Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

Astone, Matteo; Lai, Jason Kuan Han; Dupont, Sirio; Stainier, Didier Y. R.; Argenton, Francesco; Vettori, Andrea

doi:10.1038/s41598-018-27657-x

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Published: 05 July 2018

Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

Scientific Reports volume 8, Article number: 10189 (2018) Cite this article

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Abstract

As effectors of the Hippo signaling cascade, YAP1 and TAZ are transcriptional regulators playing important roles in development, tissue homeostasis and cancer. A number of different cues, including mechanotransduction of extracellular stimuli, adhesion molecules, oncogenic signaling and metabolism modulate YAP1/TAZ nucleo-cytoplasmic shuttling. In the nucleus, YAP1/TAZ tether with the DNA binding proteins TEADs, to activate the expression of target genes that regulate proliferation, migration, cell plasticity, and cell fate. Based on responsive elements present in the human and zebrafish promoters of the YAP1/TAZ target gene CTGF, we established zebrafish fluorescent transgenic reporter lines of Yap1/Taz activity. These reporter lines provide an in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Transgene expression was detected in many larval tissues including the otic vesicles, heart, pharyngeal arches, muscles and brain and is prominent in endothelial cells. Analysis of vascular development in yap1/taz zebrafish mutants revealed specific defects in posterior cardinal vein (PCV) formation, with altered expression of arterial/venous markers. The overactivation of Yap1/Taz in endothelial cells was sufficient to promote an aberrant vessel sprouting phenotype. Our findings confirm and extend the emerging role of Yap1/Taz in vascular development including angiogenesis.

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Introduction

Yes Associated Protein 1 (YAP1) and WW Domain Containing Transcription Regulator 1 (WWTR1 or TAZ), co-transcriptional effectors of Hippo signal transduction, are essential regulators of development, tissue homeostasis, and regeneration through their molecular, mechanical, and metabolic control of proliferation, migration, cell-fate, and other cellular processes^1,2. The Hippo pathway is an evolutionarily conserved tumour suppressor signal transduction pathway culminating in YAP1/TAZ phosphorylation and inhibition. In mammals, the sterile 20-like kinases MST1/STK4 and MST2/STK3, when activated by upstream signals and bound to their regulatory protein SAV1/WW45, phosphorylate and activate the LATS1/2 kinases together with their regulatory subunits MOB1A/B^3,4,5. In turn, the LATS1/2/MOB1A/B complex phosphorylates YAP1 and TAZ^6,7,8 at five and four serine/threonine residues, respectively^9,10. Active, not phosphorylated YAP1/TAZ translocate into the nucleus to drive transcription of a set of target genes as co-transcription factors of TEADs¹.

Despite a significant wealth of knowledge on these factors, one question that remains largely unaddressed is indeed whether these factors are active in physiological conditions in many adult tissues^{11,12,13,14,15}. Thus, the development of tools and techniques to follow YAP1/TAZ activity can be a significant step forward to address this issue.

While the role of YAP1/TAZ in organ growth and cancer is well established, the importance of their signaling in angiogenesis and vascular development has emerged only recently. Endothelium-specific inducible YAP1/TAZ knockout mice revealed that YAP1/TAZ activity is required for developmental sprouting angiogenesis and is controlled by VEGF¹⁶. Furthermore, YAP1/TAZ modulates actomyosin contractility for endothelial cell migration as well as establishment of tight junctions for endothelial barrier maturation¹⁷. In zebrafish it was demonstrated that Yap1 activation is induced by blood flow and required for vessel maintenance¹⁸. YAP1/TAZ expression is essential for coronary vasculature development in mouse¹⁹ while it has been shown that inhibition of Tead transcriptional activity can affect remodeling of the zebrafish caudal vein plexus (CVP) by vascular regression²⁰. A role of YAP1/TAZ in atherosclerosis and vascular smooth muscle cell proliferation, migration and differentiation has also been described^{21,22,23,24,25,26,27}.

The zebrafish vasculature and the basic vascular plan of its embryo are remarkably similar to that of other mammalian models^28,29. Developmental vasculogenesis, angiogenesis and vascular remodelling recruit the same growth and differentiation factors as those involved in mammals and the physiopathology of the vascular system is also very conserved^29,30,31. The zebrafish cardiovascular system develops considerably fast: a beating heart, a complete circulation loop and circulating erythroblasts are present by 24 hours post fertilization (hpf)^32,33. In zebrafish, the dorsal aorta (DA) and posterior cardinal vein (PCV) form between the 10 somite stage (14 hpf) and 24 hpf in a process, known as vasculogenesis, that appears to be conserved among vertebrates^34,35. From 14 hpf onward, endothelial progenitor cells located in the lateral plate mesoderm (LPM) migrate towards the midline of the embryo where they aggregate to form the DA. Shortly after the 15-somite stage, a second wave of angioblast migration follows and together with some cells from the first wave gives rise to the PCV³⁶. Consequently, the angiogenic process begins with the formation of intersegmental vessels (ISVs) of the trunk that start sprouting from the DA at 20 hpf. As ISVs reach the dorsolateral surface of the neural tube, they branch caudally and rostrally, and connect with each other to form the dorsal longitudinal anastomotic vessel (DLAV)²⁷.

In this work, we present a zebrafish fluorescent transgenic line that reports Yap1/Taz-Teads transcriptional activity in vivo. The reporter, active in many regions of embryos and larvae but largely silenced in adult fish, is prominently expressed in the cardiovascular system. To explore the role of Yap1/Taz activity in vascular development, we generated yap1 and taz mutants by CRISPR/Cas9. Interestingly, yap1^−/−;taz^+/− animals display strong alterations in PCV formation, suggesting the requirement of Yap1/Taz during the development of primary vessels.

Results

Generation of Yap1/Taz zebrafish reporter lines

In order to generate a reporter to follow YAP1/TAZ activity in vivo, we used a YAP1/TAZ reporter construct based on the −200/+27 fragment of the human CTGF gene promoter (Hsa.CTGF), which contains three YAP1/TAZ/TEAD DNA-binding sites highly conserved also in the zebrafish ctgfa promoter³⁷ (Fig. 1A). To test the responsiveness of the Hsa.CTGF-based YAP1/TAZ reporter construct, we generated a luciferase-based reporter and tested it in breast cancer cells expressing high levels of YAP1/TAZ. Transfection of the Hsa.CTGF-Lux plasmid resulted in a strong signal of the luciferase reporter gene when compared to the empty luciferase vector (not shown). Direct inhibition of YAP1/TAZ by siRNA knockdown, activation of the Hippo pathway through NF2 or treatment with latrunculin A (an F-actin inhibitor leading to YAP1/TAZ inhibition in multiple systems)³⁸ were all able to inhibit the reporter expression (Fig. S1A).

The reporter construct was used to develop a Hsa.CTGF-based YAP1/TAZ zebrafish reporter. Hsa.CTGF promoter fragment was cloned into a Gateway 5′ entry clone (p5E-MCS) and used to create the pDest(Hsa.CTGF:nlsmCherry) and pDest(Hsa.CTGF:eGFP)) (Fig. 1B). One-cell stage zebrafish embryos were injected with each destination vector together with Tol2 transposase mRNA. Mosaic transgenic fish displaying a strong fluorescence at 24 hpf were selected and raised to adulthood. An average of 76% (13/17) of the injected fish prescreened for mosaic fluorescence were found transmitting the transgene to their offspring. All the offspring from different founder fish exhibited a similar reporter protein expression pattern, displaying strong fluorescence in identical anatomical districts, such as the lens and otic vesicles, the heart, the pharyngeal arches and the vasculature (Figs 1C and S2). Two founders were selected to establish stable reporter lines containing a single allele in their germline: Tg(Hsa.CTGF:eGFP)^ia48 and Tg(Hsa.CTGF:nlsmCherry)^ia49.

