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

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.

regions (50-300 kb) contained in bacterial artificial chromosomes (BACs). State-of-the-art protocols for efficient application of BAC recombineering and transgenesis in zebrafish have been established 15,16 , paving the way for larger-scale approaches to generate reliable tools for targeted gene expression. Here we report the generation, characterization and comparison of 58 BAC-derived Gal4 and Cre constructs. This screen has led to the production of 22 new transgenic zebrafish lines, each of which allows genetic access to unique neuronal subpopulations.

Results and Discussion
Transgenesis by BAC recombineering yields 22 stable zebrafish lines. To generate an array of transgenic lines that label diverse subsets of neuronal populations, we selected candidate genes primarily based on their published spatiotemporal expression patterns ( Fig. 1a and Table 1). A large fraction of these genes had previously been shown by RNA in situ hybridization to be transcribed in small populations of retinal ganglion cells (RGCs) and/or tectal neurons. In addition, we were interested in neurons that share the transmitter GABA or acetylcholine across the brain and therefore included genes that encode for transmitter-synthesizing enzymes. A third category was genes encoding neuropeptides, since these are some of the most specific markers of defined cell types in the central nervous system 17,18 .
For each gene, we chose a BAC clone that contains both upstream and downstream sequences of the translation start site to include transcription regulatory elements on both sides of the coding sequence. BAC recombineering was performed according to the protocol of Bussmann and Schulte-Merker, which is a plasmid-based technique employing an arabinose-inducible homologous recombination 15 . The method was slightly modified by adding a stable fluorescent marker to the construct, which labels cardiac muscle cells for ease of screening and subsequent re-identification. The red-fluorescent marker "bleeding heart" (BH, cmlc2:mCherry) was linked to Gal4VP16 constructs, and "cold heart" (CH, cmlc2:Cerulean) was linked to Cre (Fig. 1b). In addition, a small subset of Gal4VP16 lines was generated without heart marker.
Recombineered BAC constructs were injected into fertilized eggs obtained from UAS:Dendra-kras transgenic fish or from wild-type fish. Potential F0 founders were selected at larval stages for their expression of the transgene and raised to fertility. Specifically, we selected F0 larvae that expressed Dendra (for Gal4VP16 and Gal4VP16-BH), the "bleeding heart" marker (for Gal4VP16-BH) or the "cold heart" marker (for Cre-CH). When the F0 fish had reached adulthood, they were crossed to wild type, UAS:Dendra or UAS:intersec (see below) and their F1 progeny were screened for inherited transgene expression in the expected pattern. Germline transformation and mosaicism rates were similar to other Tol2 based BAC transgenesis approaches previously reported 5,15 .
Out of 58 BAC constructs that were confirmed to drive expression of Gal4VP16, Gal4VP16-BH, or Cre-CH, we isolated 22 stable transgenic lines. The results are summarized in Tables 1 and 2. The success rate for obtaining stable transgenic lines was 42.9% (3/7 genes), 43.6% (17/39 genes) and 50% (6/12 genes) for Gal4VP16, Gal4VP16-BH and Cre-CH, respectively. For those stable transgenic lines, the percentage of germline transgenic founders out of all adult fish screened ("founder rate") ranged from 1.1% to 25% (average 6.3%). In general, the founder rates of different driver constructs derived from the same BAC (Gal4VP16 only, Gal4VP16-BH and Cre-CH) were found to be similar, with one exception being grin2ab (25% and 3.4% for the Gal4-BH and Cre-CH construct, respectively). Consistent with a previous report 15 , we did not find a clear correlation between transgenesis efficiency and genomic insert length of the BACs (Fig. 1c).
Gal4VP16 and Cre drivers give access to defined neuronal subsets by largely recapitulating endogenous gene expression. In accord with the RNA expression patterns of the chosen genes, the established Gal4 transgenic lines label different subsets of neurons in the brain ( Fig. 1d and Supplementary Video 1-11). To determine to what extent the BAC-driven Gal4 patterns recapitulate endogenous gene expression, we compared expression of a UAS-linked reporter (UAS:GFP or UAS:GCaMP6s) to the distribution of the selected genes by antibody staining. The Tg(chat:Gal4) and Tg(gad1b:Gal4) lines labeled cells in several brain areas that were positive for choline acetyltransferase (ChAT) and GABA, respectively (Fig. 2a,b). Out of all ChATor GABA-positive cells, a little less than half visibly expressed the UAS-linked reporter in each line (48 ± 24%, n = 105 of 231 cells out of two larvae in Tg(chat:Gal4), and 42 ± 15%, n = 150 of 355 cells out of four larvae in Tg(gad1b:Gal4)). In Tg(galn:Gal4) and Tg(sst3:Gal4) lines, antibody staining greatly overlapped with, or was identical to, the Gal4-driven reporter expression (94 ± 4%, n = 88 of 94 cells out of two larvae in Tg(galn:Gal4) line) (Fig. 2c,d). Differences in protein localizations in Tg(galn:Gal4) and Tg(sst3:Gal4) lines can be explained by the transport into axon terminals of the Galn and Sst proteins, both of which encode neuropeptide transmitters 19,20 versus the cytosolic localization of GFP. It is also possible that some of the signal from the Galn and Sst antibodies is derived from extracellular localization of these secreted factors. These results with four transgenes, for which cross-reactive antibodies are available, suggest that the BAC transgenic Gal4 lines largely reproduce the endogenous gene expression pattern and thus provide genetic access to the labeled neuronal populations. Individual transgenic lines should be tested for recapitulation of endogenous gene expression patterns for each study at hand.
We also confirmed expression of our Cre constructs, either transiently or in stable transgenic lines, using a Cre reporter line named UAS:intersec (Fig. 3a). The UAS:intersec transgene is designed to drive the expression of the reporter gene in cells in which Gal4 and Cre overlap. This enables intersectional genetic approaches as reported previously 5,21 . Taken together, the BAC transgenic driver lines established here allow experimental access to small neuronal populations, either in binary (Gal4/UAS) or ternary (Cre/Gal4/UAS:intersec) genetic configurations. We noted that Cre recombination is often incomplete in our transgenic animals ( Fig. 3b-e), similarly to a previous observation reported for a different Cre transgenic line 22 . Specifically, even though the F0 founder fish produced offspring expressing transgenesis marker Cold Heart, Cre-mediated reporter expression was often incomplete in the Cold Heart-positive offspring. The reasons for the inefficient recombination event could be due to 1) variegated Cre expression at either transcription or translation level, 2) inefficient recombinase activity of expressed Cre protein, 3) variegated expression of the UAS:intersec reporter line, or a combination of all three effects.

