Biliary organoids uncover delayed epithelial development and barrier function in biliary atresia.

BACKGROUND AND AIMS: Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. APPROACH AND RESULTS: We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, ß-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, ß-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. CONCLUSIONS: Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.

DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome.

Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.

Many essential parts of the body contain tubes: the liver for example, contains bile ducts and blood vessels. These tubes develop right next to each other, like entwined trees. To do their jobs, these ducts must communicate and collaborate, but they do not always grow properly. For example, babies with Alagille syndrome are born with few or no bile ducts, resulting in serious liver disease. Understanding the architecture of the tubes in their livers could explain why some children with this syndrome improve with time, but many others need a liver transplant. Visualising biological tubes in three dimensions is challenging. One major roadblock is the difficulty in seeing several tubular structures at once. Traditional microscopic imaging of anatomy is in two dimensions, using slices of tissue. This approach shows the cross-sections of tubes, but not how the ducts connect and interact. An alternative is to use micro computed tomography scans, which use X-rays to examine structures in three dimensions. The challenge with this approach is that soft tissues, which tubes in the body are made of, do not show up well on X-ray. One way to solve this is to fill the ducts with X-ray absorbing resins, making a cast of the entire tree structure. The question is, can two closely connected tree structures be distinguished if they are cast at the same time? To address this question, Hankeova, Salplachta et al. developed a technique called double resin casting micro computed tomography, or DUCT for short. The approach involved making casts of tube systems using two types of resin that show up differently under X-rays. The new technique was tested on a mouse model of Alagille syndrome. One resin was injected into the bile ducts, and another into the blood vessels. This allowed Hankeova, Salplachta et al. to reconstruction both trees digitally, revealing their length, volume, branching, and interactions. In healthy mice, the bile ducts were straight with uniform branches, but in mice with Alagille syndrome ducts were wiggly, and had extra branches in the centre of the liver. This new imaging technique could improve the understanding of tube systems in animal models of diseases, both in the liver and in other organs with tubes, such as the lungs or the kidneys. Hankeova, Salplachta et al. also lay a foundation for a deeper understanding of bile duct recovery in Alagille syndrome. In the future, DUCT could help researchers to see how mouse bile ducts change in response to experimental therapies.

Loss of Anks6 leads to YAP deficiency and liver abnormalities.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro.

Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.

Biliary atresia: A comprehensive review.

Biliary atresia presents as an obliterative cholangiopathy with neonatal jaundice and pale stools. The disease exhibits aetiological heterogeneity with a multiplicity of potential causative factors, both developmental and environmental. A number of clinical variants making up a minority of all cases can be defined relatively precisely which match suggested aetiology better although in most it still remains speculative. These include the syndromic form (BASM), the cystic form and those associated with CMV IgM antibodies. We review not only the clinical evidence for a developmental or an immune-mediated aetiology perhaps triggered by perinatal viral exposure but also several other recently suggested concepts such as microchimerism, gene susceptibility and environmental toxins.

Cholangiocytes derived from induced pluripotent stem cells for disease modeling.

PURPOSE OF REVIEW: Biliary diseases are a significant cause of morbidity and mortality. Challenges in establishing accurate in-vitro methods to model human bile duct diseases and evaluate therapies have contributed to a lack of effective medical treatments. The recent discovery of strategies to reprogram human somatic cells to a state of induced pluripotency has opened up new possibilities for studying both development and disease in a wide variety of human tissues. This review was undertaken to summarize the recent progress made in generating biliary tissue from induced pluripotent stem cells (iPSCs) and the application of this technology to biliary disease modeling. RECENT FINDINGS: Several groups have reported defined differentiation protocols that incorporate key signaling cues from normal biliary development to yield cholangiocyte-like cells from wild-type human iPSCs that demonstrate epithelial morphology in two and three-dimensional culture, cholangiocyte markers, biliary gene expression profiles, and functional attributes consistent with biliary epithelium. Key features of Alagille syndrome and polycystic liver disease can be modeled with iPSC-derived cholangiocytes, whereas the use of iPSCs from cystic fibrosis patients has facilitated not only modeling of cystic fibrosis biliary disease but also in-vitro correction of the disorder with pharmacological agents. SUMMARY: Mature, functional cholangiocytes can be derived from human iPSCs and utilized to model biliary diseases in vitro. These advances should facilitate further research to improve our understanding of the pathophysiology of cholangiopathies and evaluate novel treatments. In the future, this technology will likely form a key element of tissue replacement strategies.

Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.

The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and γ-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell-derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases.

Role of ß-catenin in development of bile ducts.

Beta-catenin is known to play stage- and cell-specific functions during liver development. However, its role in development of bile ducts has not yet been addressed. Here we used stage-specific in vivo gain- and loss-of-function approaches, as well as lineage tracing experiments in the mouse, to first demonstrate that ß-catenin is dispensable for differentiation of liver precursor cells (hepatoblasts) to cholangiocyte precursors. Second, when ß-catenin was depleted in the latter, maturation of cholangiocytes, bile duct morphogenesis and differentiation of periportal hepatocytes from cholangiocyte precursors was normal. In contrast, stabilization of ß-catenin in cholangiocyte precursors perturbed duct development and cholangiocyte differentiation. We conclude that ß-catenin is dispensable for biliary development but that its activity must be kept within tight limits. Our work is expected to significantly impact on in vitro differentiation of stem cells to cholangiocytes for toxicology studies and disease modeling.

Hepatic Notch2 deficiency leads to bile duct agenesis perinatally and secondary bile duct formation after weaning.

UNLABELLED: Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2(fl/fl)/Alfp-Cre(tg/-) ("Notch2-cKO") and Notch2(fl/fl)/Alfp-Cre(-/-) ("control") mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1ß, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation. CONCLUSION: Cholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.

The use of Yes-associated protein expression in the diagnosis of persistent neonatal cholestatic liver disease.

Although physiologic jaundice of neonates is common, persistent neonatal cholestasis is life-threatening and has multiple etiologies. Among these etiologies, biliary atresia (BA) requires rapid diagnosis and treatment. In diagnosing BA, the surgical pathologist must recognize subtle histologic changes, often with only a small core liver biopsy. To aid in the differential diagnosis of neonatal cholestasis, we investigated Yes-associated protein (YAP), a regulator of organ size and bile duct development. We examined whether a YAP immunostain can highlight emerging hepatobiliary epithelium in BA (n = 28) versus other causes of persistent cholestasis (non-BA; n = 15) and thus serve as a useful diagnostic marker in persistent neonatal jaundice. We show significantly (P < .01) more high-grade (<2) fibrosis and ductular proliferation among BA versus non-BA cases. Likewise, there was significantly more high-grade (2-3/3) cytoplasmic and nuclear YAP staining in BA (97% and 89%) versus non-BA (20% and 13%). High-grade nuclear YAP staining was both sensitive (88%) and specific (87%) for the diagnosis of BA. In contrast to neonatal cholestasis, the differences in YAP localization in cholestatic/obstructed versus nonobstructed adult livers were not significant. Lastly, we found that pharmacologic inhibition of the YAP complex in both cholangiocyte and cholangiocarcinoma cell lines blocked compensatory bile duct proliferation, an early marker of BA that requires nuclear YAP expression, in a time- and dose-dependent manner. In summary, we show that YAP expression modulates both bile duct proliferation and liver damage/fibrosis while acting as a sensitive and specific marker in the differential diagnosis of persistent neonatal cholestasis.

Immunohistochemical analyses of the kinetics and distribution of macrophages, hepatic stellate cells and bile duct epithelia in the developing rat liver.

Non-parenchymal cells in the liver consist mainly of Kupffer cells, hepatic stellate (HS) cells and cholangiocytes. To establish base-line data and clarify the nature, this study investigated immunohistochemically the kinetics of these cell populations in developing liver of F344 rats. Samples were collected from fetuses on days 18 and 20, neonates on days 1, 4, 8, 15, and 21, and adults at weeks 5-35. ED1 (CD68)-positive macrophages showed highest number as early as fetal day 18, and then decreased gradually until adulthood. The numbers of macrophages reacting to ED2 (CD163), OX6 (MHC II), and SRA-E5 (scavenger receptor A, CD204) increased after birth (early neonates), and ED2- and SRA-E5-positive cell numbers were maintained until adulthood, but OX6-positive cell number decreased at late stages of neonates and adulthood. ED2- and SRA-E5-positive cells appeared along sinusoids, indicating Kupffer cells, whereas OX6-positive cells were limited in Glisson's sheath. Vimentin-positive HS cells were seen consistently from fetuses to adulthood. Desmin- and glial fibrillary acidic protein (GFAP)-positive HS cells tended to be seen in fetuses and early stages of neonates. HS cells reacting to α-smooth muscle actin (α-SMA) were not detectable. Cholangiocytes, reacting to cytokeratin 19 and AE1/AE3, began to be seen on fetus day 18 with faint reaction, and interlobular bile ducts were completed in Glisson's sheath by neonatal day 8. This study shows that there are heterogeneous macrophage populations and that HS cells can show various cytoskeletal proteins in rat hepatogenesis.

