Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish

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.

Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand

“…Mature CA10 contains four cysteines, of which the conserved Cys60 and Cys244 are predicted to form an intramolecular disulfide bridge (26). We thus tested the role of the two other, more C-terminal cysteines, Cys296 and Cys310, in neurexin binding.…”

“…Very little is known at present about CA10 and/or CA11, apart from studies in zebrafish using morpholino-and CRISPRmediated disruption of the zebrafish orthologs ca10a and ca10b, which resulted in developmental abnormalities and mortality of unknown mechanism (26). However, these results do not reveal the actual functions of CA10 and CA11.…”

Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

“…Patients with mutations in CA8 show phenotype in cerebellar ataxia, mental retardation, and disequilibrium syndrome [19], while CA8 −/− mice exhibit motor dysfunction and altered calcium dynamics in cerebellar granule cells [39]. Inactivation of CA10 in zebrafish leads to abnormal embryonic development and altered movement pattern [40]. …”

Regulatory network underlying alterations of gene expression in early death of Huntington's disease

“…It mediated the interaction between neurexins and certain postsynaptic molecules, thereby facilitating the formation of novel trans-synaptic complexes [40]. Evidence in zebra sh using morpholino-mediated knockdown of CA10 showed developmental retardation, aberrant behavior and early death under unknown mechanisms of CA10 de ciency [41]. Herein, the network analysis exhibited multiple indirect interactions of CA10 with the center hub gene GABRB3, suggesting that GABRB3-mediated GABAergic transmission was possibly modulated by CA10 through its adaptor function.…”