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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

Knockdown of ca10a and ca10b genes leads to apoptosis in the morphant zebrafish.Results of the TUNEL assay detecting apoptotic cells in 5 dpf morphant embryos. A) head region of a ca10a morphant; B) eye region of a ca10a morphant; C) head region of a ca10b morphant; and D) tail regions of a ca10b morphant. (Original magnification 100X).
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pone.0134263.g010: Knockdown of ca10a and ca10b genes leads to apoptosis in the morphant zebrafish.Results of the TUNEL assay detecting apoptotic cells in 5 dpf morphant embryos. A) head region of a ca10a morphant; B) eye region of a ca10a morphant; C) head region of a ca10b morphant; and D) tail regions of a ca10b morphant. (Original magnification 100X).

Mentions: The TUNEL assay on sections of ca10a-MO2 injected 5 dpf zebrafish larvae showed apoptotic cells especially in the head and eye regions (Fig 10A and 10B). Similarly, large areas of apoptotic cells were observed in the head region of 5 dpf ca10b-MO2 morphant zebrafish larvae (Fig 10C) and weaker signals were seen in the tail region (Fig 10D). The TUNEL assay did not show any signs of apoptosis in wild-type larvae or larvae injected with RC-MOs (data not shown).


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)

Knockdown of ca10a and ca10b genes leads to apoptosis in the morphant zebrafish.Results of the TUNEL assay detecting apoptotic cells in 5 dpf morphant embryos. A) head region of a ca10a morphant; B) eye region of a ca10a morphant; C) head region of a ca10b morphant; and D) tail regions of a ca10b morphant. (Original magnification 100X).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4539348&req=5

pone.0134263.g010: Knockdown of ca10a and ca10b genes leads to apoptosis in the morphant zebrafish.Results of the TUNEL assay detecting apoptotic cells in 5 dpf morphant embryos. A) head region of a ca10a morphant; B) eye region of a ca10a morphant; C) head region of a ca10b morphant; and D) tail regions of a ca10b morphant. (Original magnification 100X).
Mentions: The TUNEL assay on sections of ca10a-MO2 injected 5 dpf zebrafish larvae showed apoptotic cells especially in the head and eye regions (Fig 10A and 10B). Similarly, large areas of apoptotic cells were observed in the head region of 5 dpf ca10b-MO2 morphant zebrafish larvae (Fig 10C) and weaker signals were seen in the tail region (Fig 10D). The TUNEL assay did not show any signs of apoptosis in wild-type larvae or larvae injected with RC-MOs (data not shown).

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