Image registration and initial characterization of Gal4 patterns suggest new avenues into functional studies of neural circuits.
In order to determine the three-dimensional labeling pattern of our newly generated Gal4 lines, we scanned optical sections at high resolution with a confocal microscope and performed image registrations across age-matched specimens 23 . By registering expression patterns to a standard reference brain, it is possible to compare the distribution of labeled cells with those present in other lines. This data format can be integrated in a comprehensive brain atlas 21,24 . We found that the HuC:lynTagRFP-T marker is Cre-Cold Heart BACs) and their founder rate (percentage of germline founders out of total adult fish screened). Note that the founder rate is not correlated with the genomic insert length (R-squared = 0.0007). (d) Dorsal view of 6 dpf old larval brains showing the live expression pattern of ten selected transgenic Gal4 lines (green; Dendra-kras, GCaMP6s or EGFP). Brains have been registered via co-expression of HuC:lynTagRFP-T (magenta). Scale bar, 100 µm. excellently suited as a bridging template for across-line image registrations. This membrane-targeted red fluorescent reporter is expressed in almost all neurons and strongly labels the cell membrane-enriched neuropil areas, whose outlines in the fish brain are highly stereotyped. After crossing Gal4 carriers of selected lines, driving the expression of green-fluorescent UAS-linked reporters (UAS:Dendra, UAS:GFP, or UAS:GCaMP6s), to carriers of the HuC:lynTagRFP-T transgene, triple-transgenic larvae were identified and whole-brain images of red and green channels were obtained. These images were aligned with each other using the HuC:lynTagRFP-T pattern as a template, and the green reporter channels were superimposed using different colors to visualize the spatial relationships of the respective patterns ( Fig. 4a and Supplementary Video 12). This analysis demonstrates the utility of our new lines for functional investigations into neural circuitry. Three immediate applications come to mind. First, telencephalic inhibitory connections have to our knowledge not been studied in teleosts. Our new gad1b:Gal4 line will be particularly useful for functional studies of GABAergic cells in the subpallium, where the labeling seems particularly strong (Fig. 4b). Second, while the zebrafish olfactory bulb has been extensively investigated, recently culminating in the complete electron-microscopic reconstruction of its synaptic connectome 25 , considerably less is known about extrinsic modulation of olfactory processing. The new chat:Gal4 line may give an entry point into this question, as it reveals the ventral pallial cholinergic innervation of the olfactory bulb (Fig. 4b). Lastly, neuropeptides have been shown to present highly specific markers of neurosecretory populations in preoptic and hypothalamic areas 17,18 . Our new sst3:Gal4 and galn:Gal4 lines will allow recording and manipulation of these poorly understood modulatory systems.

