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Published on: November 13, 2013
Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in
Ashok Aspatwar1, Martti E E Tolvanen2, Markus J T Ojanen3
1BioMediTech, 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.
Insights
Carbonic anhydrase related proteins (CARPs) X and XI are crucial for zebrafish development, regulating brain and motor functions. Knocking down these genes causes developmental delays and movement disorders, establishing a new model for CARP research.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Carbonic anhydrase related proteins (CARPs) X and XI are conserved neural proteins with unknown functions.
- Ray-finned fish lack the CA11 gene but have two CA10 co-orthologs, suggesting evolutionary divergence.
- CARP X/XI-like sequences were analyzed phylogenetically to understand their evolutionary relationships.
Purpose of the Study:
- To investigate the expression patterns and developmental roles of zebrafish ca10a and ca10b genes.
- To establish a zebrafish model for studying CARP X and XI functions.
- To elucidate the conserved structural features of CARP X and XI proteins.
Main Methods:
- Sequence analysis of zebrafish ca10a and ca10b genes.
- RT-qPCR to determine gene expression in various tissues and developmental stages.
- Antisense morpholino knockdown and CRISPR/Cas9 gene inactivation to study gene function.
- Phenotypic analysis of morphant and gene-edited zebrafish, including developmental, morphological, and behavioral assessments.
Main Results:
- Zebrafish ca10a and ca10b genes encode proteins with predicted signal peptides, N-glycosylation sites, and disulfide bonds, indicating secretory and potentially dimeric functions.
- Both genes are expressed in the brain and other tissues throughout zebrafish development.
- Knockdown or knockout of ca10a and ca10b resulted in developmental delays, high mortality, curved body, pericardial edema, head and eye abnormalities, increased brain apoptosis, and abnormal swimming behavior.
Conclusions:
- Zebrafish ca10a and ca10b (CARP Xa and CARP Xb) play critical roles in embryonic development, including brain development and motor function.
- The study introduces a novel zebrafish model for investigating CARP X and XI functions.
- Suppression of CARP Xa and CARP Xb expression leads to significant developmental defects and movement disorders in zebrafish larvae.
