Gal80 intersectional regulation of cell-type specific expression in vertebrates

Abstract

Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.

Figure 1

Pan-neuronal expression of Gal80 does not affect CNS development. Whole-mount embryos (transgenic Tg(elavl3:Gal80)^zc64 or non-transgenic sibling), ventral views (except D), anterior to the top, scale bar 50 μm. Images are confocal projections (except A and B). (A-A’) Brightfield images showing expression of gal80 by in situ in wild-type (A) and transgenic embryos (A’). (B-B’) dlx2 in situ expression in wild-type and transgenic embryos is similar. (C-C’) Pattern of tyrosine hydroxylase (TH) antibody expression is similar in wild-type and transgenic embryos. (D-D’) Axon tract architecture appears similar in wild-type and transgenic embryos (lateral views, anterior to the left). (E-E’) Acridine orange staining for apoptotic cells is similar in wild-type and transgenic embryos.

Figure 2

Gal4 drives strong expression in stable transgenic lines and can be inhibited by Gal80. Confocal maximum projections of 72hpf embryos; ventral views, anterior to the top. Embryos are heterozygous for the genotypes shown. Scale bar, 50μm. Arrow points to ectopic GFP expression in eyes driven by Gal4-VP16 (A, D) not noted using native Gal4 (E). (A-C) Live embryos imaged with identical laser power settings, showing that Gal4-driven expression (B) is stronger than a direct enhancer:GFP construct (C) and is comparable to Gal4-VP16-driven expression (A). (D-F) Immunohistochemistry for GFP in fixed embryos again shows that Gal4-dependent expression (E) is comparable to Gal4-VP16 (D), but has minimal expression in other tissues (such as eye muscles, arrow). (F) The original transgenic enhancer line Tg(otpb.A:egfp)^zc48 shows dimmer expression compared to Gal4-driven expression in (E). (G) A weakly-expressing allele of elavl3:Gal80 only partially inhibits Gal4-dependent transgene expression. (H) A strongly-expressing elavl3:Gal80 allele completely inhibits Gal4-dependent expression. Insets for (G) and (H) show relative levels of Gal80 in situ expression. (I) Gal80 is unable to inhibit Gal4-VP16-driven expression.

Figure 3

Time course of Gal80-mediated inhibition of Gal4. Confocal images of live embryo GFP expression. Transgenic fish carrying Tg(hs:Gal80); Tg(otpb.A:Gal4); Tg(UAS:GFP) were imaged before a 45’ heat-shock, and then at defined time intervals following heat-shock. The same embryo is displayed in each respective set of panels (A1-A’’’’ or B-B’’’’). Arrow points to the inhibition of Gal4-dependent expression in (A’); arrowhead points to the return of GFP expression in (A’’’’) 24 hours following the end of heat-shock. 10 z-slices were used to create the image. Ventral views, rostral to the top, identical confocal settings for imaging both embryos. Scale bar, 50μm; “h”, GFP expression in the heart from the transgenesis marker.

Figure 4

Temporal control of Gal4-dependent expression using a temperature sensitive version of Gal80. Confocal maximum intensity projections, ventral views, anterior to top, 72hpf Tg(otpb.A:Gal4)^zc67; Tg(UAS:GFP) embryos stained with anti-GFP. Scale bar, 50μm. (A, B) Expression is indistinguishable between embryos without (A) or with (B) the Tg(elavl3:Gal80ts)^zc68 transgene when raised at the restrictive temperature (28.5°C) (C, D) When raised at permissive temperatures (21.5°C or 23°C) starting at 24hpf, Gal80ts is able to effectively inhibit Gal4-dependent expression. (E) When Gal4-dependent expression is initially allowed to occur from 24 to 48hpf, shifting to the permissive temperature from 48-72hpf allows Gal80ts to inhibit Gal4-dependent expression. (F) Raising at a permissive temperature until 48hpf, then relieving Gal80-mediated inhibition at 48hpf permits Gal4-dependent expression to resume by 72hpf. (G, G’) Live confocal images of the same embryo before (at 24hpf) and after (at 36hpf) shifting to the permissive temperature, shows inhibition of Gal4-dependent expression. (H) In comparison, a control embryo raised at the restrictive temperature only shows robust expression. Inset shows this same embryo at 24hpf.

Figure 5

Nuclear localization and codon optimization improves function of Gal80. Confocal maximum projections, lateral views, anterior to left, dorsal up, of eyes in Tg(isl2b.3:Gal4)^zc65; Tg(UAS:GFP) 72hpf embryos. Scale bar, 50μm. Immunostaining for GFP, green; Topro3 nuclear stain, magenta. (A-A”’) Tg(isl2b.3:Gal4)^zc65; Tg(UAS:GFP) transgenic embryos with no Gal80 show GFP expression in all RGCs. Inset and A”’ shows high power magnification of single confocal slice. (B-B”’) Triple transgenic Tg(isl2b.3:Gal4)^zc65; Tg(UAS:GFP); Tg(brn3c:Gal80) shows inhibition of Gal4-dependent GFP expression in about 30% of RGCs. Inset and B”’ shows high power magnification of single confocal slice. (C-C”’) Transient injection with construct carrying “improved” Gal80 into Tg(isl2b.3:Gal4)^zc65; Tg(UAS:GFP) embryos demonstrates inhibition similar to that of stable lines carrying native Gal80. Improved Gal80 has nuclear localization signal (NLS) and is codon optimized (“opt”).

Figure 6

Subgroups of neurons can be genetically defined by expressing Gal4 and Gal80 with partially overlapping enhancers. Confocal ventral views, anterior to top, of brain in 72hpf embryos. Scale bar, 50μm, 25μm in B and C. Immunostaining for GFP, green; TagRFP, red. (A-A”) Maximum intensity projections of Tg(otpb.A:Gal4)^zc67; Tg(UAS:GFP); Tg(f.TH.m:NLS-Gal80_opt-2A-TRFP)^zc78 embryos shows non-overlap of Gal80 and GFP expression. (B-B”) Boxed inset area from A” shows single confocal slice of individual cells expressing either GFP or RFP but not both. (C) Single confocal slice of Tg(otpb.A:Gal4)^zc67; Tg(UAS:GFP) embryo and inset (C’-C”) shows full complement of cells express GFP in absence of Gal80.