Llgl1 Regulates Zebrafish Cardiac Development by Mediating Yap Stability in Cardiomyocytes Michael A

RESEARCH ARTICLE Llgl1 regulates zebrafish cardiac development by mediating Yap stability in cardiomyocytes Michael A. Flinn1,2,Cécile Otten3, Zachary J. Brandt1,2, Jonathan R. Bostrom1,2, Aria Kenarsary1,2,4,5, Tina C. Wan2,6, John A. Auchampach2,6, Salim Abdelilah-Seyfried3,7, Caitlin C. O’Meara2,4,5 and Brian A. Link1,2,*

ABSTRACT of Llgl1 also affected cardiac development, which is analyzed more The Hippo-Yap pathway regulates multiple cellular processes in deeply in this study. response to mechanical and other stimuli. In Drosophila, the polarity In Drosophila, L(2)gl has been shown to regulate the Hippo-Yap protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippo- pathway (Grzeschik et al., 2010; Parsons et al., 2014a). Hippo-Yap mediated transcriptional output. However, in vertebrates, little is known signaling is essential for the development and maintenance of many about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in organs. This pathway consists of several kinases and their associated regulating Yap stability in cardiomyocytes, which impacts heart activator proteins that form a core kinase complex that governs the development. In contrast to the role of Drosophila L(2)gl, Llgl1 activity of the transcriptional co-activators Yap and Wwtr1 (also depletion in cultured rat cardiomyocytes decreased Yap protein levels known as Taz) (Hao et al., 2008; Liu et al., 2011). The regulatory and blunted target gene transcription without affecting Yap transcript inputs that influence the activity of the kinase complex are still abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic poorly defined and likely vary among different organs and tissues. cardiomyocytes, pericardial effusion, impaired blood flow and aberrant Regulation of the Hippo-Yap pathway was first characterized in valvulogenesis. Cardiomyocyte Yap protein levels were decreased in Drosophila, and it was revealed that L(2)gl, along with other llgl1 morphants, whereas Notch, which is regulated by hemodynamic polarity factors, functions upstream of the core kinase complex and forces and participates in valvulogenesis, was more broadly activated. negatively regulates Yap/Wwtr1 transcriptional output (Parsons Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocyte- et al., 2014a). In Drosophila, l(2)gl mutations disrupt the membrane specific overexpression of Yap in Llgl1-depleted embryos ameliorated localization of both components of the core kinase complex and pericardial effusion and restored blood flow velocity. Altogether, our data Yorkie (the Drosophila Yap homolog), resulting in increased reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes nuclear enrichment of Yorkie and excessive target gene activation and its absence impairs cardiac development. (Grzeschik et al., 2010). Exactly how l(2)gl mutations result in increased nuclear Yorkie activity is unclear (Parsons et al., 2014a). KEY WORDS: Hippo-Yap pathway, Valvulogenesis, Cardiac However, the organization and composition of Hippo-Yap pathway development, Zebrafish components at cell junctions regulates Yorkie activity in other contexts, such as promoting activation of the core kinase complex INTRODUCTION (Sun et al., 2015). Whether the loss of Lgl1 in vertebrates impacts Lethal giant larvae (Lgl) proteins are regulators of cell polarity, cell Hippo-Yap signaling is currently unknown. junction stability and composition, as well as endomembrane The role of the Hippo-Yap pathway in cardiac development is well activities, including vesicle trafficking and acidification (Jossin documented (Wang et al., 2018). In cardiomyocytes, Hippo-Yap et al., 2017; Greenwood et al., 2016; Wang et al., 2011; Yamanaka signaling regulates proliferation and aspects of differentiation (Wang et al., 2003). Furthermore, within vertebrate cancer cells, the two et al., 2018). Mutations that negatively affect Hippo core kinase paralogs Llgl1 and Llgl2 affect cell migration (Greenwood et al., activity or transgenic over-expression of Yap result in cardiomegaly 2016; Kashyap et al., 2013). In zebrafish neuroepithelia, Llgl1 (Flinn et al., 2019; Heallen et al., 2011; von Gise et al., 2012; Xin et al., controls apical domain size and loss of function results in increased 2013). Conversely, the deletion of Yap or Tead1 in embryonic Notch activation (Clark et al., 2012). Although the focus of that cardiomyocytes results in decreased cardiomyocyte proliferation and a research was retinal neurogenesis, it was observed that knockdown thinned myocardial wall (Chen et al., 1994; von Gise et al., 2012; Xin et al., 2013) or cardiobifida (Miesfeld and Link, 2014). In the heart, Hippo-Yap is crucial for cardiomyocytes, as epicardial Hippo-Yap 1Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA. 2Cardiovascular Center, Medical College of signaling is essential for the proper differentiation of epicardium- Wisconsin, Milwaukee, WI 53226, USA. 3Institute for Biochemistry and Biology, derived cells, including fibroblasts and vascular smooth muscle cells University of Potsdam, 14476 Potsdam, Germany. 4Department of Physiology, (Xiao et al., 2018). Hippo-Yap signaling, like that of Notch, is also Medical College of Wisconsin, Milwaukee, WI 53226, USA. 5Genomics Sciences and Precision Medicine Center, Medical College of Wisconsin, Milwaukee, WI important for endocardial expression of neuregulin (Artap et al., 2018), 53226, USA. 6Department of Pharmacology, Medical College of Wisconsin, and has been implicated in regulating atrioventricular valve Milwaukee, WI 53226, USA. 7Institute for Molecular Biology, Hannover Medical development (Zhang et al., 2014). In humans, mutations in the School, 30625 Hannover, Germany. upstream Hippo-Yap pathway modulator dachsous (DCHS1)cause *Author for correspondence ([email protected]) mitral valve prolapse, a common cardiac valve disease (Durst et al., 2015). Considering the crucial role of Hippo-Yap signaling in cardiac B.A.L., 0000-0002-7173-2642 development and the link between L(2)gl and Hippo-Yap signaling in Handling Editor: Benoit Bruneau Drosophila,weassessedtheroleofLgl1onYapactivityinvertebrate

Received 5 June 2020; Accepted 10 July 2020 cardiomyocytes and investigated its effect on cardiac development and DEVELOPMENT

1 RESEARCH ARTICLE Development (2020) 147, dev193581. doi:10.1242/dev.193581 function in the zebrafish model. We found that depletion of Llgl1 in phenotype with heart morphogenesis (Clark et al., 2012), we vertebrate cardiomyocytes results in decreased levels of Yap protein investigated the relationship between Llgl1 function and Hippo-Yap and reduced transcription of Yap target genes. Our data indicate that signaling within cardiomyocytes. We initially depleted Llgl1,as Llgl1 is required for normal zebrafish cardiac development and well as Llgl2, in neonatal rat cardiomyocytes using siRNAs. siRNA function, as assessed by cardiomyocyte morphology, hemodynamics knockdown of Llgl1 or Llgl2, or both paralogs, did not alter Yap and valvulogenesis. Interestingly, although our study focuses on the mRNA abundance, as measured by qRT-PCR (Fig. 1A). However, systemic depletion of Llgl1, the aberrant cardiac phenotypes associated knockdown of either Llgl1 or Llgl2 did result in compensatory with Llgl1 were ameliorated with exogenous expression of Yap, upregulation of the paralog transcript. Knockdown of Yap did not specifically in cardiomyocytes. Collectively, this study is the first to affect mRNA abundance of either Llgl1 or Llgl2. Interestingly, in define the role of Llgl1 in Hippo-Yap regulation in cardiomyocytes contrast to Drosophila, in which mutation of l(2)gl results in and offers a potential mechanism for modulating Hippo-Yap signaling elevated Yorkie nuclear localization and transcriptional activity more precisely in cardiomyocytes, an area of increasing interest in the (Grzeschik et al., 2010; Parsons et al., 2014a), knockdown of Llgl1 field of cardiac regeneration (Heallen et al., 2011; Morikawa et al., or Llgl2 in primary rat cardiomyocytes resulted in decreased levels 2017; von Gise et al., 2012; Xin et al., 2013). of YAP protein, as assessed by western blot (Fig. 1B, Table S4). Consistent with lower YAP protein levels, the expression of YAP- RESULTS TEAD target genes decreased in cells treated with a combination of Llgl1 depletion in neonatal rat cardiomyocytes decreases Llgl1 and Llgl2 siRNA. Gene Set Enrichment Analysis (GSEA) on Yap protein and Yap transcriptional target expression RNA-seq data from cardiomyocytes simultaneously depleted of Llgl1 In Drosophila, L(2)gl protein modulates both the Notch and Hippo- and Llgl2 revealed a decrease in enrichment for YAP/WWTR1-TEAD Yap pathways (Grzeschik et al., 2010; Parsons et al., 2014b). In a direct transcriptional targets (Fig. 1C). This reference gene set was previous study, we found that knockdown of zebrafish Llgl1, one of defined by YAP ChIP-Seq and YAP knockdown experiments the two vertebrate homologs of Drosophila L(2)gl, resulted in performed on human MDA-MB-231 cells (Zanconato et al., 2015). increased Notch activity and reduced retinal neurogenesis (Clark We independently verified this GSEA by running the enrichment et al., 2012). In those studies, we also noted severe cardiac effusion analysis with RNA-seq data generated from cultured rat and dysmorphic hearts. Although the role of vertebrate Llgl1 on cardiomyocytes treated with either Yap siRNA or negative control Notch activity has been characterized (Clark et al., 2012), less is siRNA (Flinn et al., 2019, Fig. S1). Thus, although depletion of Llgl1 known about its potential impact on Hippo-Yap signaling. Because and Llgl2 does not alter the transcription of Yap, Llgl function is Yap signaling is known to modulate cardiomyocyte development required to maintain appropriate Yap protein levels and Yap-TEAD (von Gise et al., 2012), and given the strong Llgl1 depletion transcriptional activity in vertebrate cardiomyocytes.

