BackgroundWe have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.DescriptionTo increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish.ConclusionzTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

SummaryThe blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

We studied planktonic bacterial population dynamics in response to the changing environment in a coastal system during an observation period of over 5 years using fluorescence in situ hybridization. To estimate the environmental constraint on the bacterial community, we focused on temperature, salinity, abundance of photoplankton (chlorophyll a), and dissolved organic carbon (DOC). The total number of bacteria (TDC) amounted to 3.0×10 5 to 5.0×10 6 cells mL −1 , with 1.0×10 5 to 1.0×10 6 cells mL −1 for Bacteria, accounting for 11.8 to 74.8% of TDC, and 1.0×10 4 to 1.0×10 5 cells mL −1 for Gammaproteobacteria, 1.0 to 20.8% of TDC. The abundance of Archaea, which contributed from 0.1 to 12% to TDC, ranged from 2.0×10 3 to 3.0×10 4 cells mL −1 . We found a positive relationship between environmental parameters such as temperature, salinity, chlorophyll a, and DOC and the abundance of total bacteria and Bacteria. The number of Gammaproteobacteria correlated with temperature, salinity, and chlorophyll a, but not with DOC. We suggest that increasing the temperature under eutrophic conditions will lead to high bacterial abundance and probably a change in the bacterial community.

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