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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 ca10b and ca10b genes results in abnormal movement pattern in morphant zebrafish larvae.The swim patterns of 5 dpf larvae injected with 100 μM antisense ca10a-MO2 or ca10b-MO2 were compared to the larvae injected with RC-MO and wildtype (uninjected) controls as described in the methods. (In addition to the swim patterns and body movement videos of 10 larvae from each group created with a Zeiss microscope for close observation as included in the supporting information). Distances traveled by 457 fish in the four groups are presented as boxplots as created with matplotlib [43]. The results of two sample Kolmogorov-Smirnov statistical analyses are included between bracket linked groups.
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pone.0134263.g013: Knockdown of ca10b and ca10b genes results in abnormal movement pattern in morphant zebrafish larvae.The swim patterns of 5 dpf larvae injected with 100 μM antisense ca10a-MO2 or ca10b-MO2 were compared to the larvae injected with RC-MO and wildtype (uninjected) controls as described in the methods. (In addition to the swim patterns and body movement videos of 10 larvae from each group created with a Zeiss microscope for close observation as included in the supporting information). Distances traveled by 457 fish in the four groups are presented as boxplots as created with matplotlib [43]. The results of two sample Kolmogorov-Smirnov statistical analyses are included between bracket linked groups.

Mentions: The expression pattern of ca10a and ca10b genes suggested that the encoded proteins play important roles in the brain. Therefore, we hypothesized that down regulation of these genes with antisense MOs may lead to changes in behavior or motor coordination, which can be investigated by monitoring the swim pattern of the morphant embryos. It is obvious that the larvae with body and tail abnormalities, induced by high concentration of ca10a and ca10b antisense morpholinos, would show abnormal swimming behavior. To see any neurological effect on movement pattern as a result of down regulation of these genes, we needed morphant larvae with no obvious phenotypic defects. To test this hypothesis, we injected the larvae with lower concentrations (100 μM) of ca10a-MO2 and ca10b-MO2. With these concentrations, the 5 dpf larvae showed normal body and tail development with no visible phenotypic defects, similar to RC-MO injected and uninjected larvae (S1–S3 Movies). In the experiment, 4 groups (uninjected, 100 μM ca10a-MO injected, 100 μM ca10b-MO injected, and RC injected embryos) of larvae were analyzed for movement patterns to observe movement of the body while swimming and traced the movement of the fish (Fig 12). Similarly, we calculated the total distance traveled for all larvae (details in the methods). Two-sample Kolmogorov-Smirnov statistical analyses were performed between each of the groups to determine if they could have been drawn from the same distribution, shown in Fig 13. Both knockdown groups swam significantly smaller distances than the random control and wildtype groups. The distances of the two knockdown groups did not differ significantly. However, fish in the ca10b group showed a tendency towards less movement (Fig 13). The distances traveled by fish in the random control group were smaller than those of the wildtype controls. We assume that either the impact of the injection procedure or the MOs themselves account for this difference.


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 ca10b and ca10b genes results in abnormal movement pattern in morphant zebrafish larvae.The swim patterns of 5 dpf larvae injected with 100 μM antisense ca10a-MO2 or ca10b-MO2 were compared to the larvae injected with RC-MO and wildtype (uninjected) controls as described in the methods. (In addition to the swim patterns and body movement videos of 10 larvae from each group created with a Zeiss microscope for close observation as included in the supporting information). Distances traveled by 457 fish in the four groups are presented as boxplots as created with matplotlib [43]. The results of two sample Kolmogorov-Smirnov statistical analyses are included between bracket linked groups.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134263.g013: Knockdown of ca10b and ca10b genes results in abnormal movement pattern in morphant zebrafish larvae.The swim patterns of 5 dpf larvae injected with 100 μM antisense ca10a-MO2 or ca10b-MO2 were compared to the larvae injected with RC-MO and wildtype (uninjected) controls as described in the methods. (In addition to the swim patterns and body movement videos of 10 larvae from each group created with a Zeiss microscope for close observation as included in the supporting information). Distances traveled by 457 fish in the four groups are presented as boxplots as created with matplotlib [43]. The results of two sample Kolmogorov-Smirnov statistical analyses are included between bracket linked groups.
Mentions: The expression pattern of ca10a and ca10b genes suggested that the encoded proteins play important roles in the brain. Therefore, we hypothesized that down regulation of these genes with antisense MOs may lead to changes in behavior or motor coordination, which can be investigated by monitoring the swim pattern of the morphant embryos. It is obvious that the larvae with body and tail abnormalities, induced by high concentration of ca10a and ca10b antisense morpholinos, would show abnormal swimming behavior. To see any neurological effect on movement pattern as a result of down regulation of these genes, we needed morphant larvae with no obvious phenotypic defects. To test this hypothesis, we injected the larvae with lower concentrations (100 μM) of ca10a-MO2 and ca10b-MO2. With these concentrations, the 5 dpf larvae showed normal body and tail development with no visible phenotypic defects, similar to RC-MO injected and uninjected larvae (S1–S3 Movies). In the experiment, 4 groups (uninjected, 100 μM ca10a-MO injected, 100 μM ca10b-MO injected, and RC injected embryos) of larvae were analyzed for movement patterns to observe movement of the body while swimming and traced the movement of the fish (Fig 12). Similarly, we calculated the total distance traveled for all larvae (details in the methods). Two-sample Kolmogorov-Smirnov statistical analyses were performed between each of the groups to determine if they could have been drawn from the same distribution, shown in Fig 13. Both knockdown groups swam significantly smaller distances than the random control and wildtype groups. The distances of the two knockdown groups did not differ significantly. However, fish in the ca10b group showed a tendency towards less movement (Fig 13). The distances traveled by fish in the random control group were smaller than those of the wildtype controls. We assume that either the impact of the injection procedure or the MOs themselves account for this difference.

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