Search

Background: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4‐type gene, pou5f3, in this process in zebrafish mainly by dominant‐interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question. Results: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen‐ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud‐related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down‐regulated in embryos with disrupted pou5f3 activity. Conclusions: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain‐of‐function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube. Key Findings: We confirmed possible involvement of Oct4‐type POU transcription factor (PouV) in elongation of the spinal cord in zebrafish embryos.Phenotypes of pou5f3 mutation and pou5f3 activation were assessed in the neural tube.pou5f3 regulates retinoic acid metabolism, contributing to neurogenesis in the posterior neural tube. [ABSTRACT FROM AUTHOR]

In the vertebrate body, a metameric structure is present along the anterior–posterior axis. Zebrafish tbx6−/− larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three‐dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6−/− larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two‐colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6−/−. The parapophyses in tbx6−/− juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish. [ABSTRACT FROM AUTHOR]

In the zebrafish segmentation clock, hairy/enhancer of split‐related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy‐related mRNAs are subject to strict post‐transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4‐NOT deadenylase complex lengthens poly(A) tails of hairy‐related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFPmRNAs with 3′UTRs of hairy‐related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4‐NOT deadenylase complex possibly through their 3′UTRs in the zebrafish PSM. [ABSTRACT FROM AUTHOR]

Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.