Transcriptomic analysis of zebrafish prion protein mutants supports conserved cross-species function of the cellular prion protein

Abstract

Cellular Prion Protein (PrP^C) is a well-studied protein as the substrate for various progressive untreatable neurodegenerative diseases. Normal functions of PrP^C are poorly understood, though recent proteomic and transcriptomic approaches have begun to reveal common themes. We use our compound prp1 and prp2 knockout mutant zebrafish at three days post fertilization to take a transcriptomic approach to investigating potentially conserved PrP^C functions during development. Gene ontology analysis shows the biological processes with the largest changes in gene expression include redox processing, transport and cell adhesion. Within these categories several different gene families were prevalent including the solute carrier proteins, cytochrome p450 enzymes and protocadherins. Continuing from previous studies identifying cell adhesion as an important function of PrP^C we found that in addition to the protocadherins there was a significant reduction in transcript abundance of both ncam1a and st8sia2. These two genes are involved in the early development of vertebrates. The alterations in cell adhesion transcripts were consistent with past findings in zebrafish and mouse prion protein mutants; however E-cadherin processing after prion protein knockdown failed to reveal any differences compared with wild type in either our double prp1/prp2 mutant fish or after prp1 morpholino knockdown. Our data supports a cross species conserved role for PrP^C in the development and maintenance of the central nervous system, particularly by regulating various and important cell adhesion processes.

Keywords: Prion knockout; RNA-sequencing; cell adhesion; scrapie; transcriptome.

Figure 1.

Prion proteins of zebrafish Prp1, Prp2, human PrP^C and Prp1^{ua5003/ua5003} and Prp2^{ua5001/ua5001} mutant proteins. Zebrafish prion protein genes are larger however have the same conserved domains as human PRNP: the repeat domain (green), hydrophobic centre region (orange), hydrophobic tail (blue), N-linked glycosylation sites (blue lines) and di-sulphide bridge (orange lines). The prp1^{ua5003/ua5003} and prp2^{ua5001/ua5001} alleles have frameshift deletions near the beginning of the coding exon, leading to a missense sequence of amino acids (yellow), pre-mature stop codons (red) and a shortened, nonsense transcript

Figure 2.

RNA-Sequencing show 1249 genes with an increase or decrease of log₂ fold change of 0.5 between wild-type and compound homozygous prion mutant zebrafish larvae. (A) Scatter graph showing relative FPKM values for wild-type (X-axis) and mutant (Y-axis) genes after RNA-sequencing. (B) Methodology diagram showing RNA-sequencing workflow. Two groups, wild-type and prion mutant (prp1^{ua5003/ua5003}; prp2^{ua5001/ua5001}) homozygous fish, each with three replicates containing a pool of 50 3dpf larvae were processed and sent for RNA-sequencing. Heatmap displays sample of the 25 genes with a biggest differential abundance in FPKM in wild-types compared to mutants. (C) Relative transcript abundance between wild-type and prion mutant fish comparing prp1 and prp2 through both RNA-sequencing and RT-qPCR analysis. D) Relative transcript abundance of ncam1a and st8sia2 through both RNA-sequencing and RT-qPCR analysis. * = P < 0.05

Figure 3.

Biological Process Gene Ontologies most affected in 3dpf prion mutant (prp1^{ua5003/ua5003}; prp2^{ua5001/ua5001}) zebrafish compared to wild type. Biological processes showing genes with the greatest increase in transcript abundance are shown on the left (red), and genes with the biggest decrease in transcript abundance are shown on the right (blue)

Figure 4.

(A–C) There does not appear to be a difference in the maturation or localization of e-cadherin in either 5ng prp1 morpholino injected AB zebrafish or uninjected prp1^{ua5003/ua5003}; prp2^{ua5001/ua5001} homozygous mutant fish compared to uninjected wild-type controls. (F–E) After 3dpf wild-type larvae injected with prp1 MO (f) show significant signs of necrosis and developmental abnormalities compared to uninjected and control injected larvae (d, e)

Figure 5.

Snapshot of the Focal Adhesion Kinase KEGG pathway. Gene products highlighted in red show those with a significant decrease in transcript abundance, with the specific gene italicized underneath