Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one?carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.     

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine¹. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans¹. The zebrafish has become a powerful animal model for the study of vertebrate cell biology². There has been extensive documentation and analysis of embryonic development in the zebrafish³. Only recently have scientists sought to document adult anatomy and surgical dissection techniques⁴, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer⁵. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish^6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate⁸. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species^9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals. Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)^11,12, expression profiling^13,14, and transplantation^11,15. We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.     

Fit for consumption: zebrafish as a model for tuberculosis

Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish–M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis.KEY WORDS: Disease models, Genetics, Mycobacterium, Pathogenesis, Tuberculosis, Zebrafish     

A Zebrafish Model of Myelodysplastic Syndrome Produced through tet2 Genomic Editing

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2^m/m (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2^m/m mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2^m/m zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells.     

Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene

Background

Animal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing.

Results

The human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human SMN2 gene (hSMN2), which produces only a low amount of full-length SMN, and crossed this onto the smn ^-/- background. We show that human SMN2 is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct hSMN2 splicing increases full-length hSMN RNA in this model. When we placed this transgene on the smn mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in smn mutants and increased their survival.

Conclusions

We have generated a transgenic fish carrying the human hSMN2 gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the smn mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.     

Embryonic senescence and laminopathies in a progeroid zebrafish model

Background

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist.

Principal Findings

We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape.

Conclusion

We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening

Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS.KEY WORDS: MND, ALS, SOD1, Zebrafish     

Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.     

Zebrafish Models of p53 Functions

Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease. The growing number of genetic tools available in zebrafish research has resulted in the identification of many genes involved in developmental and disease processes. In particular, studies in the zebrafish have clarified roles of the p53 tumor suppressor in the formation of specific tumor types, as well as roles of p53 family members during embryonic development. The zebrafish has also been instrumental in identifying novel mechanisms of p53 regulation and highlighting the importance of these mechanisms in vivo. This article will summarize how zebrafish models have been used to reveal numerous, important aspects of p53 function.The zebrafish, Danio rerio, is a small model organism that has long been used to study vertebrate development. Zebrafish embryos are optically clear and develop externally to the mother, facilitating the study of early developmental processes. In addition, zebrafish have increasingly been used in modeling human diseases, including a number of cancers. The availability of forward and reverse genetic tools in the zebrafish has resulted in the identification and characterization of many genes involved in development and disease. One gene that has been extensively studied is the p53 tumor suppressor gene, which is structurally and functionally conserved in the zebrafish. This article will discuss how studies in the zebrafish have increased our understanding of how p53 contributes to the formation of specific tumor types, resulted in the identification of novel mechanisms of p53 regulation, and showed how p53 and p53 family members are involved in embryonic development.     

Identification and expression analysis of kcnh2 genes in the zebrafish

Long QT syndrome is a disorder that is characterised by a prolonged QT-interval and can lead to fatal cardiac arrhythmias. Many animal models have been created to study congenital long QT syndrome. Of these, zebrafish models have involved targeting two different KCNH2 gene (long QT syndrome 2) orthologues, termed zerg-2 and zerg-3, with differing cardiac phenotypes. In order to clarify this situation, this study uses a bioinformatic approach to search the current zebrafish genome sequence (Zv7 and Zv8 builds) to investigate and locate all likely zebrafish orthologues of the human KCNH2 gene. Quantitative real-time RT-PCR was also used to determine the temporal and spatial gene expression profile of the zebrafish orthologues. The data support the conclusion that zerg-2 and zerg-3 are apparent orthologues of different human genes encoding potassium ion channels, but that their functions have switched compared to the respective human proteins.     

Zebrafish Tg(hb9:MTS-Kaede): a new in vivo tool for studying the axonal movement of mitochondria

ObjectivesDeregulation of axonal transport in neurons is emerging as the major cause of many neurodegenerative diseases in human, such as Charcot-Marie-Tooth (CMT) neuropathy. However, little is known about how mitochondria move in vivo and whether cell culture systems truly represent what happens in living animals. Here we describe the generation of a new zebrafish transgenic line that specifically allows to study mitochondrial dynamics in motor neurons and its application to analyse mitochondrial movement in zebrafish models expressing CMT2A causing mutations.MethodsThe Tol2 transposon system was used to generate a transgenic zebrafish line expressing the photoconvertible fluorescent protein Kaede in mitochondria of motor neurons. Mitochondrial shape and movement were monitored by time-lapse confocal live imaging and measured by kymograph analysis. The effects of two well-known CMT causing mutations, L76P and R94Q substitutions in MFN2, were then investigated with the same methods.ResultsWe generated the transgenic zebrafish Tg(hb9:MTS-Kaede) line with genetically labelled mitochondria in motor neurons. Kaede protein was correctly and stably targeted to mitochondrial matrix while retaining its photoconvertibility, thus qualifying this model for in vivo studies. Expression of the L76P and R94Q mutations reduced mitochondrial movement in axons and altered mitochondrial distribution in distinct ways.Conclusions and general significanceThese findings confirm previously published data obtained in cell cultures and strengthen the hypothesis of different mechanism of action of the two MFN2 mutations. Considering the number of neurodegenerative diseases associated to mitochondrial dynamics, the Tg(hb9:MTS-Kaede) zebrafish line is a promising model to study in vivo alterations of mitochondrial transport underlying human diseases.     

