Early calcium and cardiac contraction defects in a model of phospholamban R9C mutation in zebrafish.

The phospholamban mutation Arg 9 to Cys (R9C) has been found to cause a dilated cardiomyopathy in humans and in transgenic mice, with ventricular dilation and premature death. Emerging evidence suggests that phospholamban R9C is a loss-of-function mutation with dominant negative effect on SERCA2a activity. We imaged calcium and cardiac contraction simultaneously in 3 and 9 days-post-fertilization (dpf) zebrafish larvae expressing plnbR9C in the heart to unveil the early pathological pathway that triggers the disease. We generated transgenic zebrafish lines expressing phospholamban wild-type (Tg(myl7:plnbwt)) and phospholamban R9C (Tg(myl7:plnbR9C)) in the heart of zebrafish. To measure calcium and cardiac contraction in 3 and 9 dpf larvae, Tg(myl7:plnbwt) and Tg(myl7:plnbR9C) fish were outcrossed with a transgenic line expressing the ratiometric fluorescent calcium biosensor mCyRFP1-GCaMP6f. We found that PlnbR9C raised calcium transient amplitude, induced positive inotropy and lusitropy, and blunted the ß-adrenergic response to isoproterenol in 3 dpf larvae. These effects can be attributed to enhanced SERCA2a activity induced by the PlnbR9C mutation. In contrast, Tg(myl7:plnbR9C) larvae at 9 dpf exhibited ventricular dilation, systolic dysfunction and negative lusitropy, hallmarks of a dilated cardiomyopathy in humans. Importantly, N-acetyl-L-cysteine rescued this deleterious phenotype, suggesting that reactive oxygen species contribute to the pathological pathway. These results also imply that dysregulation of calcium homeostasis during embryo development contributes to the disease progression at later stages. Our in vivo model in zebrafish allows characterization of pathophysiological mechanisms leading to heart disease, and can be used for screening of potential therapeutical agents.

Modeling Human Cardiac Arrhythmias: Insights from Zebrafish.

Cardiac arrhythmia, or irregular heart rhythm, is associated with morbidity and mortality and is described as one of the most important future public health challenges. Therefore, developing new models of cardiac arrhythmia is critical for understanding disease mechanisms, determining genetic underpinnings, and developing new therapeutic strategies. In the last few decades, the zebrafish has emerged as an attractive model to reproduce in vivo human cardiac pathologies, including arrhythmias. Here, we highlight the contribution of zebrafish to the field and discuss the available cardiac arrhythmia models. Further, we outline techniques to assess potential heart rhythm defects in larval and adult zebrafish. As genetic tools in zebrafish continue to bloom, this model will be crucial for functional genomics studies and to develop personalized anti-arrhythmic therapies.

Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling.

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The ß-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

Hhex regulates the specification and growth of the hepatopancreatic ductal system.

Significant efforts have advanced our understanding of foregut-derived organ development; however, little is known about the molecular mechanisms that underlie the formation of the hepatopancreatic ductal (HPD) system. Here, we report a role for the homeodomain transcription factor Hhex in directing HPD progenitor specification in zebrafish. Loss of Hhex function results in impaired HPD system formation. We found that Hhex specifies a distinct population of HPD progenitors that gives rise to the cystic duct, common bile duct, and extra-pancreatic duct. Since hhex is not uniquely expressed in the HPD region but is also expressed in endothelial cells and the yolk syncytial layer (YSL), we tested the role of blood vessels as well as the YSL in HPD formation. We found that blood vessels are required for HPD patterning, but not for HPD progenitor specification. In addition, we found that Hhex is required in both the endoderm and the YSL for HPD development. Our results shed light on the mechanisms directing endodermal progenitors towards the HPD fate and emphasize the tissue specific requirement of Hhex during development.

Early sarcomere and metabolic defects in a zebrafish pitx2c cardiac arrhythmia model.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c+/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c-/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.

Correction: Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

[This corrects the article DOI: 10.1371/journal.pgen.1007743.].

Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.

HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Pitx2c orchestrates embryonic axis extension via mesendodermal cell migration.

Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.

The Hippo pathway effector Wwtr1 regulates cardiac wall maturation in zebrafish.

Cardiac trabeculation is a highly regulated process that starts with the delamination of compact layer cardiomyocytes. The Hippo signaling pathway has been implicated in cardiac development but many questions remain. We have investigated the role of Wwtr1, a nuclear effector of the Hippo pathway, in zebrafish and find that its loss leads to reduced cardiac trabeculation. However, in mosaic animals, wwtr1-/- cardiomyocytes contribute more frequently than wwtr1+/- cardiomyocytes to the trabecular layer of wild-type hearts. To investigate this paradox, we examined the myocardial wall at early stages and found that compact layer cardiomyocytes in wwtr1-/- hearts exhibit disorganized cortical actin structure and abnormal cell-cell junctions. Accordingly, wild-type cardiomyocytes in mosaic mutant hearts contribute less frequently to the trabecular layer than when present in mosaic wild-type hearts, indicating that wwtr1-/- hearts are not able to support trabeculation. We also found that Nrg/Erbb2 signaling, which is required for trabeculation, could promote Wwtr1 nuclear export in cardiomyocytes. Altogether, these data suggest that Wwtr1 establishes the compact wall architecture necessary for trabeculation, and that Nrg/Erbb2 signaling negatively regulates its nuclear localization and therefore its activity.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure.

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Organ Function as a Modulator of Organ Formation: Lessons from Zebrafish.

