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Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell–matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor–endothelial cell interactions.Significance:A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor–endothelial cell interactions.

Effects of positive hits on endothelial barrier, cytoskeletal organization and tumor cell motility

Effect of drug hits on cortical actin and tumor cell migration and invasion

Phenotypes of the human pancreatic cell Ines and effects on endothelium

Methods and Reagents

Determination of niclosamide stability and approach for counting lung metastatic nodules

Analysis of scRNAseq findings

Work flow to assess effects of drug hits in vivo

Mitochondria critically regulate cellular processes such as bioenergetics, metabolism, calcium homeostasis and apoptosis. VDAC proteins are abundant proteins that control the passage of ions and metabolites across the outer mitochondrial membrane. We have previously shown that activation of VDAC2, is able to buffer excess calcium and thereby suppress calcium overload induced arrhythmogenic events in vitro and in vivo. However, the mechanism by which VDAC2 regulates calcium transport and cardiac contractions remained unclear. It is also unclear whether all three VDAC isoforms (VDAC1,2 and 3) possess similar cardioprotective activity. The zebrafish tremblor/ncx1 mutant lacks functional NCX1 in cardiomyocytes leading to calcium overload, and the manifestation of fibrillation-like phenotypes. Using the tremblor/ncx1 mutant as a model, we observed isoform-specific differences between the VDAC family members. VDAC1 and VDAC2 enhanced mitochondrial calcium trafficking and restore rhythmic contraction in tremblor mutants, whereas, VDAC3 did not. We found that the differing rescue capabilities of VDAC proteins were dependent upon residues in their N-terminal halves. Phylogenetic analysis further revealed the presence of an evolutionarily conserved glutamate at position 73 (E73) within VDAC1 and VDAC2, but a glutamine (Q73) in VDAC3. Excitingly, we showed that replacing VDAC2 E73 with Q73 ablated its calcium transporting activity. Conversely, substituting the Q73 with E73 allows VDAC3 to gain calcium trafficking and cardioprotective abilities. Overall, our study demonstrates an essential role for the evolutionarily conserved glutamate-73 in determining the anti-arrhythmic effect of VDAC isoforms through their regulation of mitochondrial calcium uptake.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell–matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor–endothelial cell interactions. Significance: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor–endothelial cell interactions.

Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.

Remodeling of the extracellular matrix plays an important role in vascular homeostasis in the basal chordate Botryllus schlosseri, whose large, transparent, extracorporeal vascular network makes it an ideal system for studies of vascular mechanotransduction., We investigated the physical role of the extracellular matrix (ECM) in vascular homeostasis in the basal chordate Botryllus schlosseri, which has a large, transparent, extracorporeal vascular network encompassing an area >100 cm2. We found that the collagen cross-linking enzyme lysyl oxidase is expressed in all vascular cells and that in vivo inhibition using β-aminopropionitrile (BAPN) caused a rapid, global regression of the entire network, with some vessels regressing >10 mm within 16 h. BAPN treatment changed the ultrastructure of collagen fibers in the vessel basement membrane, and the kinetics of regression were dose dependent. Pharmacological inhibition of both focal adhesion kinase (FAK) and Raf also induced regression, and levels of phosphorylated FAK in vascular cells decreased during BAPN treatment and FAK inhibition but not Raf inhibition, suggesting that physical changes in the vessel ECM are detected via canonical integrin signaling pathways. Regression is driven by apoptosis and extrusion of cells through the basal lamina, which are then engulfed by blood-borne phagocytes. Extrusion and regression occurred in a coordinated manner that maintained vessel integrity, with no loss of barrier function. This suggests the presence of regulatory mechanisms linking physical changes to a homeostatic, tissue-level response.