Hsa.CTGF-based zebrafish transgenic lines are bona fide Yap1/Taz reporter

To validate whether the Hsa.CTGF-based zebrafish reporter lines can be used to visualize the endogenous activity of YAP1/TAZ in vivo, we knocked down zygotic expression of both yap1 and taz by co-injecting one-cell stage embryos with two splice morpholinos targeting respectively yap1 and taz pre-mRNAs. mCherry expression of the Tg(Hsa.CTGF:nlsmCherry)^ia49 reporter line was significantly reduced throughout the entire embryo compared to control morpholino-injected embryos (Fig. 2A). The knockdown of endogenous Yap1 and Taz proteins was confirmed by Western blotting using whole embryo extracts (Fig. S1B).

To test whether the reporter line was able to visualize increased YAP1/TAZ activities, we injected in one-cell stage embryos of the Tg(Hsa.CTGF:nlsmCherry)^ia49 line the mRNAs coding for a constitutively active form of YAP1, TAZ or TEAD (YAP-5SA, TAZ-4SA, and TEAD-VP16 respectively). As expected, the injection of any of these mRNAs significantly increased Hsa.CTGF:nlsmCherry expression (Fig. 2B–D). A similar responsiveness of the reporter signal upon Yap1/Taz activity modulation was found for Tg(Hsa.CTGF:eGFP)^ia48 (data not shown). We also validated more specifically that the reporter signal is dependent on Yap/Taz in a number of different tissues, such as the eye, the heart, the floorplate, and the major axial vessels (Fig. S3).

We further tested the Hsa.CTGF:nlsmCherry transgene by treating the reporter embryos with Dexamethasone (DEX), a synthetic glucocorticoid recently shown to promote the nuclear activity of YAP1 in vitro³⁹. 24 hpf Tg(Hsa.CTGF:nlsmCherry)^ia49 embryos were exposed to a solution containing 25 μM of DEX and analyzed after 24 h. DEX treated embryos displayed a significant increase of fluorescence when compared with controls, confirming in-vivo that glucocorticoids activate Yap1/Taz/Tead mediated transcription. Interestingly, we also observed that DEX treatment does not increase the reporter signal in Yap1/Taz knockdown embryos, thus confirming that the response induced by glucocorticoids is mediated by Yap/Taz activation. (Fig. S4A-A”).

Recently, it has been shown that blood flow can induce nuclear import of YAP1 and its transcriptional activity¹⁸. We thus tested Hsa.CTGF zebrafish reporter with silent heart (sih/tnnt2a) morpholino to block cardiac contractility and blood flow; sih morphants showed a significant reduction of mCherry transgene expression in endothelial cells when compared to control morpholino-injected embryos (Fig. S4B).

Taken together, these experiments demonstrate that the Tg(Hsa.CTGF:nlsmCherry)^ia49 and Tg(Hsa.CTGF:eGFP)^ia48 lines can be used to analyze Yap1/Taz activity in vivo.

Tg(Hsa.CTGF:nlsmCherry) ^ia49 zebrafish reveals in vivo the spatio-temporal activation of Yap1/Taz

We next sought to identify the embryonic stages and tissues in which the reporter is active. Due to maternal expression, fluorescence is ubiquitously detectable at the dome stage and during epiboly in embryos derived from Tg(Hsa.CTGF:nlsmCherry)^ia49 females crossed to wild-type males (Fig. S5A). Consistently, mCherry expression is prominent in the adult ovary (the organ with the strongest reporter signal in adults), where the mCherry protein is clearly visible in the nuclei of the oocytes and the accompanying follicle cells (Fig. S5B,C).

The first zygotic expression of the reporter protein in transgenic embryos (derived from Tg(Hsa.CTGF:nlsmCherry)^ia49 males crossed to wild-type females) is detectable at late somitogenesis. At 20 hpf fluorescence is found widely across the developing embryo, with the strongest signal localized in the mesenchyme of the tail bud (Fig. 3A). By 24 hpf, fluorescence is observed in many tissues and organs, such as eyes, heart, midbrain-hindbrain boundary (MHB), rhombencephalon, epidermis, muscles, neural tube, notochord, floorplate and vasculature. Reporter expression is persistent in those districts even during later developmental stages (Fig. 3B–H). In the eye the signal is strong in the lens and is also present in the neural retina (Fig. 3B). The lens remains strongly fluorescent until adulthood (Fig. 3C). In the dorsal portion of the head, two regions display transgene activation by 24 hpf: the MHB and the rhombencephalon. In the rhombencephalic region Hsa.CTGF:nlsmCherry fluorescence is present in six stripes of cells that follow the metameric organization of rhombomeres (Fig. 3D). At 72 hpf, besides the lens, the reporter signal is particularly strong in the pharyngeal arches (mainly in the mandibular one), otic vesicles, pectoral fins and heart (Fig. 3I,J). A time series of the reporter expression during embryonic and early larval stage is shown in Fig. S6.

In the heart, fluorescence is already visible at early stages and persists throughout adulthood (Fig. 3K–M). To confirm the expression in the cardiac progenitors and specifically in the cardiomyocytes, we outcrossed the Tg(Hsa.CTGF:nlsmCherry)^ia49 line to the Tg(myl7:GFP) line⁴⁰. As indicated by the co-localization between mCherry and eGFP from 22 hpf, the Yap1/Taz reporter is active in cardiac progenitors as well as in differentiated cardiomyocytes (Fig. 3K,L), in agreement with previously described zebrafish reporter lines^41,42 and with genetic data in mammals, where YAP1 is active together with TEADs to promote the growth of embryonic and fetal cardiomyocytes^{11,43,44,45,46}. Hsa.CTGF:nlsmCherry fluorescence is visible also in the intestinal epithelium along the whole intestine (Fig. 3N–P). Specific expression in the epithelium of the intestine is demonstrated by fluorescence co-localization at cellular resolution in Tg(Hsa.CTGF:nlsmCherry)^ia49/Tg(gut:GFP)^s854 ⁴⁷ double transgenic embryos (Fig. 3P).

Finally, one prominent site of Hsa.CTGF:nlsmCherry expression is the vascular network, where the reporter signal is visible as soon as the vasculature starts to develop and preserved during adulthood. The co-localization of Tg(Hsa.CTGF:nlsmCherry)^ia49/Tg(kdrl:GFP) double transgenics indicates specific activation of Yap1/Taz in endothelial cells throughout the 20 hpf and 48 hpf embryos, as well as in adult fish (Fig. 4A–I).

