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Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish.

Aspatwar A, Tolvanen ME, Ojanen MJ, Barker HR, Saralahti AK, Bäuerlein CA, Ortutay C, Pan P, Kuuslahti M, Parikka M, Rämet M, Parkkila S - PLoS ONE (2015)

Bottom Line: The biological role of these proteins is still an enigma.The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system.In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech, University of Tampere, Tampere, Finland; School of Medicine, University of Tampere, Tampere, Finland.

ABSTRACT
Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.

No MeSH data available.


Related in: MedlinePlus

Silencing of ca10a and ca10b in zebrafish larvae.A) Schematic presentation of matured ca10a mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). B) Schematic structure of unprocessed mRNA for ca10a with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. C) Schematic depiction of unprocessed mRNA for ca10b and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). D) Gel electrophoresis showing RT-PCR analysis of ca10a morphant mRNA injected with MO2. E) RT-PCR gel image of ca10b morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in D and Lane 3 and 4 in E) compared with wild type mRNAs of ca10a and ca10b in wild type fish (Lane 3 in D and lane 2 E). F) The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both ca10a and ca10b, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.
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pone.0134263.g007: Silencing of ca10a and ca10b in zebrafish larvae.A) Schematic presentation of matured ca10a mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). B) Schematic structure of unprocessed mRNA for ca10a with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. C) Schematic depiction of unprocessed mRNA for ca10b and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). D) Gel electrophoresis showing RT-PCR analysis of ca10a morphant mRNA injected with MO2. E) RT-PCR gel image of ca10b morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in D and Lane 3 and 4 in E) compared with wild type mRNAs of ca10a and ca10b in wild type fish (Lane 3 in D and lane 2 E). F) The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both ca10a and ca10b, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.

Mentions: Evolutionary conservation of CA10-like genes, their ubiquitous expression pattern in different tissues, and high mRNA levels during embryonic development suggest a crucial role for CARP X-like proteins in vertebrates [41]. To shed light to the biological significance of CARP X-like proteins in embryogenesis, we silenced zebrafish ca10a and ca10b genes with MOs (Fig 7A, 7B and 7C).


Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish.

Aspatwar A, Tolvanen ME, Ojanen MJ, Barker HR, Saralahti AK, Bäuerlein CA, Ortutay C, Pan P, Kuuslahti M, Parikka M, Rämet M, Parkkila S - PLoS ONE (2015)

Silencing of ca10a and ca10b in zebrafish larvae.A) Schematic presentation of matured ca10a mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). B) Schematic structure of unprocessed mRNA for ca10a with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. C) Schematic depiction of unprocessed mRNA for ca10b and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). D) Gel electrophoresis showing RT-PCR analysis of ca10a morphant mRNA injected with MO2. E) RT-PCR gel image of ca10b morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in D and Lane 3 and 4 in E) compared with wild type mRNAs of ca10a and ca10b in wild type fish (Lane 3 in D and lane 2 E). F) The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both ca10a and ca10b, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4539348&req=5

pone.0134263.g007: Silencing of ca10a and ca10b in zebrafish larvae.A) Schematic presentation of matured ca10a mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). B) Schematic structure of unprocessed mRNA for ca10a with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. C) Schematic depiction of unprocessed mRNA for ca10b and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). D) Gel electrophoresis showing RT-PCR analysis of ca10a morphant mRNA injected with MO2. E) RT-PCR gel image of ca10b morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in D and Lane 3 and 4 in E) compared with wild type mRNAs of ca10a and ca10b in wild type fish (Lane 3 in D and lane 2 E). F) The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both ca10a and ca10b, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.
Mentions: Evolutionary conservation of CA10-like genes, their ubiquitous expression pattern in different tissues, and high mRNA levels during embryonic development suggest a crucial role for CARP X-like proteins in vertebrates [41]. To shed light to the biological significance of CARP X-like proteins in embryogenesis, we silenced zebrafish ca10a and ca10b genes with MOs (Fig 7A, 7B and 7C).

Bottom Line: The biological role of these proteins is still an enigma.The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system.In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech, University of Tampere, Tampere, Finland; School of Medicine, University of Tampere, Tampere, Finland.

ABSTRACT
Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.

No MeSH data available.


Related in: MedlinePlus