Introduction: Neural crest (NC) cells are involved in cardiac development, and NC defects account for over 10% of all congenital heart disease. The migratory and morphogenic patterns of these NC cells are not well understood because conventional optical microscopes struggle to acquire images of live embryos with sufficient temporal and spatial resolution without causing major photo-damage. To address these issues, the lab has designed and built a light-sheet fluorescence microscope (LSFM) that is capable of acquiring dual-channel signals with high spatial and temporal resolution. Hypothesis: We hypothesize that the LSFM system will capture heart tube development at a cellular level and show the time point and location at which the NC cells integrate with the heart tube. Methods and Results: The transgenic line that is manipulated for this study is the Tg(neural crest:nfsB-mCherry, cardiac: eGFP) in which the heart tube fluoresces green while NC-derived cells fluoresce red. 10 zebrafish were imaged using the 3-D time-lapse LSFM setup from 15 hours post fertilization (hpf) to 36 hpf. The imaging data was qualitative and consistent across fish. Fig. 1 (a) shows mCherry NC-derived cells (red) integrating with eGFP cardiomyocytes (green) and areas of colocalization (yellow). Fig 1 (b) and (c) show the time course of the NC cell migration into the heart tube. By 26-27 hpf, the NC cells contacted the dorsal surface of the heart tube. By the 29 hpf, the NC-derived cells integrated into the heart tube and expressed nuclear eGFP, which is visualized by the yellow fluorescing portions of the image. Conclusions: We conclude that the LSFM was successful in acquiring distinct signals from the heart tube cells and the NC-derived cells. The temporal and spatial resolution was sufficient to observe NC migration at the single cell level and map out exactly where along the heart tubule the NC-cells entered.

P-Chiral [2.2.1] bicyclic phosphines (HypPhos catalysts) have been applied to reactions between α-alkylallenoates and imines, producing guvacine derivatives. These HypPhos catalysts were assembled from trans-4-hydroxyproline, with the modular nature of the synthesis allowing variations of the exocyclic P and N substituents. Among them, exo-( p-anisyl)-HypPhos was most efficacious for [4 + 2] annulations between ethyl α-methylallenoate and imines. Through this method, ( R)-aplexone was identified as being responsible for the decrease in the cellular levels of cholesterol.

The ability to image tissue morphogenesis in real-time and in 3-dimensions (3-D) remains an optical challenge. The advent of light-sheet fluorescence microscopy (LSFM) has advanced developmental biology and tissue regeneration research. In this review, we introduce a LSFM system in which the illumination lens reshapes a thin light-sheet to rapidly scan across a sample of interest while the detection lens orthogonally collects the imaging data. This multiscale strategy provides deep-tissue penetration, high-spatiotemporal resolution, and minimal photobleaching and phototoxicity, allowing in vivo visualization of a variety of tissues and processes, ranging from developing hearts in live zebrafish embryos to ex vivo interrogation of the microarchitecture of optically cleared neonatal hearts. Here, we highlight multiple applications of LSFM and discuss several studies that have allowed better characterization of developmental and pathological processes in multiple models and tissues. These findings demonstrate the capacity of multiscale light-sheet imaging to uncover cardiovascular developmental and regenerative phenomena.

Highly regulated transcriptional control of cardiac gene networks is critical for heart development and function. Rtf1 is a critical regulator of multiple transcription processes including elongation and co-transcriptional histone modification. Rtf1 ablation in zebrafish and mice eliminates the cardiac progenitor population, producing embryos without hearts, and knockdown of Rtf1 in neonatal rat ventricular myocytes decreases the expression of sarcomere component genes, suggesting that Rtf1 is indispensable for both establishing the myocardial lineage and maintaining normal physiological homeostasis in differentiated cardiomyocytes. To explore the function of Rtf1 in the adult heart, we generated an inducible knockout of Rtf1 in the adult myocardium. Loss of Rtf1 function in adult cardiomyocytes results in left ventricular systolic dysfunction and pathological features of heart failure. The Rtf1 deficient hearts exhibit a gene expression profile characteristic of heart failure, and display disrupted myofibrils and cell-cell junctions. Intriguingly, and in line with Rtf1’s established role in promoting histone H2B ubiquitination, diminished H2BK120 ubiquitination was also observed in Rtf1 deficient adult hearts. Collectively, our findings show that Rtf1 and Rtf1 mediated H2BK120 ubiquitination are essential epigenetic regulators of both myocardial lineage determination during development as well as maintaining cellular homeostasis, integrity and function in mature cardiomyocytes.

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis.

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. The influences of Ca2+ signaling on cardiac function have been examined extensively, but whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a mutant zebrafish model lacking NCX1 activity in the heart, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. Gene expression profiling analysis revealed that the E3 ubiquitin ligase MuRF1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down MuRF1 activity or inhibiting proteasome activity preserved myofibril integrity in ncx1 deficient hearts, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the MuRF1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced MuRF1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated MuRF1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts the myofibril integrity in heart muscle cells by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.