Yap1/Taz are required for posterior cardinal vein development

To study the role played by Yap1/Taz in zebrafish development, and more specifically in the vasculature, yap1 and taz mutants (yap1^bns19 and taz^bns35, respectively) generated by CRISPR/CAS9 were used^48,49. The yap1^bns19 allele contains a 41 bp deletion in exon 1 of yap1, while the taz^bns35 allele contains a 29 bp insertion in exon 2 of taz. Both frame-shift mutations are predicted to encode a truncated protein(Fig. S7A,B). The reduction of Yap1/Taz activity in the mutants was confirmed by the analysis of the Tg(Hsa.CTGF:nlsmCherry)^ia49 reporter line. As expected, we observed a decrease of Hsa.CTGF:nlsmCherry expression in the endothelium of 48 hpf yap1^bns19 and taz^bns35 mutant embryos (Fig. S7C,C’).

While we did not observe striking vascular phenotypes in taz mutants, the primary vascular phenotypes of yap1 mutants have been described recently¹⁸. Here, we report additional alterations in the cranial and ocular vasculature: the mesencephalic vein (MsV) and the dorsal longitudinal vein (DLV) were truncated in more than 50% (5/9) of yap1^−/− larvae analyzed at 72 hpf; at 5 dpf, fewer hyaloid vessels could be observed in the eyes (Fig. S8), a number further reduced in colobomatous eyes.

Double yap1;taz homozygous mutants exhibit severe developmental defects and die by 30 hpf as previously reported^18,48,50. Therefore, we chose to analyze the animals retaining one allele of taz. yap1^−/−;taz^+/− embryos exhibited an undulating notochord at 20 hpf and curving of the tail by 32 hpf (Fig. 5A,A’). Moreover, about 16% (7/45) of yap1^−/−;taz^+/− embryos had bifid hearts and 76% (34/45) did not exhibit blood circulation; the remaining 9% (4/45) of mutants exhibited blood circulation, but not at WT levels.

In addition, we observed an intriguing phenotype at the level of the axial vessels in the yap1^−/−;taz^+/−animals. At 30 hpf, when analyzed in transverse confocal sections, embryos were found to exhibit a posterior cardinal vein (PCV) deviating from the midline (Fig. S9). This phenotype worsened and was fully penetrant at 48 and 72 hpf (Figs 5B–D and S10). Interestingly, in 67% (4/6) of 48 hpf yap^−/−;taz^+/−embryos the PCV appeared to exhibit two distinct lumens (Fig. 5B).

We aimed to investigate whether these PCV alterations might be associated with defects of arterial/venous specification during axial vessel development. In situ hybridization for expression of the arterial marker efnb2a showed that it was not affected in yap1^−/−;taz^+/− embryos at 24 and 32 hpf. On the contrary, expression of mrc1a (a marker for veins and lymphatics⁵¹) was altered in yap1^−/−;taz^+/− embryos when compared to WT siblings (Fig. 5E). During development, mrc1a is initially expressed in the presumptive venous progenitors localized in the axial vessel, becoming restricted to the PCV and other veins as well as in lymphatic vessels⁵¹. In 70% (7/10) of 32 hpf yap1^−/−;taz^+/− embryos, some mrc1a expression was observed in the DA, unlike in WT animals (Fig. 5E), suggesting a defect in DA specification.

Taken together, these findings confirm the role of Yap1/Taz in vascular development and reveal its specific requirement for the cranial and ocular vascular networks, as well as for the specification and organization of the axial vessels.

The upregulation of Yap1/Taz/Tead-mediated transcription causes aberrant vessel sprouting

The vascular developmental defects observed in yap1;taz mutants and the emerging role of Yap1/Taz in developmental angiogenesis^16,17,18, prompted us to explore the effect of the upregulation of Yap1/Taz activity on embryonic angiogenesis in zebrafish. Therefore, we injected a constitutively active form of Taz (TAZ-4SA mRNA) in one-cell stage Tg(kdrl:GFP)^s843 embryos. While no alteration was observed in PCV development, at 32 hpf we observed the appearance of abnormal sprouts emerging horizontally from some ISVs in TAZ-4SA injected embryos (Fig. 6A,B–D). These abnormal sprouts pointed mostly toward the adjacent ISVs, and often gave rise to complete anastomosis between two adjacent ISVs (Fig. 6B’). Despite the low frequency of aberrant vessel sprouting in TAZ-4SA injected embryos, this phenomenon was never observed in control fish. In particular, complete anastomosis between adjacent ISVs was never detected in more than 50 injected control embryos analysed (Fig. 6A,B’,E). To further confirm the aberrant sprouting phenotype, the experiment was repeated using YAP-5SA mRNA, obtaining similar results (data not shown).

We next asked whether the effect on ISVs angiogenesis was due to enhanced Yap1/Taz/Tead-mediated transcription, or to other non-transcriptional functions of Yap1 and Taz proteins. To answer this question, Tg(kdrl:GFP)^s843 embryos were injected with TEAD-VP16 mRNA, which constitutively activates Tead target gene transcription independently of Yap1/Taz. Aberrant vessel sprouting and anastomosis between ISVs were found also after the injection of TEAD-VP16 mRNA, thus confirming the vessel sprouting-promoting ability of Yap1/Taz/Tead-mediated transcription (Fig. 6E).

To address whether the effects of Yap1 and Taz on ISVs was specifically due to their activity in endothelial cells, we designed a new construct, placing TAZ-4SA expression under the control of the fli1a endothelial-specific promoter. To perform this experiment, we used the pDestTol2CG2 transposon backbone, containing the cardiac myl7:GFP transgenesis marker, in order to detect the mosaicism levels of injected fish⁵². We speculated that with these experimental conditions we would have been able to detect the presence of aberrant sprouting phenotype only if the vessel sprouting-promoting ability of Yap1/Taz had been due to its transcriptional activity in endothelial cells. After the injection of pDestTol2CG2-fli1a-TAZ-4SA plasmid in one-cell stage Tg(kdrl:GFP) embryos, we observed that about 44% (7/16) of the mosaic embryos bearing endothelial-specific TAZ overexpression exhibited the aberrant vessel sprouting effect induced by YAP-5SA or TAZ-4SA mRNAs injection (Fig. 6C,E). Moreover, by injecting pDestTol2CG2-fli1a-TAZ-4SA plasmid in Tg(Hsa.CTGF:nlsmCherry)^ia49; Tg(kdrl:GFP)^s843 embryos, we detected a strong induction of Hsa.CTGF:nlsmCherry reporter expression (i.e. Yap1/Taz nuclear activity) specifically in endothelial cells undergoing sprouting (Fig. 6F). These data indicate that the upregulation of Yap1/Taz activity in single endothelial cells is sufficient to promote aberrant ISVs sprouting.

We further confirmed the autonomous nature of the phenotype and ruled out the possibility that mosaic expression outside of the promoter-specific domains upon DNA injection might be partially responsible for the observed sprouting. We carried out an endothelium-specific Yap1/Taz upregulation experiment by transiently expressing an activated form of Yap1 (yap1-1β^S87A or CAYAP1)⁵³ fused with the fluorescent protein mKate. The injection of the PCS2-fli1a-mKate-CAYAP1 plasmid in one-cell stage Tg(kdrl:GFP) embryos allowed us to detect, with one-cell resolution, the vessel domains in which we were actually upregulating the pathway and to demonstrate the correlation between vascular abnormalities and vascular-specific CAYAP1 expression. Similar to the pDestTol2CG2-fli1a:TAZ-4SA system, in the mosaic PCS2-fli1a-mKate-CAYAP1 injected embryos we observed a specific expression of the mKate-CAYAP1 in conjunction with the ISVs undergoing anomalous endothelial sprouting (Fig. 6G).

