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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

The ca10a and ca10b genes play an important role during embryonic development in zebrafish.Left panel: Developmental images of ca10a and ca10b morphant zebrafish injected with two different sets of antisense MOs (MO1 and MO2) over the period of 0 hpf to 5 dpf. A) The top row images show 0 hpf embryos and lateral views of 1–5 dpf uninjected zebrafish larvae with a normal development of organs,. B)ca10a morphant zebrafish injected with 200 μM translational blocking antisense morpholinos (MO1). C) Images of ca10b morphant zebrafish. D) and E) Images of ca10a and ca10b morphants. The images in first panel of D and E show the 1 dpf larvae with rhodamine fluorescence throughout the larvae (indicating successful MO injection) and second panel (D and E) shows the 5 dpf larvae with abnormal appearance. Right panel: Developmental images of ca10a and ca10b CRISPR mutated fish and gRNA controls. F) The top row images show 0 dpf embryos and lateral views of 1–5 dpf gRNA injected zebrafish larvae (control) with a normal development of organs. G)ca10a mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 3 (target site shown in Fig 7B). H)ca10b mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 1(target site shown in Fig 7C).
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pone.0134263.g008: The ca10a and ca10b genes play an important role during embryonic development in zebrafish.Left panel: Developmental images of ca10a and ca10b morphant zebrafish injected with two different sets of antisense MOs (MO1 and MO2) over the period of 0 hpf to 5 dpf. A) The top row images show 0 hpf embryos and lateral views of 1–5 dpf uninjected zebrafish larvae with a normal development of organs,. B)ca10a morphant zebrafish injected with 200 μM translational blocking antisense morpholinos (MO1). C) Images of ca10b morphant zebrafish. D) and E) Images of ca10a and ca10b morphants. The images in first panel of D and E show the 1 dpf larvae with rhodamine fluorescence throughout the larvae (indicating successful MO injection) and second panel (D and E) shows the 5 dpf larvae with abnormal appearance. Right panel: Developmental images of ca10a and ca10b CRISPR mutated fish and gRNA controls. F) The top row images show 0 dpf embryos and lateral views of 1–5 dpf gRNA injected zebrafish larvae (control) with a normal development of organs. G)ca10a mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 3 (target site shown in Fig 7B). H)ca10b mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 1(target site shown in Fig 7C).

Mentions: The 1 dpf zebrafish larvae injected with 200 μM ca10a-MO1 showed defects in the head, abnormal body structure, and small eyes with a delayed hatching from the chorion (Fig 8B). As the development progressed, the abnormalities became more prominent; the larvae had long tapering curved tails, curved body structure, pericardial edema, absence of swim bladder, and otolith vesicles (Fig 8B). The embryos injected with ca10a-MO2 showed a less severe phenotype compared with the ca10a-MO1 injected larvae (Fig 8D). Injection of a higher concentration (300 μM) of ca10a-MO led to a more severe phenotype compared to the lower dose with higher mortality in the morphant larvae after 24 hpf (Fig 9 and Table 6). Further increase in the MO concentration (above 300 μM) had lethal effect on the larvae.


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)

The ca10a and ca10b genes play an important role during embryonic development in zebrafish.Left panel: Developmental images of ca10a and ca10b morphant zebrafish injected with two different sets of antisense MOs (MO1 and MO2) over the period of 0 hpf to 5 dpf. A) The top row images show 0 hpf embryos and lateral views of 1–5 dpf uninjected zebrafish larvae with a normal development of organs,. B)ca10a morphant zebrafish injected with 200 μM translational blocking antisense morpholinos (MO1). C) Images of ca10b morphant zebrafish. D) and E) Images of ca10a and ca10b morphants. The images in first panel of D and E show the 1 dpf larvae with rhodamine fluorescence throughout the larvae (indicating successful MO injection) and second panel (D and E) shows the 5 dpf larvae with abnormal appearance. Right panel: Developmental images of ca10a and ca10b CRISPR mutated fish and gRNA controls. F) The top row images show 0 dpf embryos and lateral views of 1–5 dpf gRNA injected zebrafish larvae (control) with a normal development of organs. G)ca10a mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 3 (target site shown in Fig 7B). H)ca10b mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 1(target site shown in Fig 7C).
© Copyright Policy
Related In: Results  -  Collection

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pone.0134263.g008: The ca10a and ca10b genes play an important role during embryonic development in zebrafish.Left panel: Developmental images of ca10a and ca10b morphant zebrafish injected with two different sets of antisense MOs (MO1 and MO2) over the period of 0 hpf to 5 dpf. A) The top row images show 0 hpf embryos and lateral views of 1–5 dpf uninjected zebrafish larvae with a normal development of organs,. B)ca10a morphant zebrafish injected with 200 μM translational blocking antisense morpholinos (MO1). C) Images of ca10b morphant zebrafish. D) and E) Images of ca10a and ca10b morphants. The images in first panel of D and E show the 1 dpf larvae with rhodamine fluorescence throughout the larvae (indicating successful MO injection) and second panel (D and E) shows the 5 dpf larvae with abnormal appearance. Right panel: Developmental images of ca10a and ca10b CRISPR mutated fish and gRNA controls. F) The top row images show 0 dpf embryos and lateral views of 1–5 dpf gRNA injected zebrafish larvae (control) with a normal development of organs. G)ca10a mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 3 (target site shown in Fig 7B). H)ca10b mutated zebrafish injected with gRNA and cas9 mRNA targeted to exon 1(target site shown in Fig 7C).
Mentions: The 1 dpf zebrafish larvae injected with 200 μM ca10a-MO1 showed defects in the head, abnormal body structure, and small eyes with a delayed hatching from the chorion (Fig 8B). As the development progressed, the abnormalities became more prominent; the larvae had long tapering curved tails, curved body structure, pericardial edema, absence of swim bladder, and otolith vesicles (Fig 8B). The embryos injected with ca10a-MO2 showed a less severe phenotype compared with the ca10a-MO1 injected larvae (Fig 8D). Injection of a higher concentration (300 μM) of ca10a-MO led to a more severe phenotype compared to the lower dose with higher mortality in the morphant larvae after 24 hpf (Fig 9 and Table 6). Further increase in the MO concentration (above 300 μM) had lethal effect on the larvae.

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