Efficient shRNA-mediated inhibition of gene expression in zebrafish

Abstract

Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression.

FIG. 1.

shRNA expression from miR30-backbone vectors. (A) Schematic showing promoter-driven expression of shRNAs, where the hairpin sequence is cloned into the miR30 backbone. After transcription, the priRNA is cleaved by Drosha to form an shRNA, which is further processed by Dicer into an siRNA and loaded into the RISC complex. The “guide” (antisense) strand then inhibits gene expression, leading to RNA cleavage if there is a complete match with the target. (B) Schematic of one construct employed. The b-actin promoter drives an actin-exon+intron construct including a miR30/shRNA cassette that is linked to a CFP reporter (pTolDest-bactin-actin/intron-miR30-CFP-pA). Nascent RNA is spliced and cleaved to yield shRNA and CPF, whose expression reflects extent of shRNA expression. (C) Extent of CFP expression in an F0 Tol2 transgenic embryo, using the construct shown in B. Lateral view, 24 hpf, head to top.

FIG. 2.

Inhibition of endogenous wnt5b expression. The 3′UTR1956 shRNA targeting wnt5b was expressed from an intronic miR30 backbone, under the b-actin promoter, using the vector pTolDest-bactin-actin/intron-miR30-CFP-pA (see Materials and Methods). Transient transgenesis was induced using Tol2 transposase. (Aa) Vector schematic. (Ab) Relative expression of wnt5b mRNA after inhibition by shRNA. Expression was quantified by qPCR in 24 hpf embryos expressing the wnt5b 3′UTR1965 shRNA (wnt5bh) relative to embryos injected with a control hairpin (YFPh). Data were normalized to endogenous b-actin RNA. (Ac) Northern blot analysis of RNA isolated from 24 hpf embryos demonstrating expression of the 21 base wnt5bh (antisense) guide strand (lane 1). The control is a synthetic wnt5b antisense strand DNA, run in parallel on the Northern blot (lane 2). (B) Whole mount in situ hybridization analysis of wnt5b expression in 12 hpf embryos expressing YFPh (a, a′) or wnt5bh (b, b′) and in 24 hpf embryos expressing YFPh (c) or wnt5bh (d). (a,b), dorsal views, anterior up; (a′b′) lateral views, anterior up; (c,d) lateral views. (C) Phenotype of shRNA-expressing embryos and wnt5b morphants. (a-a″) 24 hpf control YFPh embryos coinjected with mcherry RNA. (b-b″) 24 hpf wnt5bh embryos coinjected with mcherry RNA. (c-c″) 24 hpf wnt5bh embryos coinjected with wnt5b RNA. (d-d″) 24 hpf embryos injected with wnt5b MO together with the p53 MO ⁷ (see Materials and Methods). (a, b, c, d), lateral view of the head; (a′, b′, c′, d′), dorsal view of the head after brain ventricle injection; (a″, b″, c″, d″) lateral view of whole embryo. (D) Whole mount in situ hybridization analysis of myoD expression in 12 hpf embryos expressing the following RNAs: YFPh + mcherry RNA (a, a′), wnt5bh + mcherry RNA (b, b′), wnt5bh + wnt5b RNA (c, c′) or injected with wnt5b MO (d-d″). Dorsal views. Black bracket: bent tail; white bracket: compressed somites.

FIG. 3.