Discussion

YAP1/TAZ signaling has recently gained much attention in developmental, cancer and regeneration biology. However, most data in adult mammalian tissues indicate that YAP1/TAZ are dispensable for homeostatic self-renewal and become required only upon genetic deletion of inhibitory cues such as Hippo, or upon inflammatory and tumorigenic stimuli; this led to the still unresolved issue of whether YAP1/TAZ are active or not in most adult tissues. Here, we described the generation, validation and characterization of a Yap1/Taz zebrafish reporter, which represents a powerful tool to follow this signaling activity in a living vertebrate, offering also interesting applications in drug screening, cancer and regenerative biology.

Validation through knockdown and overexpression approaches demonstrated that the Hsa.CTGF-based zebrafish transgenic lines faithfully report in vivo Yap1/Taz activity, being able to reveal significant corresponding variations. A first Yap1/Taz zebrafish reporter, named 4xGTIIC, has been previously published by Miesfeld and Link⁴¹. During embryogenesis, a strong Yap1/Taz activity was observed in the same regions of the Hsa.CTGF-based reporter presented here, such as the epidermis, heart, otic and lens vesicles, midbrain-hindbrain boundary (MHB) region and striated muscle of the trunk. However, we described a much wider expression pattern for the Hsa.CTGF reporter with respect to that described for the 4xGTIIC, although the latter was mainly described with a destabilized GFP-expressing line, which makes hard to do comparisons. The Hsa.CTGF transgenic line allowed us to point out regions of Yap1/Taz nuclear activity previously not described in the other reporters for the pathway (rhombencephalon, neural tube, notochord, floorplate, pharyngeal arches, and pectoral fin). The Hsa.CTGF reporter activation in the myl7 positive cardiac precursor cells and in the heart is consistent with that of the Tg(eef1a1l1:Gal4db-TEAD2ΔN-2A-mCherry);(UAS:GFP) reporter developed by Fukui and colleagues⁴². The activity in the endothelium, not shown in the 4xGTIIC reporter, is also consistent with the Tg(eef1a1l1:Gal4db-TEAD2ΔN-2A-mCherry);(UAS:GFP) reporter¹⁸, as well as with the endothelium-specific reporter described by Nagasawa-Masuda and Terai²⁰. Yap1 and TEAD2 transcriptional activity has been shown to be modulated in vivo by blood flow¹⁸ and, similarly, we confirmed the positive regulation exerted by the circulation on Yap1/Taz activity by showing that the also the expression of Hsa.CTGF:nlsmCherry in endothelial cells is modulated by blood flow. Notably, the Hsa.CTGF reporter is responsive to the synthetic glucocorticoid Dexamethasone, confirming in vivo the recent findings of Sorrentino and colleagues³⁹, and highlighting the potential application of Hsa.CTGF reporter lines in drugs screenings.

A potential criticism might be that the Hsa.CTGF Yap1/Taz transgenic lines are reporting the expression pattern of the zebrafish Yap1/Taz target genes ctgfa rather than the global TEAD-dependent Yap1/Taz signaling activity. There are several reasons ruling out this hypothesis: (i) the reporter expression is driven by a 200 bp fragment of the human CTGF promoter, that represents only a minimal part with respect to the whole promoter regulating the expression of the CTGF gene. Pfefferli and Jazwinska analyzed all the transcription factor binding sites within the 3.18 kb upstream regulatory sequence of ctgfa, showing that the vast majority of binding sites are outside the 200 bp fragment that was used to drive the Hsa.CTGF transgene expression⁵⁴. (ii) The zebrafish Tg(ctgfa:EGFP) reporter based on the 3.18 kb ctgfa promoter described by Pfefferli and Jazwinska showed a different expression pattern from the Hsa.CTGF reporter, being limited to the notochord, the heart and the connective tissue of regenerating fins^54,55. (iii) In spite of a partial overlap (lens, otic vesicles, heart, pharyngeal arches, pectoral fin, and floorplate), the expression of the ctgfa/b gene and the Hsa.CTGF:nlsmCherry transgene is different. For instance, in situ hybridization performed to detect the mCherry expression on 48 and 72 hpf Tg(Hsa.CTGF:nlsmCherry) reporter embryos (data not shown) clearly labels the MHB and the rhombencephalic regions, which are not positive for ctgfa/b expression⁵⁶. On the contrary, ctgfa is expressed in the pancreatic bud (https://zfin.org/ZDB-FIG-060130-1737) while the Hsa.CTGF reporter doesn’t exhibit transgene activity in this region.

The analysis of the spatio-temporal activation of Hsa.CTGF reporter revealed a wide activation of Yap1/Taz signaling during early embryonic development, with a stronger signal in the proliferating and undifferentiated tail bud mesenchyme. This is likely reflecting the main function for YAP1/TAZ as transducers of the Hippo pathway: promotion of cell proliferation and organ growth during development^44,57,58. In adulthood, YAP1/TAZ expression is strongly limited, being enriched in the stem/progenitor cells niches⁵⁹. Consistently, the Hsa.CTGF reporter is largely silenced in the fully-grown fish with respect to the embryonic and larval development. Highly positive organs or tissues in the adult include the ovary, the lens, the heart and the endothelium. While in the lens the presence of the reporter protein could be simply due to almost absent protein turnover of these cells⁶⁰, the persistence of Yap1/Taz activity in the cardiovascular system suggests its important role in the maintenance of cardiac and vascular functions. This is in agreement with the requirement of Yap1 for the maintenance of blood vessels during zebrafish development¹⁸.

yap1 and taz single knockouts, unlike the double mutants, do not exhibit significant vascular phenotypes, implying a functional redundancy of the two genes during vascular development. Nevertheless, we described slight defects in the cranial and ocular vasculatures of the yap1^−/− embryos, phenotypes not reported in previous works. While the specific reduction of the number of hyaloid vessels in yap1 mutants might be a consequence of coloboma, yap1^−/−;taz^+/− animals exhibited an unusual phenotype during the formation of the axial vessels: the PCV deviates intermittently from the midline, and occasionally exhibits two distinct lumens. Analysis of arterial/venous-specific markers revealed that defective specification of the DA is also observed. Specifically, the expression of mrc1a in 32 hpf yap1^−/−;taz^+/− embryos is not restricted to the PCV and venous/lymphatic vessels as in WT⁵¹, but is still observed in the DA.

Notably, the overactivation of Yap1/Taz in the endothelium was sufficient to cause an abnormal vessel sprouting phenotype with formation of atypical anastomosis between adjacent ISVs, a result consistent with recent evidences in vitro and in mouse models on the role of YAP1/TAZ in angiogenesis^{16,17,61,62,63}.

Altogether, our results present a novel comprehensive in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Together with the vascular phenotypes displayed by yap1/taz mutants and upon endothelium-specific upregulation of Yap1/Taz/Tead-mediated transcription, it confirms and further extends the emerging role of Yap1/Taz signaling in vessel maintenance and developmental angiogenesis.

Material and Methods

Animals

All live animal procedures were approved by the institutional ethics committee for animal testing of the University of Padua and the Max Planck Society as well as in accordance with the relevant guidelines and regulations of Italy, Germany and European Union.