Background: PPM1G is a nuclear localized serine/threonine phosphatase implicated to be a regulator of chromatin remodeling, mRNA splicing, and DNA damage. However, its in vivo function is unknown. Results: Here we show that ppm1g expression is highly enriched in the central nervous system during mouse and zebrafish development. ppm1g−/− mice were embryonic lethal with incomplete penetrance after E12.5. Rostral defects, including neural tube and craniofacial defects were observed in ppm1g−/− embryos associated with increased cell death in the neural epithelium. In zebrafish, loss of ppm1g also led to neural defects with aberrant neural marker gene expression. Primary fibroblasts from ppm1g−/− embryos failed to grow without immortalization while immortalized ppm1g−/− fibroblasts had increased cell death upon oxidative and genotoxic stress when compared to wild type fibroblasts. Conclusions: Our in vivo and in vitro studies revealed a critical role for PPM1G in normal development and cell survival. Developmental Dynamics 242:1101–1109, 2013. © 2013 Wiley Periodicals, Inc.

Sarcomere integrity must be continuously maintained in cardiomyocytes to ensure contractile function; however, the genetic programs controlling this process are not completely understood. We have previously shown that the RNA polymerase II associated protein Rtf1 directs cardiac progenitor cell formation during early cardiogenesis. Here we report that in mature cardiomyocytes, Rtf1 controls gene programs essential for the maintenance of sarcomere structure and cardiac function. Cardiomyocyte-specific ablation of Rtf1 in mice results in dilated cardiomyopathy characterized by thinning of the myocardial walls, dilation of the ventricular cavity and impaired left ventricle contractile function. Rtf1 knockdown in neonatal rat ventricular myocytes model results in the breakdown of sarcomeres and downregulation of myofibrillar gene programs accompanied by reduced abundance of monoubiquitinated H2BK120 and epigenetic marks associated with actively transcribed genes. In zebrafish embryos, mutation in Rtf1’s histone modification domain arrests cardiomyocyte differentiation and heart tube formation. Together these findings demonstrate that Rtf1 controls cardiomyocyte maturation, myofibril integrity and cardiac function by modulating cardiac epigenetic landscapes.

Background Germ cells are specified during early development and are responsible for generating gametes in the adult. After germ cells are specified, they typically migrate to a particular niche in the organism where they reside for the remainder of its lifetime. For some model organisms, the specification and migration of germ cells have been extensively studied, but how these events occur in animals that reproduce both sexually and asexually is not well understood. Results We have identified a novel TGF-β family member in Botryllus schlosseri, tgfβ-f, and found that it is expressed by follicle cell progenitors and the differentiated follicle and support cells surrounding the maturing gametes. Using the expression of tgfβ-f and the germ cell marker vasa, we have found that nearly all germ cells in Botryllus are associated with tgfβ-f-expressing follicle progenitors in clusters consisting solely of those two cell types. These clusters were mostly small, consisting of ten or fewer cells, and generally contained between a 2:1 and 1:1 ratio of follicle progenitors to germ cells. Clusters of germ and follicle progenitor cells were primarily localized to niches in the primary and secondary buds, but could also be found in other locations including the vasculature. We analyzed the location of germ cell clusters throughout the asexual life cycle of Botryllus and found that at the stage when germ cells are first detected in the secondary bud niche, a dramatic change in the size and location of germ/follicle cell clusters also occurred. Conclusions Our findings suggest that germ/follicle cell clusters have predictable migratory patterns during the weekly asexual developmental cycle in Botryllus. An increased number of small clusters and the presence of clusters in the vasculature coinciding with the appearance of clusters in the secondary bud suggest that fragmentation of clusters and the migration of smaller clusters through the vasculature may be an important aspect of Botryllus reproductive biology, ensuring the transmission of the germline to subsequent asexual generations. Electronic supplementary material The online version of this article (doi:10.1186/s13227-016-0047-5) contains supplementary material, which is available to authorized users.