Image registration reveals new aspects of the layered architecture of the larval brain.
Our initial characterization also revealed interesting architectonic principles that invite future investigations. We identified Gal4 lines with clustered, but mutually exclusive expression in the rhombencephalon (Fig. 4c). Specifically, chat:Gal4, gad1b:Gal4 and cxcr4b:Gal4 exhibited non-overlapping expression in rostral to caudal stripes in horizontal sections (Fig. 4d,e), and dorsomedial to ventrolateral stripes in transverse sections, respectively (Fig. 4f). This observation is consistent with the previously reported striped organization of neurotransmitter and transcription factor expression in the hindbrain 26 . Our analysis now shows that this developmental patterning not only holds true for glutamatergic, GABAergic, and glycinergic groups of neurons, as reported by Kinkhabwala et al. 26 , but also for cholinergic neurons. A multi-label image registration approach, such as the one used here, is uniquely suited to uncover principles of mesoscale spatial organization.
The tectum is an intensively studied, multisensory processing area in the vertebrate midbrain 27 . Its densely packed neuropil is subdivided into layers, originally defined by the stratification pattern of incoming RGC axons [28][29][30] . RGC axons project to the following layers (from superficial to deep): Stratum opticum (SO); stratum fibrosum et griseum superficiale (SFGS, with its six sublayers, SFGS1 through SFGS6); the retinorecipient stratum griseum centrale (SGC); and the boundary between stratum album centrale and stratum periventriculare (SAC/SPV). From among our collection of BAC lines, several showed expression in the tectal neuropil (Fig. 5a). Co-registration of the patterns of our newly generated lines with that of an RGC-specific Gal4 line (isl2b:Gal4) 31 allowed us to further explore the laminar architecture of the tectum beyond RGCs (Fig. 5a,b). A densitometric analysis revealed that cxcr4b:Gal4 consistently but sparsely labeled SFGS3, SFGS4 and SAC/SPV (Fig. 5c). While the labeling of SFGS arises from cxcr4b + RGC axons, arborizations in the SAC/SPV layer originate from processes of deep midbrain neurons (data not shown). Neuropil labelings in chat:Gal4, dmbx1b:Gal4 and gad1b:Gal4 do not derive from RGC axons, but from neurites of specific classes of periventricular tectal neurons and incoming axons from various other brain areas (Fig. 5d-f). The chat:Gal4 pattern showed highest fluorescence intensities in the deep stratum griseum centrale (SGC) (Fig. 5d). Fluorescence signals in gad1b:Gal4, on the other hand, are strongest in the superficial layers (Fig. 5e), including superficial interneuron (SIN) cell bodies, which are GABAergic 32 . Neurites labeled by dmbx1b:Gal4 arborize in SFGS and SGC sublayers that are superficial to the retinorecipient SGC (Fig. 5f). As a rule, neuropil stratifications in these three lines are not restricted to single layers, but differences in labeling intensities rather reflect graded preferences for layer positioning. In summary, we isolated an array of transgenic lines, which allow targeting of different components of the layer-specific circuitry in the zebrafish tectum.