To inhibit pigment formation, embryos and larvae were incubated in 0.003% 1-phenyl-2-thiourea (PTU). The following fish lines were used and outcrossed either to wild-type fish or to the Tg(Hsa.CTGF:nlsmCherry)^ia49 Yap1/Taz reporter line: Tg(myl7:GFP)⁶⁴, Tg(Xla.Eef1a1:GFP)^s854⁴⁷ (gut/GFP), and Tg(kdrl:GFP)^s843⁶⁵ Tg(−5.2lyve1b:dsRed)^nz101 ⁶⁶, TgBAC(etv2:EGFP)^ci1 ⁶⁷, Tg(fli1a:EGFP)^y1 ⁶⁸, and Tg(2xID1BRE:nlsmCherry)^ia17 ⁶⁹. In the TgBAC(etv2:EGFP)^ci1 line, the GFP marks the whole vascular system under the promoter control of the early regulator of vascular and myeloid development etsrp/etv2. In Tg(−5.2lyve1b:DsRed)^nz101 line the DsRed is expressed in major axial veins and the lymphatic system under the control of the lyve1 promoter. Mutant alleles used in this paper are: yap1^bns19⁴⁸ and taz^bns35^48,49.

Generation of Tg(Hsa.CTGF:eGFP) ^ia48 and Tg(Hsa.CTGF:nlsmCherry) ^ia49

The −200/+ 27-CTGF promoter fragment was amplified by PCR from a human genomic DNA using the following oligonucleotides:

Hsa.CTGF-for (5′-TCTAGAAGATCTTCTGTGAGCTGGAGTGTGC-3′) and Hsa-CTGF-rev (5′-AAGCTTCCATGGAGCGGGGAAGAGTTGTTGT-3′). The CTGF promoter fragment was subcloned in the Gateway 5′ entry vector pME-MCS (Invitrogen) using the BglII and HindIII restriction enzymes. The resulting p5E-Hsa.CTGF entry vector was recombined with the nlsmCherry, and eGFP-carrying middle entry vectors and the p3E-polyA entry clone containing the SV40 late polyA signal (Invitrogen). Entry plasmids were recombined into the Tol2 destination vector pDestTol2pA2 (Invitrogen) through a MultiSite Gateway LR recombination reaction as previously described⁵². 30 pg of Tol2 destination vectors and 25 pg of Tol2 transposase mRNA⁷⁰ were co-injected into one cell-stage wild-type zebrafish embryos. Microinjected embryos were selected for mosaic transgenic expression at 24 and 72 hpf using an epifluorescent microscope, raised to adulthood and outcrossed to wild-type fish. Overall, 13 out of 17 screened fish were identified as founders. Founder fish for each reporter line, named Tg(Hsa.CTGF:eGFP)^ia48 and Tg(Hsa.CTGF:nlsmCherry)^ia49, were selected for fluorescence level and the number of transgenic insertions in order to establish stable transgenic lines with a single insertion.

Morpholinos, mRNA and plasmids injections

The antisense morpholino oligos were obtained from Gene Tools, LLV (U.S.). The following splice blocking (MO-Yap and MO-Taz) and control morpholinos were used: MO-Yap1: 5′-GCA ACA TTA ACA ACT CAC TTT AGG A-3′⁷¹; MO-Taz: 5′-GTA TGT GTT TCA CAC TCA CCC AGG T-3′; MO-tnnt2a (sih): 5′-CAT GTT TGC TCT GAT CTG ACA CGC A-3′⁷² MO-ctrl: 5′-AGA ACA TAA TCA GTA GTG TTC GA-3′. The MO stock solution (1 mM) was diluted in Danieau’s solution, and ∼1 nL was injected per embryo as previously described⁷³.

YAP-5SA is a constitutively active version of the human YAP1 protein, which has been mutated in its five key serine residues, resulting insensitive to LATS1/2-dependent phosphorylation and sequestration in the cytoplasm. Analogously, TAZ-4SA is the constitutively active version of the murine TAZ protein, mutated in its four serine residues recognized by LATS1/2. TEAD-VP16 is a fusion protein of the N-terminal region of TEAD transcription factor and the activation domain of herpes simplex virus VP16. TEAD-VP16 does not need any transcriptional co-activator to work, leading again to a constitutive transcription of its target genes⁷⁴. pCS2-Flag-mTAZ-4SA, pCS2-TEAD-VP16 and pCSP1-Flag-YAP-5SA were digested with a specific restriction enzyme (NotI for Flag-TAZ-4SA and TEAD-VP16, AscI for Flag-YAP-5SA) and transcribed using the SP6 polymerase (AM1340, Lifetechnology). In the overexpression experiments, one cell-stage Tg(Hsa.CTGF:nlsmCherry)^ia49 embryos, Tg(kdrl:GFP) embryos or Tg(Hsa.CTGF:nlsmCherry)^ia49/Tg(kdrl:GFP) double transgenic embryos were injected with 0,2/0,4 pg of TAZ-4SA and TEAD-VP16 mRNAs and 5/10 pg of YAP-5SA mRNA per embryo. To avoid phenotypic alterations associated with RNA toxicity we injected the lowest concentration of constitutively active forms of YAP and TAZ showing biological activity. The injections of the same amount of wild type YAP/TAZ RNA (used as injection control) did not induce induced overexpression effects in zebrafish embryos. Flag-mTAZ-4SA⁷⁵ was subcloned in pME-MCS (Invitrogen) from a pCS2 vector.MultiSite Gateway LR recombination reaction was performed to recombine the obtained pME-TAZ-4SA, the p5E-fli1a and the p3E-polyA (Invitrogen) into the Tol2 destination vector pDestTol2CG2 (Invitrogen). 20–40 pg of the recombined Tol2 destination vectors were co-injected into one cell-stage Tg(kdrl:GFP) zebrafish embryos. The effect of Yap1/Taz transient overactivation in the endothelium by TAZ-4SA expression was analysed at 32 hpf by confocal microscopy. Mkate-CAYAP1 plasmid⁵³ was injected into one cell-stage Tg(kdrl:GFP) embryos that were analysed by confocal microscopy at 32 hpf.

Image acquisition and analysis

The fluorescence was visualized using 488 nm (for GFP) and 561 nm (for mCherry) lasers and 20x or 40x immersion objectives (Nikon).

Fluorescence quantification of the images acquired either with the conventional fluorescence or the confocal microscope was carried out using Fiji software, by quantifying the fluorescent signal as integrated density as described elsewhere⁷⁶. For the quantification of the reporter signal specific of the endothelium, Hsa.CTGF:nlsmCherry reporter fluorescence was acquired with the confocal microscope together with the kdrl:GFP fluorescence in 32 hpf double transgenic embryos. In order to isolate the reporter expression in the endothelium, Fiji software was used to filter the mCherry signal using the kdrl:GFP as a mask. For each embryo, a confocal Z-stack projection was realized with the obtained filtered images, and then the fluorescent signal was quantified.