Na(+),K(+) ATPase pumps Na(+) out of and K(+) into the cytosol, maintaining a resting potential that is essential for the function of excitable tissues like cardiac muscle. In addition to its well-characterized physiological role in the heart, Na(+),K(+) ATPase also regulates the morphogenesis of the embryonic zebrafish heart via an as yet unknown mechanism. Here, we describe a novel non-cell autonomous function of Na(+),K(+) ATPase/Atp1a1 in the elongation of the zebrafish heart tube. Embryos lacking Atp1a1 function exhibit abnormal migration behavior of cardiac precursors, defects in the elongation of the heart tube, and a severe reduction in ECM/Fibronectin deposition around the myocardium, despite the presence of normal cell polarity and junctions in the myocardial epithelium prior to the timeframe of heart tube elongation. Interestingly, we found that Atp1a1 is not present in the myocardium at the time when cardiac morphogenesis defects first become apparent, but is expressed in an extra-embryonic tissue, the yolk syncytial layer (YSL), at earlier stages. Knockdown of Atp1a1 activity specifically in the YSL using morpholino oligonucleotides produced heart tube elongation defects like those found in atp1a1 mutants, indicating that Atp1a1 function in the YSL is necessary for heart tube elongation. Furthermore, atp1a1 expression in the YSL was regulated by the homeobox transcription factor mxtx1. Together, these data reveal a new non-cell autonomous role for Atp1a1 in cardiac morphogenesis and establish Na(+),K(+) ATPase as a major player in the genetic pathway by which the YSL regulates embryonic ECM deposition.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Na+,K+ ATPase is an essential ion pump involved in regulating ionic concentrations within epithelial cells. The zebrafish heart and mind (had) mutation, which disrupts the α1B1 subunit of Na+,K+ ATPase, causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants, including nagie oko (nok). We demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1)–positive junction belts within myocardial cells. Functional tests and pharmacological inhibition experiments demonstrate that Na+,K+ ATPase activity is positively regulated via an N-terminal phosphorylation site that is necessary for correct heart morphogenesis to occur, and that maintenance of ZO-1 junction belts requires ion pump activity. These findings suggest that the correct ionic balance of myocardial cells is essential for the maintenance of epithelial integrity during heart morphogenesis.

The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. The germ line of the newly developing bodies is derived from migrating germ cell precursors, but the signals governing this homing process are unknown. Here we show that germ cell precursors can be prospectively isolated based on expression of aldehyde dehydrogenase and integrin alpha-6, and that these cells express germ cell markers such as vasa, pumilio and piwi, as well as sphingosine-1-phosphate receptor. In vitro, sphingosine-1-phosphate (S1P) stimulates migration of germ cells, which depends on integrin alpha-6 activity. In vivo, S1P signalling is essential for homing of germ cells to newly developing bodies. S1P is generated by sphingosine kinase in the developing germ cell niche and degraded by lipid phosphate phosphatase in somatic tissues. These results demonstrate a previously unknown role of the S1P signalling pathway in germ cell migration in the ascidian Botryllus schlosseri., The regulation of germ cell migration in the colonial ascidian Botryllus schlosseri is poorly understood. In this chordate, Kassmer et al. identify sphingosine-1-phosphate as regulating germ cell migration in vitro and homing of cells to newly developing bodies in live organisms.

Background Gonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively. Results Here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads. Conclusion We have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1183) contains supplementary material, which is available to authorized users.

Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor ( tre ) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium–calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart.

The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.

Publisher Summary Vertebrates exhibit an extensive asymmetry of the internal organs with respect to the left–right axis that is established during embryogenesis and persists in the mature organism. In several vertebrate models, calcium has been found to play an essential role in left–right patterning. This chapter discusses the conserved and divergent mechanisms regulating left–right axis development in vertebrate model organisms, and describes the role of calcium at different steps in this process. Calcium levels have been proposed to modulate dynein-regulated microtubule sliding, and thereby affect the waveform of Clamydomonas flagella and the motility of sea-urchin sperm flagella. Since CaMKII is known to be an important component of calcium signaling in zebrafish left right (LR) development, it is possible that CaMKII directly or indirectly regulates dynein activity in KV cilia. Alternatively, proper calcium homeostasis and signaling might be required for the biogenesis of ultrastructurally normal motile KV cilia. Studies indicate that it is possible that increased intracellular calcium might stimulate production of a currently unknown left-sided determinant, or enhance the activity of a particular signaling pathway. Given the ease of forward and reverse genetic techniques in the zebrafish, it is likely to serve as an excellent model for future studies aiming to tease out the intermediates between asymmetric calcium and asymmetric gene expression, and also between calcium signaling and cilia motility.

The establishment of a left–right axis during vertebrate development is essential for coordinating the relative positions of the internal organs to ensure that they function appropriately. Studies in numerous model organisms have revealed differences in regulative mechanisms upstream of nodal signaling, a conserved pathway in left–right axis specification. This review will summarize the diverse pathways involved in the break of left–right symmetry and explore in depth the multiple roles of calcium in vertebrate left–right axis specification. Developmental Dynamics 237:3491–3496, 2008. © 2008 Wiley-Liss, Inc.