Inhibition of endogenous zDisc1 expression. The ORF2654 shRNA (zDisc1h) targeting zDisc1 was expressed in Tol2 F0 transgenic embryos, under control of the b-actin promoter, using the pTolDest-bactin-actin/intron-miR30-CFP-pA vector (see Materials and Methods). (Aa) Vector schematic. (Ab) Relative expression of zDisc1 mRNA after inhibition by shRNA. Expression was quantified by qPCR in 24 hpf embryos expressing zDisc1h relative to embryos expressing a control shRNA, YFPh. Data was normalized to endogenous b-actin RNA. (Ac) Northern blot analysis of RNA isolated from 24 hpf embryos demonstrating expression of the 21 base zDisc1 (antisense) guide strand (lane 1). The control is a synthetic zDisc1 antisense strand DNA, run in parallel on the Northern blot (lane 2). (B) Whole mount in situ hybridization analysis of zDisc1 expression in 24 hpf embryos expressing YFPh (a, a′) or zDisc1h (b, b′). (a-b′) Lateral views, anterior to the left; (a′,b′) higher magnification views. (C) Phenotype of shRNA-expressing embryos and zDisc1 morphants and mutants. (a-a″) 24 hpf control YFPh embryos coinjected with mcherry RNA. (b-b″) 24 hpf zDisc1h embryos coinjected with mcherry RNA. (c-c″) 24 hpf zDisc1h embryos coinjected with hDisc1 RNA. (d-d″) 24 hpf embryos injected with zDisc1 MO together with the p53 MO ⁷ (see Materials and Methods). (e-e″) 24 hpf zDisc1^fh291 mutant embryos. (a, b, c, d, e), lateral view of the head; (a′, b′, c′, d′, e′), dorsal view of the head after brain ventricle injection; (a″, b″, c″, d″, e″), lateral view of whole embryo. (D) Forebrain neurons stained for acetylated tubulin in 36 hpf embryos expressing the following RNAs: YFPh + mcherry RNA (a), zDisc1h + mcherry (b), zDisc1h + hDisc1 (c), injected with zDisc1 MO together with the p53 MO (d) or zDisc1^fh291 (e). Lateral views, anterior to left. (E) Muscle segments stained with phalloidin in 36 hpf embryos expressing the following RNAs: YFPh + mcherry (a), zDisc1h + mcherry (b), zDisc1h + hDisc1 (c), injected with zDisc1 MO together with the p53 MO (d) or zDisc1^fh291 (e). Shape of muscle segments is indicated by dotted white lines. Black bracket: narrow brain ventricles; black asterisk: bent tail; black dotted lines: region between dotted lines was used for muscle segment analysis by phalloidin staining; ac, anterior commissure; poc, postoptic commissure; sot, supraoptic tract; tpc, tract of posterior commissure; white asterisk: defective supraoptic tract, region between dotted lines was used for muscle segment analysis by phalloidin staining.

FIG. 4.

Tissue-specific inhibition of zDisc1 expression. The ORF2654 shRNA targeted to zDisc1 (zDisc1h) was expressed in I-SceI (meganuclease)-derived F0 transgenic embryos, under control of the mir124 or ntl promoter (see Materials and Methods). (Aa) Diagram of the I-SceI-mir124-miR30-GFP-pA vector, where shRNA expression is driven by the CNS-specific miR124 promoter. (Ab) Relative expression of zDisc1 mRNA after inhibition by shRNA. Expression was quantified by qPCR in 24 hpf embryos expressing zDisc1h relative to embryos expressing a control shRNA, YFPh. Data was normalized to endogenous b-actin RNA. (Ac) Whole mount in situ hybridization analysis of GFP expression in in 48 hpf embryos expressing zDisc1h. (B) Phenotype of embryos expressing shRNA from the miR124 promoter. (a-a″) 24 hpf control YFPh embryos. (b-b″) 24 hpf zDisc1h embryos. (a, b), lateral view of the head; (a′, b′), dorsal view of the head after brain ventricle injection; (a″, b″), lateral view of whole embryo. (C) Differentiated neurons stained for acetylated tubulin in 36 hpf embryos expressing YFPh (a, b) or zDisc1h (c, d) under control of the miR124 promoter. (a, c) lateral view of the head; (b, d) dorsal view of the hindbrain. (D) Muscle segments stained with phalloidin in 36 hpf embryos expressing YFPh (a) or zDisc1h (b) under control of the miR124 promoter. Shape of muscle segments is indicated by a dotted white line. (Ea) Diagram of the vector I-SceI-ntl-miR30-CFP-pA, driven by the mesendoderm specific ntl promoter. (Eb) Relative expression of zDisc1 mRNA after inhibition by shRNA. Expression was quantified by qPCR in 24 hpf embryos expressing the zDisc1h relative to embryos expressing the control shRNA, YFPh. RNA levels were normalized to endogenous b-actin RNA. (Ec) Whole mount in situ hybridization analysis of CFP expression in in 24 hpf embryos expressing zDisc1h (F) Phenotype of embryos expressing shRNA from the ntl promoter. (a-a″) 24 hpf control YFPh embryos. (b-b″) 24 hpf zDisc1h embryos. (a, b), lateral view of the head; (a′, b′), dorsal view of the head after brain ventricle injection; (a″, b″), lateral view of whole embryo. (G) Differentiated neurons stained for acetylated tubulin in 36 hpf embryos expressing YFPh (a, b) or zDisc1h (c, d) under control of the ntl promoter. (a, c) forebrain neurons, lateral view, anterior to left ; (b, d) dorsal view of hindbrain, anterior to top. (H) Muscle segments stained with phalloidin in 36 hpf embryos expressing YFPh (a) or zDisc1h (b) under control of the ntl promoter. Shape of muscle segments is indicated by a dotted white line. Black bracket: narrow brain ventricles; black dotted lines: region between dotted lines was used for muscle segment analysis by phalloidin staining; black asterisk: bent tail; ac, anterior commissure; poc, postoptic commissure; sot, supraoptic tract; tpc, tract of posterior commissure; r, rhombomeres; white asterisk: defective supraoptic tract.