Fig. 1. Knockdown of Llgl1 in cultured neonatal rat cardiomyocytes decreases YAP protein levels. (A) qRT-PCR results denoting RNA transcript levels of Llgl1, Llgl2 or Yap in isolated neonatal rat cardiomyocytes following siRNA treatments. n=3. One-way ANOVA, Tukey’s multiple comparisons test showing difference compared with negative control. Error bars indicate s.e.m. (B) Western blot analysis of anti-YAP and anti-γ-tubulin staining in siRNA treated rat cardiomyocytes. Quantification of anti-YAP band signal normalized to anti-γ- tubulin signal. n=6 for negative sequence, n=3 for untreated, n=9 for Llgl1, n=6 for Llgl2 and n=6 for Yap siRNA-treated cells. One- way ANOVA, Tukey’s multiple comparisons test. (C) GSEA for YAP/WWTR1/TEAD target genes using RNAseq data from siRNA-treated rat cardiomyocytes targeting Llgl1 and Llgl2 or a negative control sequence. n=3. Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001. DEVELOPMENT

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Developmental defects and lethality of llgl1 mutant zebrafish (Fig. 2D, Table S5). Altogether, these observations of llgl1−/− mutant To understand how loss of Llgl1 influences Yap protein levels fish demonstrate overt morphological phenotypes and impaired in vivo and to test whether Llgl1 also has a function during cardiac survival in a subset of mutants. We next used this line to characterize development, we generated an llgl1 mutant zebrafish line. zebrafish cardiac development and function in the absence of llgl1. Transcription activator-like effector nucleases (TALENs) were used to generate a ten (llgl1mw83) nucleotide deletion in exon 1 of Llgl1 depletion impairs cardiac development and function in llgl1, resulting in a frameshift mutation and generation of an early zebrafish stop codon (Fig. 2A, Fig. S2). Homozygous llgl1 mutant (llgl1−/−) To assess how the loss of Llgl1 affects cardiac development, we embryos closely resembled the morpholino knockdown phenotype crossed the llgl1mw83 line with a cardiomyocyte-reporter transgenic characterize previously in Clark et al. (2012). (Fig. 2B, Figs S3,S4). line Tg(myl7:eGFP)mw45 to visualize cardiomyocytes during Pericardial effusion, which can result from cardiac dysfunction, was development. At 48 hours postfertilization (hpf), a subset of llgl1−/− apparent in all llgl1−/− embryos by 3 days postfertilization (dpf) but embryos failed to undergo cardiac looping, whereas llgl1−/− embryos varied in expressivity, which was similarly observed in morpholino- with slight pericardial effusion resembled llgl1+/− siblings (Fig. 3A). treated embryos (Fig. 2B, Fig. S3). Interestingly, a subset of llgl1−/− This phenotype was not due to changes in atrial or ventricular embryos recovered from pericardial effusion to resemble wild-type cardiomyocyte cell numbers, as this did not change between groups siblings by 5 dpf and survived to adulthood. Variable expressivity (Fig. 3B). However, by 99 hpf, llgl1−/− hearts appeared larger than was not the result of differential maternal contribution of Llgl1 llgl1+/− siblings (Fig. 3A). Analysis using Imaris software (Bitplane) protein or llgl1 mRNA, as either maternal or paternal llgl1−/− fish revealed that a loss of Llgl1 resulted in larger atrial cardiomyocytes, as outcrossed to an llgl1+/− heterozygote yielded the same spectrum of indicated by increased internuclear distances, which is indicative of expressivity among their offspring (Fig. 2C). Although a subset of cardiomyocyte hypertrophy (Fig. 3C). As llgl1−/− mutant embryos do llgl1−/− fish recovered by 5 dpf, those displaying more overt not withstand microinjection, potentially because of epidermal pericardial effusion at 3 dpf progressed to exhibit edema throughout adhesion/resealing defects, a previously validated llgl1 morpholino the body and died by 6 dpf. Approximately two-thirds of the was used to mediate the knockdown of Llgl1 for subsequent co- llgl1−/− fish survived to adulthood based on a comparison of injection experiments (Clark et al., 2012). We first assessed cardiac measured survival versus Mendelian-predicted proportions of phenotypes of llgl1 morphant embryos that present a strong similarity homozygous mutants from a cross of llgl1−/− and llgl1+/− parents in gross morphology to llgl1−/− mutants (Fig. S3). Embryos injected

Fig. 2. Assessment of llgl1 mutant gross morphology. (A) Diagram depicting the 10 bp deletion generated by TALENs in exon 1 of llgl1. (B) Embryos (3 dpf) from an llgl1+/−×llgl1−/− cross categorized into three grades of severity: class 0, wild-type phenotype; class 1 (mild), subtle signs of pericardial effusion at 3 dpf; class 2, pronounced pericardial effusion with a small subset displaying smaller eyes; and class 3 (severe), pronounced pericardial effusion and/or body edema, small eyes and hearts fail to undergo cardiac looping (Movie 5). At 5 dpf, llgl1−/− embryos are undistinguishable from llgl1+/− siblings. Scale bars: 1 mm. (C) Quantification of phenotype classes from llgl1+/−×llgl1−/− crosses comparing maternal contribution. Three clutches were used for each condition. n=109 for maternal llgl1−/− and n=160 for paternal llgl1−/−. χ2 test. (D) Adult genotyping from six pooled llgl1+/−×llgl1−/− crosses, greater than 4 months of age. The dashed line represents the predicted Mendelian number for each genotype. n=58 for llgl1+/− and n=31 for llgl1−/−. Two-tailed binomial test. Error bars indicate s.e.m.