Generation of yap1 and taz mutants

The generation of the taz^bns35 mutant line (a.k.a wwtr1^bns35) was described elsewhere^48,49. To generate the yap1^bns19 mutant line, a specific single guide RNA (sgRNA) 5′-ACC TCA TCG GCA CGG AAG GG; was designed (crispr.mit.edu) and cloned into the pT7-gRNA vector (Addgene plasmid #46759). yap1-sgRNA in vitro transcription was performed with the MEGAshortscript T7 transcription kit (Ambion) using as template the pT7-gRNA vector linearized with BsmBI. 100 pg of sgRNA and 150 pg of CAS9 mRNA (obtained from the Addgene plsmid #46757) were co-injected into one-cell stage AB embryos. The yap1^bns19 allele is a 41 bp deletion. Genotyping primer pair for yap1^bns19 is 5′- CTG TTT GTG GTT TCT GAG GGG-3′ and 5′-TGA GAA AGC TGC CAG ACT CA-3′. Genotyping primer pair for taz^bns35 is 5′-TTT GTT GTG CAG TCA CAT TGA G-3′ and 5′-GAG GGC GTC ATG CTC TTC-3′. These alleles can be genotyped by standard PCR and resolved with gel electrophoresis (Fig. S7). The heterozygotes were crossed to Tg(Hsa.CTGF:nlsmCherry)^ia49, TgBAC(etv2:EGFP)^ci1 and Tg(−5.2lyve1b:DsRed)^nz101 for analyses.

Quantifying Hsa.CTGF reporter signal in the endothelium of yap1/taz mutants

An incross of yap1^+/−;taz^+/− fish were performed to obtain embryos for this experiment. Double homozygous mutants die by 30 hpf and are excluded from analysis. Remaining siblings that are TgBAC(etv2:EGFP) and Tg(Hsa.CTGF:nlsmCherry) positive were embedded in 1% low melting agarose and images were acquired with spinning disk confocal microscope (25x objective) at 48 hpf. Image analyses were done with Imaris software. Firstly, EGFP positive cells were selected to delineate endothelial cell nuclei expressing mCherry. Only endothelium on the side of the embryo closest to the objective lens was analyzed. For each nucleus, average signal intensity of mCherry channel was normalized to average signal intensity of EGFP. The normalized values for all selected nuclei were averaged per animal. The calculation can be summarized by the following equation:

$${X}_{j}=\frac{{\sum }_{i=1}^{n}\frac{mcherr{y}_{ij}}{EGF{P}_{ij}}}{n}$$

where X_j is the readout for fish j = 1…N; mCherry and EGFP are average intensities of respective channels for nuclei i = 1…n.

Characterization of the PCV phenotype

yap1^−/−;taz^+/− embryos and randomly sampled siblings were embedded in 1% low melting agarose for image acquisition with an LSM800 confocal microscope (25x objective) at 30 and 48 hpf. The orthogonal projection function on the Zen software was utilized to obtain transverse sections of the trunk. For cryosection of 72 hpf, yap1^−/−;taz^+/− larvae and randomly sampled siblings animals, were fixed with 4% PFA and genotyped using a small piece of the tail. Larvae positive for the TgBAC(etv2:EGFP) transgene were selected. Only yap1^−/−;taz^+/− and WT sibling larvae were embedded in OCT using standard procedures. Each section is 12 μm thick. Sections were permeabilized with 0.1% Triton X-100 followed by blocking with 5% sheep serum. Standard immunohistochemistry was performed with anti-EGFP antibody (GFP-1020, Aves Labs, Portland, OR) and Alexa-568 conjugated phalloidin (A12380, Thermo Fisher). Sections were counterstained with DAPI and imaged with LSM800.

Characterizing cranial and hyaloid vasculature phenotype in yap1 mutants

Scoring of the cranial vasculature phenotypes in yap1 mutants (obtained from an incross of yap1 heterozygous adults) was performed blind under a Nikon SMZ25 stereomicroscope. Images of the cranial vasculature in 72 hpf yap1 mutants (Figure S8A) were obtained with an LSM700 confocal microscope (20x objective). Images of hyaloid vessels of both eyes from 5 dpf animals were taken with spinning disk confocal microscope (40x objective) from the dorsal side of the eye (Fig. S8B).

Luciferase assays

Luciferase assays were performed in MDA-MB-231 cells with the novel YAP1/TAZ-responsive reporter Hsa.CTGF-Lux. The −200/+27-CTGF promoter fragment was cloned in the pGL3-basic Luciferase Reporter Vector (Promega). Luciferase reporters (50 ng/cm²) were transfected together with CMV-β-gal (75 ng/cm²) to normalize for transfection efficiency. Each sample was transfected in duplicate and each experiment was repeated at least three times independently. NF2 is Addgene #19701. Latrunculin A was from Santa Cruz. The sequences of the siRNA used in this study are as follows (sense strand sequences are indicated): YAP1 1: GACAUCUUCUGGUCAGAGA dTdT; YAP1 2: CUGGUCAGAGAUACUUCUU dTdT; TAZ 1: ACGUUGACUUAGGAACUUU dTdT; TAZ 2: AGGUACUUCCUCAAUCACA dTdT; control: UUCUCCGAACGUGUCACGU dTdT. YAP1/TAZ siRNA 1 refers to the mix composed of oligos YAP1 1 and TAZ 1; YAP1/TAZ siRNA 2 refers to the mix composed of oligos YAP1 2 and TAZ 2.

Protein extraction and western blot analysis

For protein extraction 50 embryos per group at 72 hpf were deyolked (deyolking buffer: 55 mM NaCl, 1,8 mM KCl, 1,15 mM NaHCO₃), washed (washing buffer: 110 mM NaCl, 3,5 mM KCl, 2,7 mM CaCl₂, 10 mM Tris HCl pH 8,5) and lysed in RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitor cocktail (Sigma). Protein concentrations were determined using the Bradford assay (BioRad) and western blot was performed as previously described⁷⁷.

Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Whole-mount in situ hybridization (WISH)

Standard WISH procedure was performed as described previously⁷⁸. yap1^−/−;taz^+/− embryos from a cross between yap1^+/−;taz^+/− and yap1^+/− fish were identified by their notochord or tail phenotype (see above) and fixed with 4% PFA at 24 and 32 hpf. In parallel, WT embryos from WT fish crosses were fixed with 4% PFA at 24 and 32 hpf. Both yap1^−/−;taz^+/− and WT embryos at each developmental stage were mixed into the same reaction tube after Proteinase K permeabilization. Images were acquired with Nikon SMZ25 stereomicroscope followed by genotyping. The primers used to synthesize probes for efnb2a and mrc1a are found in Table S1.

Statistical analyses

In Figs 2, 6, S4 and S7 data are presented as mean ± SEM and statistical comparison between groups were performed using a two-tailed Student’s t-test. Statistical analyses were carried out with Prism software (GraphPad). Statistical tests for Fig. 5C,D were performed using Poisson regression with glm function in R. The cranial vasculature phenotype (Fig. S8A’) has been evaluated using a chi-square test. The number of hyaloid vessel (Fig. S8B’) was tested using standard Student T Test. Boxplots were generated with ggplot2 package in R.

