Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway

Abstract

To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.

Keywords: Eye development; Fluorescent reporter; Heart development; Hippo signaling; In vivo imaging.

Figure 1. 4xGTIIC:d2GFP expression during zebrafish development

(A) Schematic of the Yap/Taz-Tead reporter construct. (B′,D′,F′) Low magnification images show 4xGTIIC:d2GFP expression in whole embryos/larvae at 1 dpf (B′), 2 dpf (D′) and 3 dpf (F′). (C′, E′, G′) Higher magnification images show d2GFP expression in non-neural cells located at the (C′) midbrain-hindbrain boundary and around the eye at 1 dpf, within the tectum at 2 dpf (E′), and in the heart at 3 dpf (G′). Brightfield images (B,C,D,E,F,G) are placed above each fluorescent image. cTNT=chicken troponin T minimal promoter; FP= fluorescent protein; M=midbrain; H=hindbrain; E=eye; A=Atrium; V=Ventricle; Y=Yolk. B, B′, C, C′, D, D′, F, F′ = lateral view; E, E′ = dorsal view; G, G′ = ventral view.

Figure 2. 4xGTIIC:mCherry co-localizes with -5.0 foxc1b:eGFP but not -7.2sox10:eGFP positive cells

(A–A‴) Co-expression of 4xGTIIC:mCherry and -5.0 foxc1b:eGFP in superficial migratory cranial mesenchymal cells located above the midbrain/hindbrain boundary. The arrows indicate co-localzation between the two transgenes. (B–B‴) Non-overlapping expression of 4xGTIIC:mCherry and -7.2sox10:eGFP in superficial migratory neural crest cells located between the eye and midbrain/hindbrain boundary. The arrows indicate cells that only express one of the transgenes. Embryos are at the 22 somite stage.

Figure 3. Nuclear Yap protein co-localizes with 4xGTIIC:d2GFP/eGFP positive cells

(A–A‴, B–B‴) d2GFP/eGFP positive cells located at the midbrain-hindbrain boundary contain nuclear Yap (red) as detected by immunofluorescence. (C–C‴, D–D‴) Cells located in the dorsal otic vesicle express d2GFP/eGFP and nuclear Yap (red). M=midbrain; H=hindbrain; OV=otic vesicle.

Figure 4. 4xGTIIC:d2GFP expression is decreased in response to Yap knockdown and enhanced with Yap or Taz overexpression

(A′,C′) d2GFP expression levels are decreased in yap/p53 MO injected embryos (C′) compared to the p53 control embryos (A′) at 2 dpf. (B′,D′) yap/p53 MO embryos have a smaller eye and decreased RPE expression (D′) compared to p53 MO control embryos. (E–E″) Overexpression of mCherry does not cause enhanced or ectopic d2GFP expression. (F–F″) Overexpression of Yap, (G–G″) Yap constitutive active (CA) transgene (yapS87A) or (H–H″) Taz results in enhanced and ectopic d2GFP expression in retinal progenitor cells (embryos are 30 hpf). Scale bar=50 μm in B.

Figure 5. Yap/Taz-Tead nuclear activity is high in cardiac precursors and cardiomyocytes, and is important in cardiac precursor migration

(A–B‴) Expression of (A′,B′) 4xGTIIC:mCherry and (A″,B″) cmlc2:eGFP in the developing heart field at (A–A‴) 22 hpf and (B–B‴) 30 hpf. (C–D′) Cardia bifida in (C,C′) 4xGTIIC:d2GFP and (D,D′) cmlc2:eGFP positive embryos injected with NLS-YapDN mRNA (28 hpf). (E–F′) Heart expression in uninjected (E,E′) 4xGTIIC:d2GFP and (F,F′) cmlc2:eGFP positive embryos (28 hpf). Scale bar = 100 μm in C, D, E, and F.

Figure 6. Loss of crb2a results in a decrease of 4xGTIIC:d2GFP expression in the neural retina at 48 hpf and at 24, 36, 48 hpf in the RPE

(A–F′) Neural retina and RPE 4xGTIIC:d2GFP expression in crb2a +/? and crb2a −/− embryos. (G–L‴) Immunofluorescence of Yap (green) and Crb2a (red) in crb2a +/? and crb2a −/− embryos. Arrow indicates Yap positive cells in the crb2a +/? neural retina that do not appear in crb2a −/−. (M,N) Quantification of d2GFP pixel intensity in the (M) neural retina and (N) RPE of crb2a +/?/crb2a −/− embryos at 24 (n=46/n=13), 36 (n=32/n=28), and 48 (n=26/n=23) hpf. Error bars represent S.E.M. p=unpaired t-test with equal S.D. Scale bar=50μm in A,C, and E.

Figure 7. Vgll4b represses nuclear Yap/Taz-TEAD activity

(A–B″) 4xGTIIC:d2GFP positive RPE cells expressing (A′) mCherry or (B′) Vgll4b. Arrows depict the mCherry and Vgll4b positive cells. (C) Quantification of d2GFP pixel intensity in mCherry + (n=19), mCherry – (n=38), dsRed/Vgll4b + (n=28), and dsRed/Vgll4b – (n=58) cell clones. More than 12 embryos were used for each condition. Error bars represent S.E.M. p=unpaired t-test with equal S.D. (D) Diagram of the relationship between Yap-Tead and Vgll4b-Tead in RPE cells expressing endogenous or overexpressed levels of Vgll4b.