*P<0.05. DEVELOPMENT

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Fig. 3. Loss of llgl1 disrupts normal heart development. (A) Representative confocal z-stack images of myl7:eGFP expression in hearts at 48 hpf and 99 hpf in llgl1+/− and llgl1−/− siblings. A, atrium; V, ventricle. (B) Quantification of atrial and ventricular cardiomyocyte nuclei in 48 hpf llgl1 morphants versus uninjected controls. n=9 for atrial nuclei analysis, n=6 for uninjected control and n=7 for llgl1 morpholino microinjection for ventricular nuclei analysis. (C) Quantification of average minimum distance between atrial cardiomyocyte nuclei in llgl1+/− and llgl1−/− siblings at 48 hpf. n=3 for llgl1+/− hearts and n=4 for llgl1−/− hearts. (D) Representative confocal z-stack images of actin-stained 48 hpf hearts. (E) Representative single-section confocal micrograph of alcam or actin-stained 48 hpf hearts, focusing on the AVC. Red arrows depict valve leaflets, blue arrows depict large rounded ventricular cardiomyocytes and green arrows depict abnormal actin staining in the myocardium. Quantification of ventricular cardiomyocyte number and volume. n=4 for untreated embryos and n=6 for llgl1 morpholino-treated embryos. (F) Representative electron micrographs of 4 dpf wild-type and llgl1−/− cardiomyocytes. Red asterisks denote normal sarcomeres of wild type, whereas blue asterisks denote thin and disorganized sarcomeres of llgl1−/− cardiomyocytes. Red arrows illustrate normal intercalated discs of wild type, whereas blue arrows indicate elongated dysmorphic intercalated discs of llgl1−/− cardiomyocytes. The hashtag indicates loss of adhesion between cardiomyocytes. Quantification of cardiomyocyte intercalated disc (ICD) length (top right). n=30 intercalated discs per group compiled from three animals per group, and ten micrographs per animal. Two-tailed unpaired Student’s t-test. Error bars indicate s.e.m. ns, not significant, *P<0.05, **P<0.01. Scale bars: 25 μm (A); 50 μm (D,E); 500 nm (F). with the llgl1 morpholino were examined for the pericardial effusion disorganized and reduced sarcomere bundles in llgl1 morphant phenotype before analysis and the observed phenotype varied in cardiomyocytes at 48 hpf (Fig. S7). Similar phenotypes were observed severity (Fig. S3). Additionally, as with llgl1−/− mutant embryos, llgl1 in llgl1−/− mutant embryos at 4 dpf, which presented dysgenic morphants failed to undergo cardiac looping at 48 hpf, resulting in sarcomeres and enlarged dysmorphic intercalated discs (Fig. 3F, dysmorphic ventricles (Fig. 3D, Fig. S5). Confocal analysis of 48 hpf Fig. S7). In embryos with a severe pericardial effusion phenotype, llgl1 morphant hearts revealed larger rounded ventricular adhesion between cardiomyocytes and the surrounding cells was cardiomyocytes with an abnormal distribution of actin (Fig. 3E, affected. The intercalated disc phenotype is reminiscent to the Fig. S6). Before trabeculation, ventricular cardiomyocytes develop enlarged and dysmorphic adherens junctions observed in llgl1 dense cortically localized myofibrils in the basal/luminal region of the morphant retinal neuroepithelia (Clark et al., 2012). Collectively, cell (Reischauer et al., 2014). To test whether this atypical distribution these results show that Llgl1 regulates the development of of myofibrils might affect the contractile structures of the heart, we cardiomyocyte morphology and contraction in the first few days of performedtransmissionelectronmicroscopy,whichrevealed life in zebrafish. DEVELOPMENT

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Fig. 4. Adult male llgl1 mutant zebrafish display smaller hearts. Quantification of echocardiogram data from 5-month-old zebrafish. n=11 for llgl1+/− females, n=14 for llgl1−/− females, n=14 for llgl1+/− males and n=7 llgl1−/− males. Two-way ANOVA, Tukey’s multiple comparisons test. Error bars indicate s.e.m. *P<0.05.

Although ∼30% of llgl1−/− fish die, the others recover and survive microscopy revealed a significant increase in the area encompassed to adulthood. We analyzed three clutches of siblings raised from by Notch signaling within 2 dpf llgl1 morphants (Fig. 5C). Notch llgl1−/−×llgl1+/− crosses using echocardiography to assess cardiac activity was apparent throughout the ventricle, rather than localized to function between genotypes. Echocardiograms revealed variations in the atrioventricular canal (AVC) as seen in control embryos. This result cardiac physiology among adult llgl1−/− males, as indicated by is similar to the findings in llgl1 morphant retinal progenitor cells, statistically smaller end-diastolic and end-systolic volume (EDV and which also showed increased Notch activity (Clark et al., 2012). ESV, respectively) measurements when normalized to body length Finally, in situ hybridization of notch1b, bmp4 and klf2a mRNAs, (Fig. 4). Stroke volume was also significantly reduced in males. which mark valve cells, revealed disrupted valvulogenesis in llgl1 Although there was no difference in the A wave peak velocity morphants (Fig. 5D, Figs S7-S9). Collectively, our data indicate that between groups, we observed a significant decrease in maximum E deletion of Lgl1 disrupts unidirectional blood flow that is accompanied wave velocity in llgl1−/− fish compared with llgl1+/− siblings, with no by impaired valvulogenesis. Loss of hemodynamic forces likely observable differences between sexes of the same genotype. These compounds valve dysgenesis and heart chamber defects. results illustrated a dysfunction in passive filling of the ventricle during early diastole in llgl1−/− fish and a decrease in cardiac Exogenous expression of Yap in zebrafish cardiomyocytes function in llgl1−/− male fish compared with llgl1+/− male siblings. ameliorates the abnormal cardiac physiological function Altogether, our observations of llgl1 mutant and morphant fish associated with loss of Llgl1 illustrate that a loss of Llgl1 in zebrafish results in dysmorphic heart As we found that depletion of Llgl1 reduced YAP protein levels in development and impaired cardiac function in adult males. rat cardiomyocytes in vitro, we next tested whether, in contrast to Intracardiac fluid forces are essential for embryonic cardiogenesis Drosophila, in vivo depletion of Llgl1 also reduced Yap protein (Andrés-Delgado and Mercader, 2016; Haack and Abdelilah-Seyfried, levels in zebrafish cardiomyocytes. Anti-Yap staining in 2 dpf 2016; Steed et al., 2016; Paolini and Abdelilah-Seyfried, 2018). In the zebrafish showed a significant decrease in Yap protein signature in developing zebrafish heart, valve leaflets are normally formed by the tropomyosin-expressing ventricular cardiomyocytes of llgl1 96 hpf, ensuring unidirectional blood flow (Steed et al., 2016). Given morphants compared with controls (Fig. 6A). Ventricular size was the abnormal morphology of cardiomyocytes and pericardial effusion measured using confocal microscopy and quantified with Imaris phenotype in Llgl1-depleted embryos, we hypothesized that software. Furthermore, qRT-PCR analysis revealed a decrease in hemodynamics and valvulogenesis were also affected. Both llgl1−/− Yap-regulated transcripts in hearts isolated from 3 dpf llgl1−/− and llgl1+/− embryo hearts displayed oscillatory flow between the embryos compared with wild type (Fig. 6B). The expression of both heart chambers at 78 hpf (Movies 1, 2). However, at 99 hpf, we llgl1 and llgl2 transcripts was also significantly decreased. Taken observed unidirectional flow within llgl1+/− hearts, whereas llgl1−/− together, these data indicate that Llgl1 regulates Yap protein levels hearts continued to exhibit substantial oscillatory flow (Movies 3-5). and associated transcriptional activity in zebrafish cardiomyocytes. To assess ventricular regurgitation, the degree of anterograde or To assess the extent to which the loss of Llgl1 cardiac phenotype retrograde flow during diastasis was quantified, revealing a significant was caused by decreased levels of Yap protein, we addressed whether increase in retrograde flow in llgl1−/− embryos (Fig. 5A). Similarly, we exogenous expression of Yap would rescue heart development in llgl1 observed a significant decrease in blood flow velocity in llgl1 morphants. Specifically, we microinjected Tol2-based plasmid DNA morphants (Fig. 5B, Movies 6, 7). As impaired blood flow can encoding myc-tagged Yap driven by a cardiomyocyte-specific suppress valvulogenesis, we analyzed the developing valve using a promoter (myl7:Yap-myc). This plasmid has been used previously Notch1 reporter transgenic line, Tg(tp1-MmHbb:d2GFP)mw43,which and the expressed protein has been verified at 2 dpf in cardiomyocytes was used to identify the developing valve (Samsa et al., 2015). using western blot (Flinn et al., 2019). Interestingly, llgl1−/− embryos

Quantification of the short-lived d2GFP protein by confocal were sensitive to microinjection, failing to develop after perturbance at DEVELOPMENT

