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

Nourizadeh-Lillabadi R, Seilø Torgersen J, Vestrheim O, König M, Aleström P, Syed M - PLoS ONE (2010)

Bottom Line: The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway.

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.

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Related in: MedlinePlus

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.
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pone-0013573-g002: 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.

Mentions: Optimization of the two MOs specific for the 5′UTR of GenBank sequence AJ620614.1 (Fig. 1) injections revealed that 0.2 mM prp2-MO2 resulted in 71% mortality at 24 hpf and 74% at 48 hpf. In order to reduce the mortality, 0.15 mM prp2-MO2 was chosen because it led to a mortality rate below 50%. Phase contrast microscopy of Prp2 morphants revealed a clear phenotype with midbrain and hindbrain developmental defects as compared to the control group (Fig. 2 B–D). In order to further characterize the phenotypic effects, wholemount immunofluorescence was carried out using antibodies against Prp2 and HNK-1. Fluorescence microscopy analysis of the wild type embryos at 24 hpf unveiled low but significant levels of Prp2 proteins in the telencephalon, eye and trigeminal ganglion (Fig. 3A). Investigation of HNK-1 showed strong staining to the trigeminal ganglion and neuronal extensions (Fig. 3B). In the morphants no Prp2 activity could be detected and the Zn12 antibody visualized altered trigeminal ganglion morphology and reduced number of peripheral neurons in the head (Fig. 3C). Also, nuclear staining with DAP1 revealed apoptotic cells with hyper-condensed nuclei in the trigeminal ganglion, eye, olfactory placode, and in the midbrain-hindbrain area (Fig. 3D).


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

Nourizadeh-Lillabadi R, Seilø Torgersen J, Vestrheim O, König M, Aleström P, Syed M - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962645&req=5

pone-0013573-g002: 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.
Mentions: Optimization of the two MOs specific for the 5′UTR of GenBank sequence AJ620614.1 (Fig. 1) injections revealed that 0.2 mM prp2-MO2 resulted in 71% mortality at 24 hpf and 74% at 48 hpf. In order to reduce the mortality, 0.15 mM prp2-MO2 was chosen because it led to a mortality rate below 50%. Phase contrast microscopy of Prp2 morphants revealed a clear phenotype with midbrain and hindbrain developmental defects as compared to the control group (Fig. 2 B–D). In order to further characterize the phenotypic effects, wholemount immunofluorescence was carried out using antibodies against Prp2 and HNK-1. Fluorescence microscopy analysis of the wild type embryos at 24 hpf unveiled low but significant levels of Prp2 proteins in the telencephalon, eye and trigeminal ganglion (Fig. 3A). Investigation of HNK-1 showed strong staining to the trigeminal ganglion and neuronal extensions (Fig. 3B). In the morphants no Prp2 activity could be detected and the Zn12 antibody visualized altered trigeminal ganglion morphology and reduced number of peripheral neurons in the head (Fig. 3C). Also, nuclear staining with DAP1 revealed apoptotic cells with hyper-condensed nuclei in the trigeminal ganglion, eye, olfactory placode, and in the midbrain-hindbrain area (Fig. 3D).

Bottom Line: The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway.

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.

Show MeSH
Related in: MedlinePlus