Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping

Abstract

Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies. design of intersectional genetic experiments. However, ZBB software required local installation and only included 9 Cre lines, limiting opportunities for intersectional targeting.Here, we describe the ZBB2 atlas, which comprises whole-brain expression patterns for 264 transgenic lines, including 65 Cre lines, and 158 Gal4 lines. We generated more than 100 new enhancer trap lines that express Cre or Gal4 in diverse subsets of neurons, then registered a high resolution image of each to the original ZBB atlas. For 3D visualization of expression patterns and to facilitate spatial searches for experimentally useful lines, we now provide an online interface to the atlas. Collectively, ZBB2 labels almost all cellular regions within the brain and will facilitate the reproducible targeting of neuronal subsets for circuit-mapping studies. ResultsTo accelerate discovery of functional circuits we generated a library of transgenic lines that express Cre in restricted patterns within the brain and built an online 3D atlas ( Fig. 1). New Cre lines were generated through an enhancer trap screen: we injected embryos with a Cre vector containing a basal promoter that includes a neuronal-restrictive silencing element to suppress expression outside the nervous system, and tol2 transposon arms for high efficiency transgenesis (REx2-SCP1:BGi-Cre-2a-Cer) (Bergeron et al., 2012;Kawakami, 2007;Marquart et al., 2015). In injected G0 embryos, the reporter randomly integrates into the genome such that Cre expression is directed by local enhancer elements. To isolate lines with robust brain expression in relatively restricted domains, we visually screened progeny of G0 adults crossed to the βactin:Switch transgenic line that expresses red fluorescent protein (RFP) in cells with Cre (Horstick et al., 2014). We retained 52 new lines that express Cre in restricted brain regions at 6 days post-fertilization (dpf), the stage most frequently used 4B). Gal4 lines tend to have more restricted expression than Cre lines, with a median coverage of 1% (range 0.02 to 29%, ~900 neurons per line). Collectively Gal4 lines label 91% of the cell body vol...

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