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Fig. 5. Valve morphology and hemodynamics are compromised in llgl1 mutants. (A) Representative images from videos acquired using light microscopy of 99 hpf hearts. A heart from an llgl1−/− embryo with slight pericardial effusion is displayed in the middle, whereas on the right, a heart with severe pericardial effusion is depicted. Above: blood flow was analyzed during diastasis using the particle image velocimetry in Fiji. Arrow vectors show directionality distance moved in pixels. Below: particle image velocimetry (PIV) data were analyzed using the PIV analyzer function in Fiji and qualified for anterograde or retrograde flow. Quantification of anterograde versus retrograde area within the heart chambers during diastasis is depicted in the graph (right). n=4 for llgl1+/+ embryos and n=5 for llgl1−/− embryos. (B) Blood flow velocity measurements analyzed from videos. n=10 for untreated embryos and n=5 for llgl1 morpholino-treated embryos. (C) Representative images from confocal z-stack micrographs of hearts of 2 dpf embryos carrying the tp1 transgenic Notch reporter. The area of cardiac GFP expression is depicted in the graph. n=9 for untreated embryos and n=12 for llgl1 morpholino-treated embryos. (D) Representative images of in situ hybridization of valve markers in 48 hpf fish. Ventral view focusing on the heart (red dashed line). Color model depicts the ventricle in red, atrium in blue, AVC in yellow and venous pole in purple. Two-tailed unpaired Student’s t-test. Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001. Scale bars: 100 µm. the one-cell stage, thus necessitating the use of the llgl1 morpholino. and S387, and E3ubiquitin-mediated degradation via β-TRCP (Zhao Exogenous expression of Yap in cardiomyocytes ameliorated the et al., 2010). This triple serine region is highly conserved across pericardial effusion phenotype of llgl1 morphants at 3 dpf (Fig. 6C). vertebrates (Zhao et al., 2010). Indeed, in zebrafish Yap, replacement Furthermore, analysis of hemodynamic forces showed a statistically of the Lats target residue serine S335 with non-phosphorylatable significant increase in blood flow velocity with co-injection of the alanine results in increased Yap stability and activity in myl7:Yap-myc construct in llgl1 morphants compared with llgl1 cardiomyocytes (Flinn et al., 2019). To test Yap localization and morphants alone (Fig. 6D). No statistical difference in blood flow phospho-degradation, we generated two constructs expressing eGFP- velocity was observed between the myl7:Yap-myc-treated llgl1 Yap fusion proteins either with or without the serine 355 to alanine morphants or the myl7:Yap-myc-treated wild-type embryos mutation. In addition, we generated a non-phosphorylatable serine 54 compared with the control groups. This rescue of cardiac function to alanine mutant that disrupts Tead-binding and moderates potential in llgl1 morphants by Yap overexpression in cardiomyocytes is overexpression phenotypes (Miesfeld et al., 2015). Comparisons consistent with a role for Llgl1 in controlling Yap protein levels. between the two transgenes revealed changes in the localization and degradation of Yap caused by loss of Llgl1. Loss of Llgl1 affects Yap localization and levels Membrane localization of Yap protein at both the lateral surface In vertebrates, the levels of Yap protein are partially controlled by and adherens junction-like intercalated discs of cardiomyocytes has regulated degradation. In addition to promoting 14-3-3 binding and been shown to regulate the activity of Yap (Morikawa et al., 2017; cytoplasmic retention, Lats kinases also affect Yap activity via Flinn et al., 2020). Given its role in establishing apical-basal polarity, phospho-mediated degradation (Zhao et al., 2010). We suspected we hypothesized that loss of Llgl1 would disrupt membrane that Lats activity might underlie the reduction of Yap protein levels localization of Yap, thus making it accessible for Lats-mediated within Llgl1-depleted cardiomyocytes and could explain the phosphodegradation. To assess the function of Llgl1 on Yap stability, observed differences between Drosophila and vertebrates, as the we used the epidermis as a model that shows strong membrane phosphodegron sequence is not conserved in Yorkie. Studies in localization of Yap and can be clearly and easily imaged by whole- human cell lines revealed that Ser 381 of YAP can be phosphorylated embryo confocal microscopy (Fig. S10). In an attempt to rescue by LATS, resulting in subsequent phosphorylation by CK1δ/ε at S384 embryos exhibiting severe pericardial effusion, we conducted cold DEVELOPMENT

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Fig. 6. Llgl1 promotes appropriate Yap protein levels in cardiomyocytes, and exogenous expression of Yap in cardiomyocytes ameliorates deleterious cardiac physiology associated with loss of Llgl1. (A) Representative images of 2 dpf zebrafish ventricles from single plane confocal micrographs (left). Boxes indicate area of enlargement. Arrows depict Yap within the cardiomyocytes. Quantification of the intensity of anti-Yap signature in tropomyosin+ cells normalized to the control morpholino group (right). n=8 for untreated embryos and n=15 for llgl1 morpholino-treated embryos. (B) qRT-PCR transcript analysis for llgl1, llgl2 and genes regulated by Yap activity in llgl1+/+ or llgl1−/− 3 dpf zebrafish hearts. n=10 for each group. (C) Representative images of 3 dpf embryos microinjected with llgl1 morpholino with or without Tol2-mediated integration of myl7:yap-myc (left). Arrows depict the region measured for pericardial effusion. Quantification of pericardial effusion in 3 dpf embryos (right). n=6 for uninjected control group, n=6 for the myl7:yap-myc control group, n=7 for llgl1 morphants and n=7 for llgl1 morphants with myl7:yap-myc expression. (D) Quantification of blood flow velocity in 2 dpf embryos. n=8 for the uninjected control group, n=5 for the transposase only control group, n=5 for the myl7:yap-myc control group, n=13 for llgl1 morphants and n=9 for llgl1 morphants with myl7:yap-myc expression. Two-tailed unpaired Student’s t test (A,B) and One-way ANOVA, Tukey’s multiple comparisons test (C,D). Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001. Scale bars: 50 µm (A); 1 mm (C). rearing and staged embryos to 5 dpf as described by Kimmel et al. eGFP-YapS54A and eGFP-YapS54A;S335A under the epidermal krt18 (1995). We have previously shown that loss of retinal pigmented promoter via the Tol2 transposable constructs. Expression of plasmid epithelium in yap1−/− mutant fish can be rescued by slowing DNA revealed that eGFP-YapS54A localized to epidermal cell development (Miesfeld et al., 2015). When llgl1−/− embryos were membranes in 30 hpf control embryos (Fig. 7B). Compared with reared at low temperature (20.5°C), the severity of edema was controls, the llgl1 morphant epidermis lacked membrane localization diminished and all mutants survived longer; this revealed an and, although the eGFP-YapS54A signal varied from cell to cell, the epidermal dysgenesis along their fins (Fig. S11A,B). In eGFP-YapS54A signal was significantly decreased in intensity. In comparison, cold rearing of wild-type fish did not result in contrast, expression of the eGFP-YapS54A;S335A variant, which is abnormal phenotypes. Electron micrographs of the llgl1−/− mutant resistant to phospho-degradation, showed no change in abundance fin epidermis showed an array of irregularities. Specifically, the compared with control embryos but was also delocalized from the cell density of desmosomes was reduced between the outer enveloping cortex. Similarly, an antibody that recognizes Yap revealed a layer and basal epidermal layer, and the basement membrane of subcellular displacement and reduction in expression levels in llgl1−/− mutant fin epidermis was disordered compared with the 48 hpf llgl1−/− mutants (Fig. 7C, Fig. S10). Collectively, our compact composition seen in llgl1+/− fins. Actinotrichia, collagen- results suggest that loss of Llgl1 affects Yap localization and rich skeletal components appeared dysmorphic and were trapped consequently turnover, potentially through the remodeling of apical within the basal epidermal layer (Fig. S11C). Additionally, apical junctions in epithelia and intercalated discs of cardiomyocytes junctions of the enveloping layer were elongated and more electron (Fig. 8). Furthermore, these data suggest that the loss of Yap protein dense (Fig. 7A), resembling phenotypes observed in the intercalated associated with depletion of Llgl1 is ultimately the result of Lats discs of cardiomyocytes (Fig. 3F, Fig. S7) and apical junctions of kinase activity. retinal progenitor cells in llgl1 morphants (Clark et al., 2012). As Yap localizes to cell junctions in the epidermis, endothelium and DISCUSSION cardiomyocytes (Morikawa et al., 2017; Neto et al., 2018; The Hippo pathway regulates tissue growth in the response to soluble Schlegelmilch et al., 2011), we next assayed whether loss of Llgl1 cues, cell density, mechanical force and other stimuli (Deng et al., resulted in a displacement of Yap from the cell cortex. To visualize 2015; Dupont et al., 2011; Fletcher et al., 2018). However, a great

Yap localization in the zebrafish epidermis, we expressed challenge remains in identifying those signals upstream that sense, DEVELOPMENT

