagents failed to meet the most stringent tests of reagent specificity [29], insofar as we could not rescue the prp2 knockdown phenotypes with prp2 mRNA, consistent with previous reports [27].…”

Reverse Genetic Screening Reveals Poor Correlation between Morpholino-Induced and Mutant Phenotypes in Zebrafish

“…We also observed evidence of NMD or loss of protein in selected mutants supporting the fact that many are likely null alleles, yet we still observed a high false-positive morphant phenotype rate. Consistent with these observations, recent studies have reported inconsistencies between morphants and mutants for individual genes, including phenotypes affecting lymphatic development (nr2f2, Aranguren et al, 2011; Swift et al, 2014; van Impel et al, 2014), neuronal function ( mfn2 and prp2 , Chapman et al, 2013; Fleisch et al, 2013; Nourizadeh-Lillabadi et al, 2010; Vettori et al, 2011), hindbrain patterning ( gbx2 , Kikuta et al, 2003; Su et al, 2014), and lateral line development ( tcf7 , Aman et al, 2011). Taken together with our broader findings here, these studies raise a major concern about relying solely on the application of MOs for primary characterization of gene function in the zebrafish.…”

Prion protein function and the disturbance of early embryonic development in zebrafish

“…30 In zebrafish, onset of endogenous PrP expression occurs within one day post fertilization (dpf). 2,29 From recent RNAseq (RNA deep sequencing) derived early zebrafish development transcriptomes, it can be registered that PrP-2 is present as maternal RNA and persists until early gastrula (50% gastrula epiboly at 5.3 hours post fertilization hpf), while PrP-1 is not present during this very early stage of development. 31 Morpholino knock-down experiments have demonstrated that the fish prion genes are essential for embryo survival, which is contrary to findings in knockout mice.…”

“…Thus, further studies are needed on both the molecular pathology and biological function of PrP. The nomenclature for prion protein variants and the corresponding genes can be confusing 2 and in this paper we have chosen standard mammalian prion gene and protein nomenclature for mammals (PRNP to denote the gene and PrP to denote the protein). For simplicity we here use PrP and PrP to denote the zebrafish genes and proteins, respectively.…”