Organogenesis requires an intricate balance between cell differentiation and tissue growth to generate a complex and fully functional organ. However, organogenesis is not solely driven by genetic inputs, as the development of several organ systems requires their own functionality. This theme is particularly evident in the developing heart as progression of cardiac development is accompanied by increased and altered hemodynamic forces. In the absence or disruption of these forces, heart development is abnormal, suggesting that the heart must sense these changes and respond appropriately. Here, we discuss concepts of how embryonic heart function contributes to heart development using lessons learned mostly from studies in zebrafish.

Claudin-10 is required for relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Species-specific symmetry-breaking events at the left-right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left-right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen's node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen's node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen's node prevented expression of Nodal, Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left-right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen's node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Are there conserved roles for the extracellular matrix, cilia, and junctional complexes in left-right patterning?

Many different types of molecules have essential roles in patterning the left-right axis and directing asymmetric morphogenesis. In particular, the relationship between signaling molecules and transcription factors has been explored extensively. Another group of proteins implicated in left-right patterning are components of the extracellular matrix, apical junctions, and cilia. These structural molecules have the potential to participate in the conversion of morphogenetic cues from the extracellular environment into morphogenetic patterning via their interactions with the actin cytoskeleton. Although it has been relatively easy to temporally position these proteins within the hierarchy of the left-right patterning pathway, it has been more difficult to define how they mechanistically fit into these pathways. Consequently, our understanding of how these factors impart patterning information to influence the establishment of the left-right axis remains limited. In this review, we will discuss those structural molecules that have been implicated in early phases of left-right axis development.

Claudin family members exhibit unique temporal and spatial expression boundaries in the chick embryo.

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Several members of the claudin family have been shown to be important during embryonic development and morphogenesis. However, detailed embryonic expression patterns have been described for only a few claudins. Here, we provide a phylogenetic analysis of the chicken claudins and a comprehensive analysis of their mRNA expression profiles. We found that claudin family members exhibit both overlapping and unique expression patterns throughout development. Especially striking were the distinct expression boundaries observed between neural and non-neural ectoderm, as well as within ectodermal derivatives. Claudins were also expressed in endodermally-derived tissues, including the anterior intestinal portal, pharynx, lung and pancreas and in mesodermally derived tissues such as the kidney, gonad and heart. The overlapping zones of claudin expression observed in the chick embryo may confer distinct domains of ion permeability within the early epiblast and in epithelial, mesodermal and endothelial derivatives that may ultimately influence embryonic patterning and morphogenesis during development.

Claudin-5 expression in the vasculature of the developing chick embryo.

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Studies in human, mouse, Xenopus, and zebrafish have shown that only a limited number of claudins are expressed in endothelial cells. Here, we report the expression pattern of Claudin-5 during chick development. Between HH stage 4 and 6 Claudin-5 expression was observed exclusively in extraembryonic tissue. Claudin-5 expression was not observed in the embryo until HH stage 8, coincident with the onset of embryonic vascularization. Claudin-5 expression was maintained in the developing vasculature in the embryonic and extraembryonic tissue throughout organogenesis (HH stage 19-35), including the vasculature of the ectoderm and of organs derived from the mesoderm and endoderm lineages. These data describe a conserved expression pattern for Claudin-5 in the endothelial tight junction barrier and is the first report of the onset of Claudin-5 expression in a vertebrate embryo.

Manipulating claudin expression in avian embryos.

Since the discovery of Claudin-1 and -2 by Tsukita and colleagues in the late 1990s [Furuse et al. J Cell Biol 141:1539-50,1998], claudin family members have been found to have critical roles in maintaining the integrity of epithelial and endothelial tight junctions [Furuse and Moriwaki Ann N Y Acad Sci 1165:58-61, 2009; Morita et al. Proc Natl Acad Sci USA 96:511-6, 1999; Tsukita and Furuse Ann N Y Acad Sci 915:129-35, 2000; Turksen and Troy J Cell Sci 117:2435-47, 2004]. The properties of distinct claudin family members in tight junction permeability and specificity have been extensively studied in vitro using cell culture models. In vivo, claudin family members are dynamically regulated during embryogenesis and alterations in their expression patterns can have detrimental effects on the formation and physiological function of the tissues in which they are expressed. The chick embryo provides an excellent system to dissect the roles of specific family members in vivo and to explore the effects of modulating claudin expression during the epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions that are associated with tissue morphogenesis and differentiation. We are using the chick embryo to understand the roles of the claudin family of tight junction proteins during gastrulation and left-right patterning during embryogenesis. Here, we describe methodologies for manipulating claudin gene expression in specific target tissues during chick embryogenesis.

Expression patterns of hormones, signaling molecules, and transcription factors during adenohypophysis development in the chick embryo.

The chick embryo is an ideal model to study pituitary cell-type differentiation. Previous studies describing the temporal appearance of differentiated pituitary cell types in the chick embryo are contradictory. To resolve these controversies, we used RT-PCR to define the temporal onset and in situ hybridization and immunohistochemistry to define the spatial localization of hormone expression within the pituitary. RT-PCR detected low levels of Fshbeta (gonadotropes) and Pomc (corticotropes, melanotropes) mRNA at E4 and Gh (somatotropes), Prl (lactotropes), and Tshbeta (thyrotropes) mRNA at E8. For all hormones, sufficient accumulation of mRNA and/or protein to permit detection by in situ hybridization or immunohistochemistry was observed approximately 3 days later and in all cases corresponded to a notable increase in RT-PCR product. We also describe the expression patterns of signaling (Bmp2, Bmp4, Fgf8, Fgf10, Shh) and transcription factors (Pitx1, Pitx2, cLim3) known to be important for pituitary organogenesis in other model organisms.