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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

Preliminary model of human CARP X.The model showing a disulfide bridge between conserved cysteines C60 and C223 (marked in red).
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pone.0134263.g003: Preliminary model of human CARP X.The model showing a disulfide bridge between conserved cysteines C60 and C223 (marked in red).

Mentions: All 61 CARP protein sequences in our study possess three conserved cysteines, corresponding to the positions 60, 244, and 310 in human CARP X. In addition, there is a fourth cysteine residue (296 in human CARP X) which is conserved in CARP X in tetrapods, coelacanth (Latimeria), the CARPs in lamprey, and CARP Xa and Xb. However, the fourth cysteine residue is not found in CARP XI or in the invertebrate CARP X-like proteins. In addition, there are several non-conserved cysteines in the predicted signal peptide regions. The conserved cysteines C60 and C223 are predicted to be within the CA domain. Their positions in the sequence alignment, and preliminary protein model correspond approximately to the position of the conserved disulfide seen in the structures of CA IV, VI, IX, XII, and XIV, and in sequence alignments of CA XV and XVII [4], but which is absent from other, intracellular CA isoforms (Fig 3) A disulfide with a reasonable geometry could be formed easily, even though the model displaces the first cysteine by one turn of a helix relative to the position seen in other CAs.


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)

Preliminary model of human CARP X.The model showing a disulfide bridge between conserved cysteines C60 and C223 (marked in red).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134263.g003: Preliminary model of human CARP X.The model showing a disulfide bridge between conserved cysteines C60 and C223 (marked in red).
Mentions: All 61 CARP protein sequences in our study possess three conserved cysteines, corresponding to the positions 60, 244, and 310 in human CARP X. In addition, there is a fourth cysteine residue (296 in human CARP X) which is conserved in CARP X in tetrapods, coelacanth (Latimeria), the CARPs in lamprey, and CARP Xa and Xb. However, the fourth cysteine residue is not found in CARP XI or in the invertebrate CARP X-like proteins. In addition, there are several non-conserved cysteines in the predicted signal peptide regions. The conserved cysteines C60 and C223 are predicted to be within the CA domain. Their positions in the sequence alignment, and preliminary protein model correspond approximately to the position of the conserved disulfide seen in the structures of CA IV, VI, IX, XII, and XIV, and in sequence alignments of CA XV and XVII [4], but which is absent from other, intracellular CA isoforms (Fig 3) A disulfide with a reasonable geometry could be formed easily, even though the model displaces the first cysteine by one turn of a helix relative to the position seen in other CAs.

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