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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]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.01.013 Byline: Fumitaka Inoue, Mst. Shahnaj Parvin, Kyo Yamasu Keywords: Brain formation; Conserved sequence; Enhancer; fgf8; Isthmus; Midbrain-hindbrain boundary; Pax2; Transcription; Transgenic fish; Zebrafish Abstract: Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator. Author Affiliation: Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan Article History: Received 7 November 2007; Revised 18 December 2007; Accepted 5 January 2008

Egg activation at fertilization requires the release of [Ca.sup.2+] from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an ~58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of [Ca.sup.2+] at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and [Ca.sup.2+] release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLC[gamma]-dependent [Ca.sup.2+] release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLC[gamma]-dependent [Ca.sup.2+] release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause [Ca.sup.2+] release at fertilization and is capable of stimulating [Ca.sup.2+] release in the unfertilized egg via PLC% as at fertilization. Keywords: Fertilization; Src family kinase; Calcium; Egg activation

Development of the adult rudiment and the adult-type skeleton and the resorption of larval tissues of the sea urchin involves the action of thyroid hormone (THs), found in the larval body and the algae on which the larvae feed. THs accumulate after the growth of the larva and attain peak levels at the eight-armed phase when the adult rudiment is finished. THs accumulation in the larval body influences larval growth.

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.