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Fig. 7. Loss of Llgl1 in zebrafish epidermis results in dysmorphic junctions and increased Yap phospho-degradation. (A) Representative transmission electron micrographs of fin epidermis from 5 dpf stage cold-reared embryos (left). Arrows indicate apical junctions. Quantification of apical junction length of the outer epidermal layer and the abundance of desmosomes between the epidermal layers (right). n=11 for llgl1+/− and 14 for llgl1−/− embryos. (B) Representative confocal z-stack micrographs of 30 hpf zebrafish epidermis expressing either eGFP-YapS54A or eGFP-YapS54,355A. Arrowheads indicate membrane localization of the transgene (left). Quantification of the intensity of the transgene signal in positively expressed cells is depicted (right). n=13 for eGFP-YapS54A intensity quantification. n=10 for untreated and n=12 llgl1 morpholino-treated embryos for eGFP-YapS54,335A intensity. (C) Representative confocal z-stack micrographs of 2 dpf embryos immunostained with an antibody against Yap. Two-tailed unpaired Student’s t-test. Error bars indicate s.e.m. ns, not significant, *P<0.05, **P<0.01, ***P<0.001. Scale bars: 500 nm (A); 50 µm (B,C). mediate and modify the physiological inputs into this pathway. We delocalized Yap in Mdx mice was shown by cardiomyocyte-specific found that Llgl1 regulates Yap protein levels and transcriptional knockout of Sav, a positive mediator of Lats activity. These double activity. However, as we could not find a Llgl1 antibody specific for mutant mice showed a striking increase in Yap localization and zebrafish, we cannot rule out splicing variants or alternative start sites transcriptional activity compared with wild-type littermates. This accounting for some diminished form of Llgl1 expression in llgl1mw83 observation is in line with the role of Lats kinases in modulating the mutant fish. Nonetheless, we show that depletion of Llgl1 in cultured levels of Yap following delocalization from the cell membrane. Other rat and zebrafish cardiomyocytes, and epidermal cells in vivo, resulted cell junction-associated factors have been implicated in modulating in lower levels of total Yap protein. Furthermore, depletion of Llgl1 the activity of Lats kinases, including those of the Ajuba protein also resulted in diminished expression of Yap-TEAD target genes in family (Sun and Irvine, 2013). Work with Drosophila and cultured both llgl1−/− mutant zebrafish and cultured rat cardiomyocytes. Our mammalian cells, demonstrated that both activators and inhibitors of in vivo data show that loss of Llgl1 resulted in the delocalization of Hippo pathway activity localize to apical cell junctions, and that Yap from the membrane in epidermal cells, rendering Yap junction reorganization is associated with changes in Yorkie/Yap susceptible to degradation. The effect of Llgl1 on Yap levels, but activity (Sun et al., 2015). Furthermore, in cardiomyocytes Amotl1 not cell junction localization, depended on serine 335 of Yap, which has been implicated in shuttling Yap from the intercalated disc is a known target of Lats kinases that prime Yap for degradation. As junction to the nucleus in a manner that depends on the activity of opposed to the function of L(2)gl in Drosophila, our collective Fat4, an atypical cadherin that localizes to cell junctions and is observations illustrate a role for Llgl1 as a positive mediator of Yap essential for intercalated disc integrity (Ragni et al., 2017). Altogether, activity. these published data, coupled with our observations, indicate that Several observations suggest that Llgl1 affects Yap stability through Llgl1 promotes the protein composition of cell junction complexes cell junction remodeling. Similar to the llgl1 morphant retinal that facilitate Yap localization and protection from phospho-mediated neuroepithelium (Clark et al., 2012), we found that apical junctions proteasome targeting and degradation. in the epidermis of the llgl1−/− mutant and intercalated discs in We further identified a novel role for Llgl1 as a physiological cardiomyocytes of llgl1 mutants were elongated and more electron modifier of zebrafish cardiac development. Phenotypic dense compared with wild-type cells. Multiple junction-associated consequences of Llgl1 disruption on heart morphogenesis proteins have been implicated in regulating Yap activity. These included cardiac looping defects, abnormal cardiomyocyte include α-catenin in the intercalated discs of cardiomyocytes (Li et al., morphology and atrioventricular valve dysgenesis. Our global 2015; Vite et al., 2018) and apical junctions of skin keratinocytes Llgl1 depletion model precludes us from determining whether (Schlegelmilch et al., 2011). In addition, Yap is associated at the lateral abnormal myocardial development subsequently impaired valve cell cortex in cardiomyocytes by the dystrophin-glycoprotein complex development, or whether Llgl1 contributes to these developmental (Morikawa et al., 2017). Although disruption of the dystrophin- processes independently. Evidence from the literature demonstrates glycoprotein complex in neonatal Mdx (Dmd) null mice, which are that altered hemodynamics can impair valvulogenesis (Donat et al., deficient in dystrophin, displaces Yap from the lateral membrane 2018; Haack and Abdelilah-Seyfried, 2016; Steed et al., 2016; regions, nuclear localization of Yap is unaltered (Morikawa et al., Paolini and Abdelilah-Seyfried, 2018). Thus, a primary input to

2017). However, a role for Lats kinases in modulating the stability of myocardial contractility and blood flow velocity could secondarily DEVELOPMENT

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of Scrib affects Yap activity was not evaluated in that study. Additionally, loss of Llgl2, the paralog of Llgl1, in zebrafish causes a lack of hemidesmosome formation between the basal epidermis and the underlying basement membrane (Sonawane et al., 2005). This results in a catastrophic loss of epidermal integrity. Similarly, llgl1−/− mutants with severe pericardial effusion show a variety of abnormalities within the developing epidermis. Collectively, our data show that Llgl1 mediates cardiomyocyte development and valvulogenesis, and thus influences heart development in zebrafish, at least in part, through Yap availability in cardiomyocytes. Although variants in human Llgl1 have not been found to associate with cardiovascular anomalies, our studies provide additional evidence for the role of Hippo-Yap pathway regulation in congenital heart disease.

MATERIALS AND METHODS Animals All protocols used in these studies were approved by the local Animal Care and Use Committee, and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The llgl1mw83 knockout mutant was generated using a pair of TALENs (Table S1), producing a 10 bp deletion (c. 55_64del) that resulted in frameshift mutations and the generation of an early stop codon (Fig. S2). A transgenic zebrafish line expressing enhanced GFP under the myl7 cardiomyocyte-specific promoter Tg(myl7:eGFP)mw45 was used to visualize cardiomyocytes (Miesfeld and Link, 2014). A Notch reporter transgenic zebrafish line, Tg(tp1-MmHbb: d2GFP)mw43, was used to visualize valve development (Clark et al., 2012). Sprague Dawley rat neonates (Charles River Laboratories) were used for cardiac cell isolation. Zebrafish were euthanized by anesthetization with tricaine followed by an ice bath, and rat pups were euthanized by decapitation.

Genotyping The presence or absence of the llgl1mw83 allele was verified by PCR amplification using the following primers: F, 5′-ACCGACTGACGCCTA- Fig. 8. A model for the role of Llgl1 in modulating Yap stability. Cartoon ACGGAA-3′; and R, 5′-CATGCGGTGGTCGTTGATGCCT-3′. Genomic depicting the proposed mechanism by which Llgl1 prevents Yap DNA was extracted using the Puregene Core Kit A (Qiagen) according to the phosphodegradation. Llgl1 is necessary to maintain appropriate protein manufacturer’s protocol. Primers flanking the mutation were used to amplify a composition of cell junctions, such as intercalated discs in cardiomyocytes or 375 bp product. Digestion with AlwNI (New England BioLabs) yielded 140 adherens junctions of epithelia in which Yap colocalizes with cell-junctional and 240 bp fragments for the wild-type allele, whereas the llgl1mw83 amplicon α components such as -catenin. Loss of Llgl1 disrupts the normal composition remained undigested. of intercalated discs between cardiomyocytes, resulting in the displacement of Yap and subsequent phosphorylation by Lats kinases, leading to targeted degradation via ubiquitin ligation. Microinjections All plasmids were generated using Gateway (Invitrogen) entry clones in contribute to impaired valvulogenesis, and therefore compound conjunction with the Tol2kit (Kwan et al., 2007). Terminally myc-tagged hemodynamic-dependent phenotypes through a feed-forward loop. Yap was inserted downstream of the cardiomyocyte-specific promoter myl7, Our data demonstrate that cardiac hemodynamics is at least partially and eGFP-Yap fusion proteins containing mutations at S54A or S54A; S335A were inserted downstream of the krt18 promoter for expression in mediated by Llgl1 and Yap activity in cardiomyocytes themselves, epithelia cells. ZDR embryos were microinjected at the one-cell stage with as cardiomyocyte-specific rescue of Yap in llgl1 morphants restored 4.6 nl of a solution containing 8 ng/µl plasmid and 15 ng/µl transposase blood flow velocity to baseline levels. We have yet to determine mRNA. The llgl1 ATG (5′-CCGTCTGAACCTAAACTTCATCATC-3′) whether Llgl1 has a direct role in regulating valvulogenesis. and control (5′-CCTCTTACCTCAGTTACAATTTATA-3′) morpholinos However, because Llgl1 can control signaling factors such as Yap were microinjected as described by Clark et al. (2012). and Notch, both of which are known to contribute to atrioventricular valve development, this is a possibility. Future studies will address Zebrafish immunohistochemistry this outstanding issue. Epidermal anti-Yap staining was performed on 48 hpf embryos using anti- The role for an apico-basal cell polarity factor in regulating cardiac Yap antibody (Cell Signaling Technology, 4912S) as described by Miesfeld morphogenesis is not unprecedented. Scribble (Scrib), which et al. (2015). To image hearts, zebrafish embryos were fixed at 48 hpf and complexes with Llgl1 and Discs Large family members to regulate stained for actin using rhodamine phalloidin (Sigma-Aldrich, 658740; basolateral cell identity in many cell types, results in cardiac dilution) at 1:100 stock concentration, and anti-alcam [Developmental dysgenesis when deleted from mouse cardiomyocytes (Boczonadi Studies Hybridoma Bank (DHSB)] before heart extraction for confocal et al., 2014). In these Scrib-conditional depletion mice, differentiation imaging on a LSM 710 microscope (Zeiss) as described by Renz et al. (2015). Anti-Yap staining in the heart was accomplished using anti-Yap (Abcam, of cardiomyocytes was compromised, resulting in structural ab81183) and anti-tropomyosin (DSHB, CH1) antibodies. Antibody dilutions abnormalities of the ventricular myocardium, such as ventricular are provided in Table S4. Similarly, whole-mount in situ hybridization was −/− septal defects. Interestingly, like llgl1 mutant zebrafish, most performed on 48 hpf zebrafish embryos as described by Renz et al. (2015) Scrib-deficient embryos recovered and survived to adulthood, using the following probes: klf2a (Renz et al., 2015); notch1b (Walsh and although the mature hearts showed elevated fibrosis. Whether loss Stainier, 2001); and bmp4 (Walsh and Stainier, 2001). Both notch1b and DEVELOPMENT