References

Piccolo, S., Dupont, S. & Cordenonsi, M. The Biology of YAP/TAZ: Hippo Signaling and Beyond. Physiol. Rev. 94, 1287–1312 (2014).
Article PubMed CAS Google Scholar
Santinon, G., Pocaterra, A. & Dupont, S. Control of YAP/TAZ Activity by Metabolic and Nutrient-Sensing Pathways. Trends in Cell Biology 26, 289–299 (2016).
Article PubMed CAS Google Scholar
Praskova, M., Xia, F. & Avruch, J. MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr. Biol. 18, 311–321 (2008).
Article PubMed PubMed Central CAS Google Scholar
Chan, E. H. Y. et al. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24, 2076–2086 (2005).
Article PubMed CAS Google Scholar
Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).
Article PubMed CAS Google Scholar
Dong, J. et al. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130, 1120–1133 (2007).
Article PubMed PubMed Central CAS Google Scholar
Zhao, B. et al. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747–2761 (2007).
Article PubMed PubMed Central CAS Google Scholar
Lei, Q.-Y. et al. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426–2436 (2008).
Article PubMed PubMed Central CAS Google Scholar
Yu, F. X. & Guan, K. L. The Hippo pathway: Regulators and regulations. Genes and Development 27, 355–371 (2013).
Article PubMed CAS Google Scholar
Zhao, B., Li, L., Lei, Q. & Guan, K.-L. The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. Genes Dev. 24, 862–874 (2010).
Article PubMed PubMed Central CAS Google Scholar
von Gise, A. et al. YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy. Proc. Natl. Acad. Sci. USA 109, 2394–9 (2012).
Article ADS Google Scholar
Hu, J. et al. Yes-associated protein (yap) is required for early embryonic development in zebrafish (Danio rerio). Int. J. Biol. Sci. 9, 267–278 (2013).
Article PubMed PubMed Central CAS Google Scholar
Wang, Y. et al. Deletion of yes-associated protein (YAP) specifically in cardiac and vascular smooth muscle cells reveals a crucial role for YAP in mouse cardiovascular development. Circ. Res. 114, 957–965 (2014).
Article PubMed PubMed Central CAS Google Scholar
Varelas, X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development 141, 1614–26 (2014).
Article PubMed CAS Google Scholar
Azzolin, L. et al. YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170 (2014).
Article PubMed CAS Google Scholar
Wang, X. et al. YAP/TAZ Orchestrate VEGF Signaling during Developmental Angiogenesis. Dev. Cell 42, 462–478 (2017).
Article PubMed CAS Google Scholar
Kim, J. et al. YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation. J. Clin. Invest. 127, 3441–3461 (2017).
Article PubMed PubMed Central Google Scholar
Nakajima, H. et al. Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance. Dev. Cell 40, 523–536.e6 (2017).
Article PubMed CAS Google Scholar
Singh, A. et al. Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development. Cell Rep. 15, 1384–1393 (2016).
Article PubMed PubMed Central CAS Google Scholar
Nagasawa-Masuda, A. & Terai, K. Yap/Taz transcriptional activity is essential for vascular regression via Ctgf expression and actin polymerization. PLoS One 12, (2017).
Wang, K.-C. et al. Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis. Proc. Natl. Acad. Sci. 113, 11525–11530 (2016).
Article PubMed CAS Google Scholar
Huang, Y. et al. Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow. Nature 540, 579–582 (2016).
Article ADS CAS Google Scholar
wang, lunchang et al. Yes-Associated Protein Inhibits Transcription of Myocardin and Attenuates Differentiation of Vascular Smooth Muscle Cell from Cardiovascular Progenitor Cell Lineage. Stem Cells 35, 351–361 (2017).
Article PubMed CAS Google Scholar
Feng, X. et al. Thromboxane A2 Activates YAP/TAZ Protein to Induce Vascular Smooth Muscle Cell Proliferation and Migration. J. Biol. Chem. 291, 18947–58 (2016).
Article PubMed PubMed Central CAS Google Scholar
Kimura, T. E. et al. The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP. J. Mol. Cell. Cardiol. 90, 1–10 (2016).
Article PubMed PubMed Central CAS Google Scholar
Xie, C. et al. Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin. J. Biol. Chem. 287, 14598–14605 (2012).
Article PubMed PubMed Central CAS Google Scholar
Isogai, S., Horiguchi, M. & Weinstein, B. M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev. Biol. 230, 278–301 (2001).
Article PubMed CAS Google Scholar
Roman, B. L. & Weinstein, B. M. Building the vertebrate vasculature: research is going swimmingly. Bioessays 22, 882–893 (2000).
Article PubMed CAS Google Scholar
Lawson, N. D. & Weinstein, B. M. Arteries and veins: making a difference with zebrafish. Nat. Rev. Genet. 3, 674–682 (2002).
Article PubMed CAS Google Scholar
Lambrechts, D. & Carmeliet, P. Genetics in zebrafish, mice, and humans to dissect congenital heart disease: insights in the role of VEGF. Curr. Top. Dev. Biol. 62, 189–224 (2004).
Article PubMed CAS Google Scholar
Dejana, E., Tournier-Lasserve, E. & Weinstein, B. M. The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev. Cell 16, 209–221 (2009).
Article PubMed CAS Google Scholar
Jensen, L. D. et al. Zebrafish models to study hypoxia-induced pathological angiogenesis in malignant and nonmalignant diseases. Birth Defects Res. Part C - Embryo Today Rev. 93, 182–193 (2011).
Article CAS Google Scholar
Stainier, D. Y. Zebrafish genetics and vertebrate heart formation. Nat. Rev. Genet. 2, 39–48 (2001).
Article PubMed CAS Google Scholar
Coffin, J. D. & Poole, T. J. Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos. Development 102, 735–748 (1988).
PubMed CAS Google Scholar
Herbert, S. P. & Stainier, D. Y. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol 12, 551–564 (2011).
Article PubMed PubMed Central CAS Google Scholar
Herbert, S. P. et al. Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation. Science 326, 294–298 (2009).
Article ADS PubMed PubMed Central CAS Google Scholar
Zhao, B. et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22, 1962–1971 (2008).
Article PubMed PubMed Central CAS Google Scholar
Aragona, M. et al. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154, 1047–1059 (2013).
Article PubMed CAS Google Scholar
Sorrentino, G. et al. Glucocorticoid receptor signalling activates YAP in breast cancer. Nat. Commun. 8, 140173 (2017).
Article CAS Google Scholar
Burns, C. G. et al. High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat. Chem. Biol. 1, 263–264 (2005).
Article PubMed CAS Google Scholar
Miesfeld, J. B. & Link, Ba 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. Mech. Dev. 133, 177–88 (2014).
Article PubMed CAS Google Scholar
Fukui, H. et al. S1P-Yap1 signaling regulates endoderm formation required for cardiac precursor cell migration in zebrafish. Dev. Cell 31, 128–136 (2014).
Article PubMed CAS Google Scholar
Chen, Z., Friedrich, G. A. & Soriano, P. Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice. Genes Dev. 8, 2293–2301 (1994).
Article PubMed CAS Google Scholar
Sawada, A. et al. Redundant roles of Tead1 and Tead2 in notochord development and the regulation of cell proliferation and survival. Mol. Cell. Biol. 28, 3177–3189 (2008).
Article PubMed PubMed Central CAS Google Scholar
Xin, M. et al. Regulation of Insulin-like growth factor signaling by Yap governrs cardiomyocyte proliferation and embryonic heart size. Sci. Signal. 4, ra70 (2011).
Article PubMed PubMed Central CAS Google Scholar
Heallen, T. et al. Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size. Science. 332, 458–461 (2011).
Article ADS PubMed PubMed Central CAS Google Scholar
Field, H. A., Ober, E. A., Roeser, T. & Stainier, D. Y. R. Formation of the digestive system in zebrafish. I. Liver morphogenesis. Dev. Biol. 253, 279–290 (2003).
Article PubMed CAS Google Scholar
Kimelman, D., Smith, N. L., Lai, J. K. H. & Stainier, D. Y. R. Regulation of posterior body and epidermal morphogenesis in zebrafish by localized Yap1 and Wwtr1. Elife 6, (2017).
Lai, J. K. H. et al. The Hippo pathway effector Wwtr1 regulates cardiac wall maturation in zebrafish. Development 145, (2018).
Miesfeld, J. B. et al. Yap and Taz regulate retinal pigment epithelial cell fate. Development 142, 3021–3032 (2015).
Article PubMed PubMed Central CAS Google Scholar
Jung, H. M. et al. Development of the larval lymphatic system in zebrafish. Development 144, 2070–2081 (2017).
Article PubMed PubMed Central CAS Google Scholar
Kwan, K. M. et al. The Tol2kit: A multisite gateway-based construction Kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).
Article PubMed CAS Google Scholar
Cox, A. G. et al. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nat. Cell Biol. 18, 886–96 (2016).
Article PubMed PubMed Central CAS Google Scholar
Pfefferli, C. & Jaźwińska, A. The careg element reveals a common regulation of regeneration in the zebrafish myocardium and fin. Nat. Commun. 8, (2017).
Chiou, M. J., Chao, T. T., Wu, J. L., Kuo, C. M. & Chen, J. Y. The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region. Biochem. Biophys. Res. Commun. 349, 750–758 (2006).
Article PubMed CAS Google Scholar
Fernando, C. A. et al. Temporal and spatial expression of CCN genes in zebrafish. Dev. Dyn. 239, 1755–1767 (2010).
Article PubMed PubMed Central CAS Google Scholar
Wu, S., Huang, J., Dong, J. & Pan, D. hippo Encodes a Ste-20 Family Protein Kinase that Restricts Cell Proliferation and Promotes Apoptosis in Conjunction with salvador and warts. Cell 114, 445–456 (2003).
Article PubMed CAS Google Scholar
Lu, L. et al. Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver. Proc. Natl. Acad. Sci. USA 107, 1437–1442 (2010).
Article ADS PubMed Google Scholar
Ramos, A. & Camargo, F. D. The Hippo signaling pathway and stem cell biology. Trends Cell Biol. 22, 339–346 (2012).
Article PubMed PubMed Central CAS Google Scholar
Stewart, D. N. et al. Carbon turnover in the water-soluble protein of the adult human lens. Mol. Vis. 19, 463–475 (2013).
PubMed PubMed Central CAS Google Scholar
Choi, H.-J. et al. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2. Nat. Commun. 6, 6943 (2015).
Article PubMed CAS Google Scholar
Mascharak, S. et al. YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography. Biomaterials 115, 155–166 (2017).
Article PubMed CAS Google Scholar
Wei, H. et al. Verteporfin suppresses cell survival, angiogenesis and vasculogenic mimicry of pancreatic ductal adenocarcinoma via disrupting the YAP-TEAD complex. Cancer Sci. 108, 478–487 (2017).
Article PubMed PubMed Central CAS Google Scholar
Burns, C. E., Traver, D., Mayhall, E., Shepard, J. L. & Zon, L. I. Hematopoietic stem cell fate is established by the Notch – Runx pathway. 2331–2342 https://doi.org/10.1101/gad.1337005.binds (2005).
Beis, D. et al. Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development 132, 4193–4204 (2005).
Article PubMed CAS Google Scholar
Okuda, K. S. et al. Lyve1 Expression Reveals Novel Lymphatic Vessels and New Mechanisms for Lymphatic Vessel Development in Zebrafish. Development 139, 2381–91 (2012).
Article PubMed PubMed Central CAS Google Scholar
Proulx, K., Lu, A. & Sumanas, S. Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis. Dev. Biol. 348, 34–46 (2010).
Article PubMed CAS Google Scholar
Lawson, N. D. & Weinstein, B. M. In Vivo Imaging of Embryonic Vascular Development Using Transgenic Zebrafish. Dev. Biol. 248, 307–318 (2002).
Article PubMed CAS Google Scholar
Moro, E. et al. Generation and application of signaling pathway reporter lines in zebrafish. Mol. Genet. Genomics 288, 231–242 (2013).
Article PubMed PubMed Central CAS Google Scholar
Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).
Article PubMed CAS Google Scholar
Mateus, R. et al. Control of tissue growth by Yap relies on cell density and F-actin in zebrafish fin regeneration. Development 142, 2752–2763 (2015).
Article PubMed CAS Google Scholar
Sehnert, A. J. et al. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat. Genet. 31, 106–110 (2002).
Article PubMed CAS Google Scholar
Vettori, A. et al. Developmental defects and neuromuscular alterations due to mitofusin 2 gene (MFN2) silencing in zebrafish: A new model for Charcot-Marie-Tooth type 2A neuropathy. Neuromuscul. Disord. 21, 58–67 (2011).
Article PubMed Google Scholar
Cao, X., Pfaff, S. L. & Gage, F. H. YAP regulates neural progenitor cell number via the TEA domain transcription factor. Genes Dev. 22, 3320–3334 (2008).
Article PubMed PubMed Central CAS Google Scholar
Azzolin, L. et al. Role of TAZ as mediator of wnt signaling. Cell 151, 1443–1456 (2012).
Article PubMed CAS Google Scholar
Facchinello, N., Schiavone, M., Vettori, A., Argenton, F. & Tiso, N. In Wnt Signaling: Methods and Protocols (eds Barrett, Q. & Lum, L.) 1481, 81–94 (Springer New York, 2016).
Vettori, A. et al. Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization. Proc. Natl. Acad. Sci. 114, 9948–9953 (2017).
Article PubMed CAS Google Scholar
Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).
Article PubMed CAS Google Scholar

