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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

Expression analysis of ca10a and ca10b mRNAs during embryonic development.The expression levels of ca10a and ca10b were measured from the total mRNA isolated from the developing larvae of 0–168 hpf. A) Expression values of ca10a mRNA. B) Expression values of ca10b mRNA. The expression values were normalized to the beta actin control according to the equation of Pfaffl [34]. The expression of ca10a gene at 0 hpf and ca10b at 96 hpf assigned a relative value of 1.
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pone.0134263.g006: Expression analysis of ca10a and ca10b mRNAs during embryonic development.The expression levels of ca10a and ca10b were measured from the total mRNA isolated from the developing larvae of 0–168 hpf. A) Expression values of ca10a mRNA. B) Expression values of ca10b mRNA. The expression values were normalized to the beta actin control according to the equation of Pfaffl [34]. The expression of ca10a gene at 0 hpf and ca10b at 96 hpf assigned a relative value of 1.

Mentions: In addition, we used RT-qPCR to investigate the expression of ca10a and ca10b during the development of 0–168 hpf (0–7 dpf) zebrafish larvae (Fig 6). Expression of both ca10a and ca10b were highest at 96 hpf (4 hpf) and remained high thereafter. Low levels of ca10a and ca10b expression was seen at earlier time points. The expression pattern suggests that the ca10a gene product is required throughout embryonic development, especially after 72 hpf (3 dpf). Interestingly, ca10b mRNA was present at a very high level at 0 hpf, suggesting maternal origin (Fig 6B) and this is also supported by the high expression in the ovary of the adult fish (Fig 5B). The expression pattern of the ca10b gene suggests that this gene plays a role at the beginning of development and is required throughout embryonic development in zebrafish.


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)

Expression analysis of ca10a and ca10b mRNAs during embryonic development.The expression levels of ca10a and ca10b were measured from the total mRNA isolated from the developing larvae of 0–168 hpf. A) Expression values of ca10a mRNA. B) Expression values of ca10b mRNA. The expression values were normalized to the beta actin control according to the equation of Pfaffl [34]. The expression of ca10a gene at 0 hpf and ca10b at 96 hpf assigned a relative value of 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134263.g006: Expression analysis of ca10a and ca10b mRNAs during embryonic development.The expression levels of ca10a and ca10b were measured from the total mRNA isolated from the developing larvae of 0–168 hpf. A) Expression values of ca10a mRNA. B) Expression values of ca10b mRNA. The expression values were normalized to the beta actin control according to the equation of Pfaffl [34]. The expression of ca10a gene at 0 hpf and ca10b at 96 hpf assigned a relative value of 1.
Mentions: In addition, we used RT-qPCR to investigate the expression of ca10a and ca10b during the development of 0–168 hpf (0–7 dpf) zebrafish larvae (Fig 6). Expression of both ca10a and ca10b were highest at 96 hpf (4 hpf) and remained high thereafter. Low levels of ca10a and ca10b expression was seen at earlier time points. The expression pattern suggests that the ca10a gene product is required throughout embryonic development, especially after 72 hpf (3 dpf). Interestingly, ca10b mRNA was present at a very high level at 0 hpf, suggesting maternal origin (Fig 6B) and this is also supported by the high expression in the ovary of the adult fish (Fig 5B). The expression pattern of the ca10b gene suggests that this gene plays a role at the beginning of development and is required throughout embryonic development in zebrafish.

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