9 RESEARCH ARTICLE Development (2020) 147, dev193581. doi:10.1242/dev.193581 bmp4 plasmids were gifts from Didier Stainier (Max Planck Institute for Heart Strand Synthesis System RT-PCR Kit (Invitrogen) according to the and Lung Research, Bad Nauheim, Germany). manufacturer’s instructions. qRT-PCR was performed using a CFX96 and CFX Connect Real-Time System (Bio-Rad) using SsoAdvanced SYBR Confocal microscopy Green Supermix (Bio-Rad) or TaqMan Gene Expression Master Mix Zebrafish embryos were fixed in a solution of 4.0% paraformaldehyde at 4°C (Thermo Fisher Scientific). Primers used are listed in Table S3. overnight. Embryos were then embedded in 1.0% low-melt agarose and Quantifications were normalized to TATA-binding protein (Tbp) for imaged using a D-Eclipse C1 Microscope System (Nikon) with EZ-C1 in vitro rat cell cultures and eef1a1l1 for zebrafish hearts. Product size software (version 4.11). The resulting images were analyzed using Fiji ImageJ and uniformity was assessed via melt curve analysis. to determine the signal intensity of compiled z-stack images, or Imaris (Bitplane) for nearest neighbor nuclear distance, cell number and cell size. Western blotting Cell lysates were acquired from siRNA-treated primary rat cardiomyocytes as Echocardiography described by O’Meara et al. (2015). Gel electrophoresis was performed using a Echocardiographs were obtained from 5-month-old zebrafish using a Vevo 4-15% Mini-PROTEAN TGX precast gel (Bio-Rad) followed by blotting onto 770 with an RMV 708 probe (VisualSonics). Zebrafish were anesthetized in Immobilon-FL PVDF membranes (Millipore). Antibodies and concentrations 150 ppm tricane and 45 ppm isoflurane before imaging. Scans were taken in used for immunochemistry are described in Table S4. Protein detection was triplicate and measured in a genotype-masked manner using the Vevo 770 performed using an Odyssey infrared imager (LI-COR Biosciences). software package (ver 3.0.0 build 55.00) as demonstrated by Lee et al. (2016). Statistical analysis Embryonic blood flow imagining Data were analyzed using Prism 7 (GraphPad). Two-way ANOVA followed Blood flow was analyzed using Fiji ImageJ from captured video. Particle by Tukey’s multiple comparisons tests were performed on samples with two image velocimetry was used to show the direction of blood flow during experimental factors. Statistical comparisons between two groups were diastasis, whereas blood velocity was determined by the distance analyzed by Student’s t-test, and for three or more groups by one-way erythrocytes traveled in the dorsal aorta during a single pulse. ANOVA followed by Tukey’s multiple comparisons test. A χ2 test of independence was used to compare the degree of embryonic pericardial Electron microscopy effusion between maternal lineages, whereas a two-tail binomial test was used Zebrafish embryos were fixed in a solution containing 1.0% to compare genotypes in adult populations with expected Mendelian ratios. paraformaldehyde and 2.5% glutaraldehyde overnight at 4°C. The embryos were then treated with 1.0% osmium tetroxide in phosphate Acknowledgements buffer, dehydrated through a series of methanol solutions, and infused with We acknowledge Shelby Hader, Dr Dawid Chabowski and Dr David Gutterman EMbed 812 resin (Electron Microscopy Sciences). Plastic sections were (Department of Physiology, The Medical College of Wisconsin) for their assistance with imaging zebrafish vasculature. stained with uranyl acetate (Electron Microscopy Sciences). The Hitachi H- 600 transmission electron microscope with an ORCA-100 digital camera Competing interests (Hamamatsu) was used for transmission electron microscopy. The authors declare no competing or financial interests. siRNA knockdown in cultured rat cardiac cells Author contributions Primary cardiac myocytes and fibroblasts were obtained from 2-day-old Conceptualization: C.C.O., B.A.L.; Methodology: M.A.F., C.O., Z.J.B.; Formal Sprague Dawley rat hearts. Hearts were excised and digested with the Neonatal analysis: M.A.F., C.O., Z.J.B., T.C.W., J.A.A., S.A.-S, C.C.O., B.A.L.; Investigation: Heart Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s M.A.F., C.O., Z.J.B., J.R.B., A.K., T.C.W., J.A.A., S.A.-S, C.C.O., B.A.L.; Writing - protocol. Cardiomyocytes were isolated through an isotonic Percoll gradient original draft: M.A.F., C.C.O., B.A.L.; Writing - review & editing: M.A.F., C.O., Z.J.B., J.R.B., A.K., T.C.W., J.A.A., S.A.-S, C.C.O., B.A.L.; Supervision: C.C.O., B.A.L.; centrifugation. Isolated cardiomyocytes were plated in Dulbecco’s modified Project administration: B.A.L.; Funding acquisition: J.A.A., S.A.-S, C.C.O., B.A.L. eagle medium (DMEM, Life Technologies) containing 1 g/l glucose, supplemented with 15% fetal bovine serum (FBS) and penicillin- Funding streptomycin (Life Technologies). After 16 h, the cells were transfected with This work was supported by the Cardiovascular Center and Research and two different siRNAs designed against the same target gene (to achieve robust Education Program Fund at the Medical College of Wisconsin (C.C.O., J.A.A. knockdown), each at a concentration of 25 nM. siRNAs designed against Yap, and B.A.L.); by Advancing a Healthier Wisconsin Co-Investigator Grant (B.A.L. Llgl1, Llgl2, a combination of Llgl1 and Llgl2, or negative control siRNA and C.C.O.); by the National Institutes of Health (R01 HL141159 to C.C.O., (Table S2) were transfected with Mission siRNA Transfection Reagent (Sigma- 5R01HL131788 and 1S10 OD025038 to J.A.A., and T32 HL134643 to M.A.F.); by ’ Aldrich). Untreated and transfected cells were cultured in DMEM the Cardiovascular Center s A.O. Smith Fellowship Scholars Program (M.A.F.); by the Medical College of Wisconsin Cardiovascular Center [FP00012308 to supplemented with 7.5% FBS for 48 hours. Cells were then collected in J.A.A.]; by Excellence cluster REBIRTH (SFB958 to S.A.-S.); by Deutsche TRIzol Reagent for RNA extraction for qRT-PCR and RNAseq, or collected in Forschungsgemeinschaft (SE2016/7-2 and SE2016/10-1 to S.A.-S.); and by the lysis buffer for western blotting as described by O’Meara et al. (2015). Deutsches Zentrum für Herz-Kreislaufforschung (S.A.-S.). Deposited in PMC for release after 12 months. RNAseq RNA was extracted from neonatal rat primary cardiomyocytes transfected Data availability The RNAseq data generated in this study are available in the GEO under accession with siRNA designed against a combination of Llgl1 and Llgl2 or negative number GSE121929. control siRNA (Table S2), using the TRIzol (Ambion) extraction protocol recommended by the manufacturer. RNA quality was determined using an Supplementary information Agilent BioAnalyzer. RNA libraries were prepared by BGI Americas using Supplementary information available online at the low-input poly-A BGISEQ-500RSRNASeq protocol. Sequencing results https://dev.biologists.org/lookup/doi/10.1242/dev.193581.supplemental were analyzed by BGI Americas. Primary sequencing data were generated with a BGISEQ-500 50SE at 20 million reads per sample. Bowtie2 was used Peer review history to map reads to a reference genome. GSEA (Broad Institute) was performed The peer review history is available online at on RNAseq results (fragments per kilobase million). https://dev.biologists.org/lookup/doi/10.1242/dev.193581.reviewer-comments.pdf

