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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

Partial rescue of ca10a and ca10b zebrafish morphants.A) The ca10a morphant (5dpf) embryos; B) The 5 dpf zebrafish ca10a morphant embryos rescued with injection of CA10 mRNA; C) The ca10b morphant (5dpf) embryos; D) Partially rescued 5dpf embryos with CA11 mRNA.
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pone.0134263.g011: Partial rescue of ca10a and ca10b zebrafish morphants.A) The ca10a morphant (5dpf) embryos; B) The 5 dpf zebrafish ca10a morphant embryos rescued with injection of CA10 mRNA; C) The ca10b morphant (5dpf) embryos; D) Partially rescued 5dpf embryos with CA11 mRNA.

Mentions: To confirm the specificity of the phenotypes produced by antisense MOs, we co-injected the ca10a and ca10b MOs with capped human mRNAs for CA10 and CA11 genes. The morphant fish injected with 80 pg of mRNA per embryo along with 300 μM antisense MOs showed observable improvement in the phenotype of morphant embryos as shown in Fig 11A–11D. The partial rescue of ca10a and ca10b morphant embryos with the injection of gene-specific human mRNAs also confirmed the specificity of the ca10a and ca10b antisense MOs used in the study.


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)

Partial rescue of ca10a and ca10b zebrafish morphants.A) The ca10a morphant (5dpf) embryos; B) The 5 dpf zebrafish ca10a morphant embryos rescued with injection of CA10 mRNA; C) The ca10b morphant (5dpf) embryos; D) Partially rescued 5dpf embryos with CA11 mRNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134263.g011: Partial rescue of ca10a and ca10b zebrafish morphants.A) The ca10a morphant (5dpf) embryos; B) The 5 dpf zebrafish ca10a morphant embryos rescued with injection of CA10 mRNA; C) The ca10b morphant (5dpf) embryos; D) Partially rescued 5dpf embryos with CA11 mRNA.
Mentions: To confirm the specificity of the phenotypes produced by antisense MOs, we co-injected the ca10a and ca10b MOs with capped human mRNAs for CA10 and CA11 genes. The morphant fish injected with 80 pg of mRNA per embryo along with 300 μM antisense MOs showed observable improvement in the phenotype of morphant embryos as shown in Fig 11A–11D. The partial rescue of ca10a and ca10b morphant embryos with the injection of gene-specific human mRNAs also confirmed the specificity of the ca10a and ca10b antisense MOs used in the study.

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