Tomoya Kotani

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.

Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.

Involvement of Xenopus Pumilio in the translational regulation that is specific to cyclin B1 mRNA during oocyte maturation

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation.

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.

Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules

Temporally regulated formation of RNA granules containing cyclin B1 transcript is critical for the precise timing of translational activation.

misty somites, a maternal effect gene identified by transposon-mediated insertional mutagenesis in zebrafish that is essential for the somite boundary maintenance

Somite boundary formation is crucial for segmentation of vertebrate somites and vertebrae and skeletal muscle morphogenesis. Previously, we developed a Tol2 transposon-mediated gene trap method in zebrafish. In the present study, we aimed to isolate transposon insertions that trap maternally-expressed genes. We found that homozygous female fish carrying a transposon insertion within a maternally-expressed gene misty somites (mys) produced embryos that showed obscure somite boundaries at the early segmentation stage (12-13 hpf). The somite boundaries became clear and distinct after this period and the embryos survived to adulthood. This phenotype was rescued by expression of mys cDNA in the homozygous adults, confirming that it was caused by a decreased mys activity. We analyzed a role of the mys gene by using morpholino oligonucleotides (MOs). The MO-injected embryo exhibited severer phenotypes than the insertional mutant probably because the mys gene was partially active in the insertional mutant. The MO-injected embryo also showed the obscure somite boundary phenotype. Fibronectin and phosphorylated FAK at the intersomitic regions were accumulated at the boundaries at this stage, but, unlike wild type embryos, somitic cells adjacent to the boundaries did not undergo epithelialization, suggesting that Mys is required for epithelialization of the somitic cells. Then in the MO-injected embryos, the boundaries once became clear and distinct, but, in the subsequent stages, disappeared, resulting in abnormal muscle morphogenesis. Accumulation of Fibronectin and phosphorylated FAK observed in the initial stage also disappeared. Thus, Mys is crucial for maintenance of the somite boundaries formed at the initial stage. To analyze the mys defect at the cellular level, we placed cells dissociated from the MO-injected embryo on Fibronectin-coated glasses. By this cell spreading assay, we found that the mys-deficient cells reduced the activity to form lamellipodia on Fibronectin while FAK was activated in these cells. Thus, we demonstrate that a novel gene misty somites is essential for epithelialization of the somitic cells and maintenance of the somite boundary. Furthermore, Mys may play a role in a cellular pathway leading to lamellipodia formation in response to the Fibronectin signaling. We propose that the Tol2 transposon mediated gene trap method is powerful to identify a novel gene involved in vertebrate development.

Possible involvement of Nemo-like kinase 1 in Xenopus oocyte maturation as a kinase responsible for Pumilio1, Pumilio2, and CPEB phosphorylation.

Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.

A Genomic Region Transcribed Into a Long Noncoding RNA Interacts With the Prss42/Tessp-2 Promoter in Spermatocytes During Mouse Spermatogenesis, and Its Flanking Sequences Can Function as Enhancers

Spermatogenesis is regulated by many meiotic stage‐specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2‐F3, and the three genes at this site (Prss42/Tessp‐2, Prss43/Tessp‐3, and Prss44/Tessp‐4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell‐specific at or close to the Prss42/Tessp‐2 promoter, and a testis‐specific lncRNA, lncRNA‐HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp‐2 gene. lncRNA‐HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage‐X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA‐HSVIII locus specifically interacted with the Prss42/Tessp‐2 promoter in primary and secondary spermatocytes. A 5.8‐kb genome sequence, encompassing the entire lncRNA‐HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp‐2 promoter activity using a reporter‐gene assay, yet this construct did not change lncRNA‐HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstream and downstream regions of the lncRNA‐HSVIII sequence significantly increased Prss42/Tessp‐2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA‐HSVIII locus with the Prss42/Tessp‐2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp‐2 gene. Mol. Reprod. Dev. 83: 541–557, 2016.

A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing.

Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturation-inducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation.

Real‐time imaging of actin filaments in the zebrafish oocyte and embryo

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real‐time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C‐terminus of Moesin, an actin filament‐binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP‐MoeC), under the control of a cyclin B1 promoter. GFP/RFP‐MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula‐stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP‐MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full‐grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.