References qRT-PCR Andrés-Delgado, L. and Mercader, N. (2016). Interplay between cardiac function RNA was extracted using TRIzol (Thermo Fisher Scientific) phenol and heart development. Biochim. Biophys. Acta Mol. Cell Res. 1863, 1707-1716. chloroform extraction. cDNA was generated using a Superscript III First- doi:10.1016/j.bbamcr.2016.03.004 DEVELOPMENT

10 RESEARCH ARTICLE Development (2020) 147, dev193581. doi:10.1242/dev.193581

Artap, S., Manderfield, L. J., Smith, C. L., Poleshko, A., Aghajanian, H., See, K., Miesfeld, J. B. and Link, B. A. (2014). Establishment of transgenic lines to monitor Li, L., Jain, R. and Epstein, J. A. (2018). Endocardial Hippo signaling regulates and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily myocardial growth and cardiogenesis. Dev. Biol. 440, 22-30. doi:10.1016/j.ydbio. conserved and divergent functions of the Hippo pathway. Mech. Dev. 133, 2018.04.026 177-188. doi:10.1016/j.mod.2014.02.003 Boczonadi, V., Gillespie, R., Keenan, I., Ramsbottom, S. A., Donald-Wilson, C., Miesfeld, J. B., Gestri, G., Clark, B. S., Flinn, M. A., Poole, R. J., Bader, J. R., Al Nazer, M., Humbert, P., Schwarz, R. J., Chaudhry, B. and Henderson, D. J. Besharse, J. C., Wilson, S. W. and Link, B. A. (2015). Yap and Taz regulate retinal (2014). Scrib:Rac1 interactions are required for the morphogenesis of the pigment epithelial cell fate. Development 142, 3021-3032. doi:10.1242/dev.119008 ventricular myocardium. Cardiovasc. Res. 104, 103-115. doi:10.1093/cvr/cvu193 Morikawa, Y., Heallen, T., Leach, J., Xiao, Y. and Martin, J. F. (2017). Dystrophin– Chen, Z., Friedrich, G. A. and Soriano, P. (1994). Transcriptional enhancer factor 1 glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation. disruption by a retroviral gene trap leads to heart defects and embryonic lethality in Nature 547, 227. doi:10.1038/nature22979 mice. Genes Dev. 8, 2293-2301. doi:10.1101/gad.8.19.2293 Neto, F., Klaus-Bergmann, A., Ong, Y. T., Alt, S., Vion, A.-C., Szymborska, A., Clark, B. S., Cui, S., Miesfeld, J. B., Klezovitch, O., Vasioukhin, V. and Link, Carvalho, J. R., Hollfinger, I., Bartels-Klein, E., Franco, C. A. et al. (2018). YAP B. A. (2012). Loss of Llgl1 in retinal neuroepithelia reveals links between apical and TAZ regulate adherens junction dynamics and endothelial cell distribution domain size, Notch activity and neurogenesis. Development 139, 1599-1610. during vascular development. eLife 7, e31037. doi:10.7554/eLife.31037 doi:10.1242/dev.078097 O’Meara, C. C., Wamstad, J. A., Gladstone, R. A., Fomovsky, G. M., Butty, V. L., Deng, H., Wang, W., Yu, J., Zheng, Y., Qing, Y. and Pan, D. (2015). Spectrin Shrikumar, A., Gannon, J. B., Boyer, L. A. and Lee, R. T. (2015). Transcriptional regulates Hippo signaling by modulating cortical actomyosin activity. eLife 4, reversion of cardiac myocyte fate during mammalian cardiac regeneration. Circ. e06567. doi:10.7554/eLife.06567 Res. 116, 804-815. doi:10.1161/CIRCRESAHA.116.304269 Donat, S., Lourenço, M., Paolini, A., Otten, C., Renz, M. and Abdelilah-Seyfried, Paolini, A. and Abdelilah-Seyfried, S. (2018). The mechanobiology of zebrafish S. (2018). Heg1 and Ccm1/2 proteins control endocardial mechanosensitivity cardiac valve leaflet formation. Curr. Opin. Cell Biol. 55, 52-58. doi:10.1016/j.ceb. during zebrafish valvulogenesis. eLife 7, e28939. doi:10.7554/eLife.28939 2018.05.007 Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Parsons, L. M., Grzeschik, N. A. and Richardson, H. E. (2014a). lgl Regulates the Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S. et al. (2011). Role of Hippo Pathway independently of Fat/Dachs, Kibra/Expanded/Merlin and YAP/TAZ in mechanotransduction. Nature 474, 179-183. doi:10.1038/ dRASSF/dSTRIPAK. Cancers (Basel) 6, 879-896. doi:10.3390/cancers6020879 nature10137 Parsons, L. M., Portela, M., Grzeschik, N. A. and Richardson, H. E. (2014b). Lgl Durst, R., Sauls, K., Peal, D. S., deVlaming, A., Toomer, K., Leyne, M., Salani, M., regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6- Talkowski, M. E., Brand, H., Perrocheau, M. et al. (2015). Mutations in DCHS1 Baz polarity complex. Curr. Biol. 24, 2073-2084. doi:10.1016/j.cub.2014.07.075 cause mitral valve prolapse. Nature 525, 109-113. doi:10.1038/nature14670 Ragni, C. V., Diguet, N., Le Garrec, J.-F., Novotova, M., Resende, T. P., Pop, S., Fletcher, G. C., Diaz-de-la-Loza, M.-C., Borreguero-Muñoz, N., Holder, M., Charon, N., Guillemot, L., Kitasato, L., Badouel, C. et al. (2017). Amotl1 Aguilar-Aragon, M. and Thompson, B. J. (2018). Mechanical strain regulates mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict the hippo pathway in Drosophila. Development 145, dev159467. doi:10.1242/dev. heart growth. Nat. Commun. 8, 14582. doi:10.1038/ncomms14582 159467 Reischauer, S., Arnaout, R., Ramadass, R. and Stainier, D. Y. R. (2014). Actin Flinn, M. A., Jeffery, B. E., O’Meara, C. C. and Link, B. A. (2019). Yap is required binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish for scar formation but not myocyte proliferation during heart regeneration in heart and the identification of Erbb2 signaling as a remodeling factor of myofibril zebrafish. Cardiovasc. Res. 115, 570-577. doi:10.1093/cvr/cvy243 architecture. Circ. Res. 115, 845-856. doi:10.1161/CIRCRESAHA.115.304356 Flinn, M. A., Link, B. A. and O’Meara, C. C. (2020). Upstream regulation of the Renz, M., Otten, C., Faurobert, E., Rudolph, F., Zhu, Y., Boulday, G., Duchene, Hippo-Yap pathway in cardiomyocyte regeneration. Semin. Cell Dev. Biol. 100, J., Mickoleit, M., Dietrich, A.-C., Ramspacher, C. et al. (2015). Regulation of β1 11-19. doi:10.1016/j.semcdb.2019.09.004 integrin-Klf2-mediated angiogenesis by CCM proteins. Dev. Cell 32, 181-190. Greenwood, E., Maisel, S., Ebertz, D., Russ, A., Pandey, R., Schroeder, J., doi:10.1016/j.devcel.2014.12.016 Greenwood, E., Maisel, S., Ebertz, D., Russ, A. et al. (2016). Llgl1 prevents Samsa, L. A., Givens, C., Tzima, E., Stainier, D. Y. R., Qian, L. and Liu, J. (2015). metaplastic survival driven by epidermal growth factor dependent migration. Cardiac contraction activates endocardial notch signaling to modulate chamber Oncotarget 7, 60776-60792. doi:10.18632/oncotarget.11320 maturation in zebrafish. Development 142, 4080-4091. doi:10.1242/dev.125724 Grzeschik, N. A., Parsons, L. M., Allott, M. L., Harvey, K. F. and Richardson, Schlegelmilch, K., Mohseni, M., Kirak, O., Pruszak, J., Rodriguez, J. R., Zhou, H. E. (2010). Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway D., Kreger, B. T., Vasioukhin, V., Avruch, J., Brummelkamp, T. R. et al. (2011). through two distinct mechanisms. Curr. Biol. 20, 573-581. doi:10.1016/j.cub.2010. Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell 144, 01.055 Haack, T. and Abdelilah-Seyfried, S. (2016). The force within: endocardial 782-795. doi:10.1016/j.cell.2011.02.031 development, mechanotransduction and signalling during cardiac Sonawane, M., Carpio, Y., Geisler, R., Schwarz, H., Maischein, H.-M. and morphogenesis. Development 143, 373-386. doi:10.1242/dev.131425 Nuesslein-Volhard, C. (2005). Zebrafish penner/lethal giant larvae 2 functions in Hao, Y., Chun, A., Cheung, K., Rashidi, B. and Yang, X. (2008). Tumor hemidesmosome formation, maintenance of cellular morphology and growth suppressor LATS1 Is a negative regulator of oncogene YAP. J. Biol. Chem. 283, regulation in the developing basal epidermis. Development 132, 3255-3265. 5496-5509. doi:10.1074/jbc.M709037200 doi:10.1242/dev.01904 Heallen, T., Zhang, M., Wang, J., Bonilla-Claudio, M., Klysik, E., Johnson, R. L. Steed, E., Boselli, F. and Vermot, J. (2016). Hemodynamics driven cardiac valve and Martin, J. F. (2011). Hippo pathway inhibits wnt signaling to restrain morphogenesis. Biochim. Biophys. Acta Mol. Cell Res. 1863, 1760-1766. doi:10. cardiomyocyte proliferation and heart size. Science 332, 458-461. doi:10.1126/ 1016/j.bbamcr.2015.11.014 science.1199010 Sun, G. and Irvine, K. D. (2013). Ajuba family proteins link JNK to Hippo signaling. Jossin, Y., Lee, M., Klezovitch, O., Kon, E., Cossard, A., Lien, W.-H., Fernandez, Sci. Signal. 6, ra81. doi:10.1126/scisignal.2004324 T. E., Cooper, J. A. and Vasioukhin, V. (2017). Llgl1 connects cell polarity with Sun, S., Reddy, B. V. V. G. and Irvine, K. D. (2015). Localization of Hippo signalling cell-cell adhesion in embryonic neural stem cells. Dev. Cell 41, 481-495.e5. complexes and Warts activation in vivo. Nat. Commun. 6, 8402. doi:10.1038/ doi:10.1016/j.devcel.2017.05.002 ncomms9402 Kashyap, A., Zimmerman, T., Ergül, N., Bosserhoff, A., Hartman, U., Alla, V., Vite, A., Zhang, C., Yi, R., Emms, S. and Radice, G. L. (2018). Alpha-catenin- Bataille, F., Galle, P. R., Strand, S. and Strand, D. (2013). The human Lgl dependent cytoskeletal tension controls Yap activity in the heart. Development polarity gene, Hugl-2, induces MET and suppresses snail tumorigenesis. 145, dev149823. doi:10.1242/dev.149823 Oncogene 32, 1396-1407. doi:10.1038/onc.2012.162 von Gise, A., Lin, Z., Schlegelmilch, K., Honor, L. B., Pan, G. M., Buck, J. N., Ma, Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. Q., Ishiwata, T., Zhou, B., Camargo, F. D. et al. (2012). YAP1, the nuclear target (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation 253-310. doi:10.1002/aja.1002030302 but not hypertrophy. Proc. Natl. Acad. Sci. USA 109, 2394-2399. doi:10.1073/ Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, pnas.1116136109 D. S., Parant, J. M., Yost, H. J., Kanki, J. P. and Chien, C.-B. (2007). The Tol2kit: Walsh, E. C. and Stainier, D. Y. R. (2001). UDP-glucose dehydrogenase required a multisite gateway-based construction kit for Tol2 transposon transgenesis for cardiac valve formation in zebrafish. Science 293, 1670-1673. doi:10.1126/ constructs. Dev. Dyn. 236, 3088-3099. doi:10.1002/dvdy.21343 science.293.5535.1670 Lee, L., Genge, C. E., Cua, M., Sheng, X., Rayani, K., Beg, M. F., Sarunic, M. V. and Wang, T., Liu, Y., Xu, X.-H., Deng, C.-Y., Wu, K.-Y., Zhu, J., Fu, X.-Q., He, M. and Tibbits, G. F. (2016). Functional assessment of cardiac responses of adult zebrafish Luo, Z.-G. (2011). Lgl1 activation of Rab10 promotes axonal membrane (Danio rerio) to acute and chronic temperature change using high-resolution trafficking underlying neuronal polarization. Dev. Cell 21, 431-444. doi:10.1016/ echocardiography. PLoS ONE 11, e0145163. doi:0.1371/journal.pone.0145163 j.devcel.2011.07.007 Li, J., Gao, E., Vite, A., Yi, R., Gomez, L., Goossens, S., van Roy, F. and Radice, Wang, J., Liu, S., Heallen, T. and Martin, J. F. (2018). The Hippo pathway in the G. L. (2015). Alpha-catenins control cardiomyocyte proliferation by regulating Yap heart: pivotal roles in development, disease, and regeneration. Nat. Rev. Cardiol. activity. Circ. Res. 116, 70-79. doi:10.1161/CIRCRESAHA.116.304472 15, 672. doi:10.1038/s41569-018-0063-3 Liu, C., Huang, W. and Lei, Q. (2011). Regulation and function of the TAZ Xiao, Y., Hill, M. C., Zhang, M., Martin, T. J., Morikawa, Y., Wang, S., Moise, A. R.,