Molecular mechanisms of liver and bile duct development.

The liver is derived from the ventral foregut endoderm. After hepatic specification, liver progenitor cells delaminate from the endoderm and invade the septum transversum mesenchyme to form the liver bud. In addition to proliferation and expansion, liver progenitor cells differentiate into two epithelial cell types, each arranged into unique structures with distinctive function. Growth, morphogenesis, and differentiation during liver development are regulated by a variety of factors that are expressed in a spatially and temporally specific manner. A comprehensive understanding of the regulatory mechanisms underlying the liver development has influenced the diagnosis of liver diseases and further progress will be critical for future advances in therapy. This review highlights some of the best understood steps of liver development, summarizing progress in our understanding of the molecular mechanisms that underlie differentiation, morphogenesis, and functional integration of the liver.

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPß.

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPß. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPß, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.

DNA hypomethylation causes bile duct defects in zebrafish and is a distinguishing feature of infantile biliary atresia.

UNLABELLED: Infantile cholestatic disorders arise in the context of progressively developing intrahepatic bile ducts. Biliary atresia (BA), a progressive fibroinflammatory disorder of extra- and intrahepatic bile ducts, is the most common identifiable cause of infantile cholestasis and the leading indication for liver transplantation in children. The etiology of BA is unclear, and although there is some evidence for viral, toxic, and complex genetic causes, the exclusive occurrence of BA during a period of biliary growth and remodeling suggests an importance of developmental factors. Interestingly, interferon-γ (IFN-γ) signaling is activated in patients and in the frequently utilized rhesus rotavirus mouse model of BA, and is thought to play a key mechanistic role. Here we demonstrate intrahepatic biliary defects and up-regulated hepatic expression of IFN-γ pathway genes caused by genetic or pharmacological inhibition of DNA methylation in zebrafish larvae. Biliary defects elicited by inhibition of DNA methylation were reversed by treatment with glucocorticoid, suggesting that the activation of inflammatory pathways was critical. DNA methylation was significantly reduced in bile duct cells from BA patients compared to patients with other infantile cholestatic disorders, thereby establishing a possible etiologic link between decreased DNA methylation, activation of IFN-γ signaling, and biliary defects in patients. CONCLUSION: Inhibition of DNA methylation leads to biliary defects and activation of IFN-γ-responsive genes, thus sharing features with BA, which we determine to be associated with DNA hypomethylation. We propose epigenetic activation of IFN-γ signaling as a common etiologic mechanism of intrahepatic bile duct defects in BA.

Biliary atresia: will blocking inflammation tame the disease?

Biliary atresia is the most common cholangiopathy of childhood. With complete obstruction of segments or the entire length of extrahepatic bile ducts, the timely pursuit of hepatoportoenterostomy is the best strategy to restore bile drainage. However, even with prompt surgical intervention, ongoing injury of intrahepatic bile ducts and progressive cholangiopathy lead to end-stage cirrhosis. The pace of disease progression is not uniform; it may relate to clinical forms of disease and/or staging of liver pathology at diagnosis. Although the etiology of disease is not yet defined, several biological processes have been linked to pathogenic mechanisms of bile duct injury. Among them, there is increasing evidence that the immune system targets the duct epithelium and disrupts bile flow. We discuss how careful clinical phenotyping, staging of disease, and basic mechanistic research are providing insights into clinical trial designs and directions for development of new therapies to block progression of disease.

Biliary differentiation and bile duct morphogenesis in development and disease.

The biliary tract consists of a network of intrahepatic and extrahepatic ducts that collect and drain the bile produced by hepatocytes to the gut. The bile ducts are lined by cholangiocytes, a specialized epithelial cell type that has a dual origin. Intrahepatic cholangiocytes derive from the liver precursor cells, whereas extrahepatic cholangiocytes are generated directly from the endoderm. In this review we discuss the mechanisms of cholangiocyte differentiation and bile duct morphogenesis, and describe how developing ducts interact with the hepatic artery. We also present an overview of the mechanisms of biliary dysgenesis in humans.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells.

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals.

The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, we demonstrate that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Our studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2.