Llgl1 Regulates Zebrafish Cardiac Development by Mediating Yap Stability in Cardiomyocytes Michael A
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© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev193581. doi:10.1242/dev.193581 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