Early embryonic gene expression profiling of zebrafish prion protein (Prp2) morphants

Abstract

Background: The 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 findings: The 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/significance: The 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.

Figure 1. Morpholino sequences.

Morpholino sequences (prp2-MO1and prp2-MO2 sequences underlined) used for knockdown of the zebrafish PrP2 gene function.

Figure 2. prp2-MO2 morphant 24 hpf larvae phenotypes.

Larvae were immobilized in CyGEL prior to microscopy. A) Non-injected wt control. B–D) three individual morphants displaying defective midbrain and hindbrain development.

Figure 3. Whole mount immunofluorescence analysis of 24 hpf wild type (A and B) and Prp2 morphants (C and D).

Neuronal structures were visualized by the Zn12 antibody (Yellow), whereas Prp2 was detected with a rabbit anti serum (Red). A) In the wild type 24 hpf Prp2 is observed in the trigeminal ganglion and telencephalon, but also present in other neuronal tissue. B) Neuronal structures visualized by HNK-1 staining (Zn-12 antibody) in the same specimen as in A. C) Aberrant morphology of the trigeminal ganglion in Prp2 morphants (arrow) as well as reduced number of peripheral neurons visualized by HNK-1 staining. D) Non deconvolved single plane image of a morphant with condensed nuclei (inside stippled ring). Abbreviations: eye (e), telencephalon (t) and trigeminal ganglion (tg).

Figure 4. IPA cluster analysis of significantly differentially expressed genes.

IPA cluster analysis of significantly differentially expressed genes for 24 hpf zebrafish prp2-MO2 morphant embryos. The clusters reveal genes involved in (A) cell death and apoptosis with functions focused on apoptosis in neural cells and (B) genes involved in nervous system development. Red color indicates up- and green downregulation.

Figure 5. A network for connecting differentially expressed genes.

A network for connecting differentially expressed genes from zebrafish 24 hpf prp2-MO2 morphant embryos was identified by IPA in which PRNP, APP, heparin and EP300 occupy a central role. The uncolored protein symbols, including PRNP, are added by IPA to fill network gaps and are not among the analyzed dataset of differentially expressed genes. Red color indicates up- and green downregulation. A significant prp2 mRNA down-regulation was experimentally demonstrated by qRT-PCR (see Fig. 6).

Figure 6. Comparison between microarray data and qRT-PCR data.

Microarray data (blue staples), qRT-PCR of RNA from 24 hpf prp2-MO2 injected embryos (red staples) and 24 hpf prp2-MO1 injected embryos (yellow staples) are presented as fold change of expression of 14 genes. Mean fold change (±SD) for qRT-PCR are based on triplicate embryo pools.