Conclusions and outlook.
Progress in the neurobiology of the zebrafish preparation has recently accelerated, driven by advances in imaging technology, behavioral analysis and computational methods 25,[33][34][35][36][37][38][39] . Optogenetics affords the ability to manipulate circuit components in the behaving animal [40][41][42] . We expect that our new set of BAC transgenic Gal4 and Cre lines will facilitate research that employs imaging or optogenetics in the zebrafish system and thus provide a valuable resource for the neuroscience community. In the future, breakthroughs are expected from the refinement of genetic tools that allow addressing specific subpopulations of neurons in the context of intact circuitry. Knock-in technology using CRISPR/Cas9 represents a promising approach to target endogenous loci with superior fidelity [43][44][45][46] and may in the future be scaled up for the systematic generation of transgenic lines.  Plasmids. To generate pCR8GW-Gal4VP16-FRT-Kan-FRT-Bleeding Heart and pCR8GW-Cre-FRT-Kan-FRT-Cold Heart plasmids, the Bleeding Heart (cmlc2:mCherry, also known as myl7:m-Cherry) and Cold Heart (cmlc2:Cerulean, also known as myl7:Cerulean) cassettes (obtained from Michael Nonet, Washington University, USA) were inserted into pCR8GW-Gal4VP16-FRT-Kan-FRT and pCR8GW-Cre-FRT-Kan-FRT 16 , in the reverse orientation relative to Gal4VP16 or Cre coding sequence.
To generate the Tol2 HuC:lynTagRFP-T plasmid, lynTagRFP-T (TagRFP-T tagged with lyn kinase membrane targeting sequence) 47 was PCR amplified and cloned downstream of the HuC promoter. For the intersectional reporter, the loxP-tdTomato-CAAX-loxP-EGFP-CAAX cassette was codon optimized and synthesized by Genscript (Piscataway, NJ), and subsequently cloned into a pTol2-14xUAS vector to obtain UAS:loxP-tdTomato_CAAX-loxP-EGFP_CAAX. BAC recombineering. BAC recombineering was performed as described previously 15,16 . Briefly, in the first step, BAC clones were transformed with the pRedET plasmid (Gene Bridges), which enables the arabinose-inducible homologous recombination. In the second step, Tol2 arms in opposing directions flanking an ampicillin resistance cassette were PCR amplified from piTol2_amp plasmid 16 and inserted into the BAC backbone. In the third and final step, either Gal4VP16 only, Gal4VP16-BH or Cre-CH cassettes were PCR amplified and inserted into the BAC, such that the start-ATG site of the gene of interest was replaced by that of Gal4VP16 or Cre. For th:Cre-CH line, the GFP coding sequence of the th:GFP BAC 48 was replaced with the Cre-CH cassette. After we confirmed successful insertions of the cassette by PCR, the final BAC DNA was purified using NucleoBond XTra BAC kit (Machery Nagel), and correct insertions were verified by sequencing.
Transgenesis. Tg(HuC:lynTagRFP-T)mpn404 49 and Tg(UAS:intersec)mpn128 were created using the standard Tol2 transposon system. BAC DNAs were injected at 100 ng/µl together with zebrafish codon-optimized Tol2 transposase mRNA (50-100 ng/µl), synthesized from pCS-zT2TP plasmid 16 . Gal4VP16 and Gal4VP16-BH BAC DNAs were injected into Tg(UAS:Dendra-kras)s1998t transgenic embryos. Cre-CH BAC DNAs were injected into wild-type TL embryos. After injection, embryos expressing Dendra, Bleeding Heart, or Cold Heart were screened and raised to sexual maturity. Injected fish were either incrossed or outcrossed with wild-type or Tg (UAS:Dendra-kras)s1998t transgenic adult fish to identify transgenic carriers. The germline mosaicism rate was defined as a percentage of transgenic offspring (F1) out of all offspring born from F0 founder fish. Outcrossing of F1 transgenic lines (and of following generations) revealed segregation of Gal4 expression at Mendelian ratios, suggesting that they carry a single insertion. In cases where multiple founders were identified, the founders which showed the most complete expression patterns in their F1 offspring were selected and maintained. We noted that the transgene expression of different larvae derived from the same transgenic founders was variable in some of our BAC transgenic lines, as is known for the Gal4/UAS system in general 50,51 . Nevertheless, among larvae that were pre-screened for the high-level expression of the transgene, the expression patterns were largely consistent across different larvae (Supplementary Figure S1).

Immunohistochemistry.
Immunostaining was performed according to ref. 24, with slight modifications.
Fish were fixed in 4% paraformaldehyde (PFA) in PBS overnight. For the initial antigen retrieval, fish were heated to 63 °C for 15 minutes in 150 mM Tris-HCl. The time for staining with primary antibody was increased to at least 5 days and with secondary antibody to at least 2 days. Secondary antibodies were diluted in only PBT. After staining, samples were washed with PBT and postfixed for 30 minutes in 4% PFA, thereafter briefly washed in PBT and then soaked in 85% glycerol. ChAT antibody staining was performed according to ref. 52, with the proteinase K treatment prolonged to 120 min for 6 dpf larvae. For a list of antibodies used, see Supplementary Table S1.
Image acquisition. For live imaging, 6-7 dpf larvae were anesthetized in 0.016% tricaine and embedded in 2% low-melting-point agarose. Imaging was performed on a Zeiss LSM-780 or LSM-700 confocal microscope, using 20×/1.0 NA water-dipping objectives. For imaging fixed samples, larvae were embedded in 85% glycerol, and imaging was performed using a 25×/0.8 NA multi-immersion objective. Whole-brain images were acquired by tiling the brain into three individual image stacks and subsequent stitching, using ZEN software (black edition, v8.0; Zeiss). Images were corrected for fluorescence attenuation in the z-dimension using the brightness correction function in the ZEN software.
Image registration. Registration was performed using the Computational Morphometry Toolkit (CMTK) 53 .
Whole-brain images of living 6 dpf old zebrafish larvae were co-registered into one reference brain using expression of HuC:lynTagRFP-T as a template. For visualization of RGC innervation strata in the tectum, fish expressing isl2b:Gal4 UAS:GCaMP6s and HuC:lynTagRFP-T were co-registered into the reference brain.