transcription co-activator. Int. J. Biochem. Mol. Biol. 2, 247-256. Wythe, J. D. and Martin, J. F. (2018). Hippo signaling plays an essential role in DEVELOPMENT

11 RESEARCH ARTICLE Development (2020) 147, dev193581. doi:10.1242/dev.193581

cell state transitions during cardiac fibroblast development. Dev. Cell 45, Zanconato, F., Forcato, M., Battilana, G., Azzolin, L., Quaranta, E., Bodega, B., 153-169.e6. doi:10.1016/j.devcel.2018.03.019 Rosato, A., Bicciato, S., Cordenonsi, M. and Piccolo, S. (2015). Genome-wide Xin, M., Kim, Y., Sutherland, L. B., Murakami, M., Qi, X., McAnally, J., Porrello, association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic E. R., Mahmoud, A. I., Tan, W., Shelton, J. M. et al. (2013). Hippo pathway growth. Nat. Cell Biol. 17, 1218-1227. doi:10.1038/ncb3216 effector Yap promotes cardiac regeneration. Proc. Natl. Acad. Sci. USA 110, Zhang, H., von Gise, A., Liu, Q., Hu, T., Tian, X., He, L., Pu, W., Huang, X., He, L., 13839-13844. doi:10.1073/pnas.1313192110 Cai, C.-L. et al. (2014). Yap1 is required for endothelial to mesenchymal transition Yamanaka, T., Horikoshi, Y., Sugiyama, Y., Ishiyama, C., Suzuki, A., Hirose, T., of the atrioventricular cushion. J. Biol. Chem. 289, 18681-18692. doi:10.1074/jbc. Iwamatsu, A., Shinohara, A. and Ohno, S. (2003). Mammalian Lgl forms a M114.554584 protein complex with PAR-6 and aPKC independently of PAR-3 to regulate Zhao, B., Li, L., Tumaneng, K., Wang, C.-Y. and Guan, K.-L. (2010). A coordinated epithelial cell polarity. Curr. Biol. 13, 734-743. doi:10.1016/S0960- phosphorylation by Lats and CK1 regulates YAP stability through SCFbeta-TRCP. 9822(03)00244-6 Genes Dev. 24, 72-85. doi:10.1101/gad.1843810 DEVELOPMENT