In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths) 1. Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.

Leo1 is a component of the Polymerase-Associated Factor 1 (PAF1) complex, an evolutionarily conserved protein complex involved in gene transcription regulation and chromatin remodeling. The role of leo1 in vertebrate embryogenesis has not previously been examined. Here, we report that zebrafish leo1 encodes a nuclear protein that has a similar molecular structure to Leo1 proteins from other species. From a genetic screen, we identified a zebrafish mutant defective in the leo1 gene. The truncated Leo1 LA1186 protein lacks a nuclear localization signal and is distributed mostly in the cytoplasm. Phenotypic analysis showed that while the initial patterning of the primitive heart tube is not affected in leo1 LA1186 mutant embryos, the differentiation of cardiomyocytes at the atrioventricular boundary is aberrant, suggesting a requirement for Leo1 in cardiac differentiation. In addition, the expression levels of markers for neural crest-derived cells such as crestin , gch2 , dct and mitfa are greatly reduced in leo1 LA1186 mutants, indicating a requirement for Leo1 in maintaining the neural crest population. Consistent with this finding, melanocyte and xanthophore populations are severely reduced, craniofacial cartilage is barely detectable, and mbp -positive glial cells are absent in leo1 LA1186 mutants after three days of development. Taken together, these results provide the first genetic evidence of the requirement for Leo1 in the development of the heart and neural crest cell populations.

The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education [1,2]. In functional genomics, we have found an ideal platform to simultaneously benefit students and contribute to scientific discovery. The sequencing of eukaryotic genomes has facilitated the identification of complete sets of genes in humans and model genetic organisms. This has allowed many forms of high-throughput analyses of transcriptional profiles, protein interactions, structural motifs, and even genome-wide knock-downs in cell lines or in selected organisms. However, one of the best tools to provide functional information about gene action— obtaining in vivo evidence about the phenotype resulting from heritable loss of function—is difficult and less amenable to high-throughput research. We were able to achieve a large-scale in vivo analysis with a significant number of undergraduate students at UCLA, called the UCLA Undergraduate Consortium for Functional Genomics. This work, a practical manifestation of policy positions proposing discoverybased education, is described in summary form here (and in Box 1) and in detail online at http://www.bruinfly.ucla.edu. This effort combines professional-quality research with a strategy for research-based undergraduate education.

Ca2+ homeostasis is essential for rhythmic contractions of the adult myocardium. In the developing embryo, a role of Ca2+ in establishing cardiac rhythmicity has been proposed, but the underlying mechanism has yet to be fully explored. We have previously shown that zebrafish tremblor mutant embryos develop a fibrillating heart and that the tremblor locus encodes a cardiac-specific isoform of the Na+/Ca2+ exchanger, NCX1h. While rhythmic Ca2+ waves were detected with each cardiac contraction in wild type hearts, only local, unsynchronized Ca2+ signals together with sporadic contractions were observed in tremblor hearts. These data suggest that loss of function of NCX1h induces cardiac fibrillation in tremblor and that tremblor mutants can serve as a model for cardiac arrhythmia induced by aberrant calcium homeostasis.From a collection of small molecules, we found a novel compound, OK-F7, that could restore rhythmic cardiac contractions in tremblor embryos at low micromolar concentrations in a reversible and dosage-dependent manner. We tested the effect of OK-F7 on isolated cardiomyocytes under Ca2+ overload conditions and found that treatment with 1 µM OK-F7 reduces the frequency of spontaneously propagating Ca2+ waves by approximately half, while 25 µM OK-F7 almost entirely abolishes Ca2+ waves. A biochemical pull-down assay using an OK-F7 affinity column isolated a mitochondrial membrane protein that selectively bound to OK-F7. These findings suggest that OK-F7 suppresses cardiac fibrillation by reducing the frequency of arrhythmogenic Ca2+ waves in Ca2+-overloaded cardiomyocytes and indicate a novel role of mitochondria in cardiac Ca2+ handling and control of rhythmicity. Further validation of the target protein and biophysical experiments confirming a role of mitochondria in the mechanism of OK-F7 action are underway and will be discussed.Supported by NIH-1R01HL096980-01A1 and FWF-J3065-B11.