Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg

Peroxiredoxin5 controls vertebrate ciliogenesis by modulating mitochondrial ROS

Aims: Peroxiredoxin5 (Prdx5) - a thioredoxin peroxidase is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. The present study is aimed to investigate the functional significance of Prdx5 in mitochondria and to analyze its roles in ciliogenesis during the process of vertebrate development. Results: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular ROS, consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation upon Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced upon Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells. Innovation: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion resulting in excessive ROS levels suggesting cilia as vulnerable targets of ROS. Conclusion: Prdx5 play protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS resulting in mitochondrial dysfunction and aberrant ciliogenesis