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Acknowledgements

We would like to thank Luigi Pivotti and Martina Milanetto for their professional help in managing Padua Zebrafish Facility. We also thank Sébastien Gauvrit, Christian Helker, Alethia Villasenor and Kaska Koltowska for insightful discussions. The work of MA, FA and AV has been supported by AIRC grant IG 10274 and European Union Grant ZF-HEALTH CT-2010-242048 to FA. AV is supported by the Italian National Institute of Health Grant GR-2011-02346749. Work in the Stainier lab is supported by funds from the Max Planck Society, EU (ERC), DFG and Leducq Foundation.

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University of Padova, Department of Biology, Padova, Italy
Matteo Astone, Francesco Argenton & Andrea Vettori
Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
Jason Kuan Han Lai & Didier Y. R. Stainier
University of Padova, Department of Molecular Medicine, Padova, Italy
Sirio Dupont

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Matteo Astone
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Jason Kuan Han Lai
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Didier Y. R. Stainier
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M.A., A.V., J.K.H.L., F.A. and D.Y.R.S. designed experiments. M.A., A.V., J.K.H.L., S.D. performed experiments. M.A., A.V., J.K.H.L., D.Y.R.S. and F.A. performed data analysis and wrote the manuscript. All authors reviewed the manuscript.

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Astone, M., Lai, J.K.H., Dupont, S. et al. Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development. Sci Rep 8, 10189 (2018). https://doi.org/10.1038/s41598-018-27657-x

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Received: 15 January 2018
Accepted: 05 June 2018
Published: 05 July 2018
DOI: https://doi.org/10.1038/s41598-018-27657-x

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