Genetic targeting and anatomical registration of neuronal populations in the zebrafish brain with a new set of BAC transgenic tools

Abstract

Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.

Neuron types in the zebrafish optic tectum labeled by an id2b transgene

“…The id2b gene was selected based on published in situ hybridization data demonstrating sparse expression in the tectum (Thisse & Thisse, 2004). As previously described (Förster et al, 2017), the CH211-175H7 BAC clone was used as a template containing 137 kb of genomic sequence flanking the id2b start site and recombineered by established techniques (Bussmann & Schulte-Merker, 2011).…”

“…In order to visualize anatomical landmarks to annotate brain regions containing cells labeled in Id2b:gal4 transgenics, we created triple transgenic Tg(id2b:gal4, uas:egfp, HuC:lynTagRFP-t) larvae. In addition to EGFP as a fluorescent reporter for gal4 expression, these larvae were also labeled with pan-neuronal expression of axonally targeted TagRFP (Förster et al, 2017). As shown in Figure 2a (Bergeron et al, 2012;Goll, Anderson, Stainier, Spradling, & Halpern, 2009).…”

“…Tg(id2b:Gal4-VP16)mpn215, Tg(HuC:lynTagRFP-t)mpn404, Tg (14xUAS-mCherry), Tg(14xUAS:UAS:syp-GFP), and Tg(shh:GFP) transgenic lines have been previously described (Förster et al, 2017;Heap, Goh, Kassahn, & Scott, 2013;Nikolaou et al, 2012;Xiao et al, 2011).…”

Prepontine non-giant neurons drive flexible escape behavior in zebrafish

“…The LC was visualized using Et(gata2a:EGFP)pku2 (vmat2:GFP) (Wen et al, 2008). Images of TgBAC(chata:Gal4-vp16)mpn202 (chata-Gal4) were as published (Forster et al, 2017), registered to ZBB.…”

Multiple overlapping hypothalamus-brainstem circuits drive rapid threat avoidance

“…We tested this idea by manipulating these cell types in behaving animals, using the Gal4/UAS system (Kawakami et al, 2016) to drive transgene expression in restricted subsets of peptidergic neurons in the preoptic hypothalamus ( Figure 5a,b, Figure S5a, Methods). We used three different transgenic lines that labeled neurons according to the expression of a specific neuropeptide: oxytocin/isotocin (Tg(oxt:Gal4), Wee et al, 2019), corticotrophin-releasing factor (Tg(crf:Gal4), Methods), and somatostatin (Tg(sst3:Gal4), Förster et al, 2017), as well as a line that labels multiple peptidergic neurons (Tg(otpba:Gal4), Fujimoto et al, 2011, Herget & Ryu, 2015. We first asked whether artificial stimulation of these cell types would induce avoidance-like motor actions in the absence of a stressor.…”