Systematic transcriptome analysis of the zebrafish model of diamond-blackfan anemia induced by RPS24 deficiency

Background

Diamond–Blackfan anemia (DBA) is a class of human diseases linked to defective ribosome biogenesis that results in clinical phenotypes. Genetic mutations in ribosome protein (RP) genes lead to DBA phenotypes, including hematopoietic defects and physical deformities. However, little is known about the global regulatory network as well as key miRNAs and gene pathways in the zebrafish model of DBA.

Results

In this study, we establish the DBA model in zebrafish using an RPS24 morpholino and found that RPS24 is required for both primitive hematopoiesis and definitive hematopoiesis processes that are partially mediated by the p53 pathway. Several deregulated genes and miRNAs were found to be related to hematopoiesis, vascular development and apoptosis in RPS24-deficient zebrafish via RNA-seq and miRNA-seq data analysis, and a comprehensive regulatory network was first constructed to identify the mechanisms of key miRNAs and gene pathways in the model. Interestingly, we found that the central node genes in the network were almost all targeted by significantly deregulated miRNAs. Furthermore, the enforced expression of miR-142-3p, a uniquely expressed miRNA, causes a significant decrease in primitive erythrocyte progenitor cells and HSCs.

Conclusions

The present analyses demonstrate that the comprehensive regulatory network we constructed is useful for the functional prediction of new and important miRNAs in DBA and will provide insights into the pathogenesis of mutant rps24-mediated human DBA disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-759) contains supplementary material, which is available to authorized users.     

Impact of a Combined High Cholesterol Diet and High Glucose Environment on Vasculature

Aims

Vascular complications are the leading cause of mortality and morbidity in patients with diabetes. However, proper animal models of diabetic vasculopathy that recapitulate the accelerated progression of vascular lesions in human are unavailable. In the present study, we developed a zebrafish model of diabetic vascular complications and the methodology for quantifying vascular lesion formation real-time in the living diabetic zebrafish.

Methods and Results

Wild type zebrafish (AB) and transgenic zebrafish lines of fli1:EGFP, lyz:EGFP, gata1:dsRed, double transgenic zebrafish of gata1:dsRed/fli1:EGFP were exposed to high cholesterol diet and 3% glucose (HCD-HG) for 10 days. The zebrafish model with HCD-HG treatment was characterized by significantly increased tissue levels of insulin, glucagon, glucose, total triglyceride and cholesterol. Confocal microscopic analysis further revealed that the diabetic larvae developed clearly thickened endothelial layers, distinct perivascular lipid depositions, substantial accumulations of inflammatory cells in the injured vasculature, and a decreased velocity of blood flow. Moreover, the vascular abnormalities were improved by the treatment of pioglitazone and metformin.

Conclusion

A combination of high cholesterol diet and high glucose exposure induces a rapid onset of vascular complications in zebrafish similar to the early atherosclerotic vascular injuries in mammalian diabetic models, suggesting that zebrafish may be used as a novel animal model for diabetic vasculopathy.     

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.Podocyte dysfunction, injury or loss is a common and decisive cause of various glomerular diseases and understanding the molecular mechanism underlying podocyte response to stress will be very helpful to undermine the pathogenesis of podocyte injury and the targeted therapy for glomerular diseases.The members of Gadd45 gene family, Gadd45a, Gadd45b and Gadd45r have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA damage repair, cell survival, senescence and apoptosis.¹ Recently, this gene family has been found differentially expressed in several podocyte injury models. Zhang et al.² observed an induction of GADD45β mRNA expression by lipopolysaccharide in the lung, kidney and spleen, which had the highest GADD45β mRNA expression among all of the tissues examined. Jeffrey W Pippin reported that protein expression of GADD45 was increased in glomeruli from passive Heymann nephritis rats and cultured podocytes exposed in vitro to C5b-9. ³ More recently, Shi et al.⁴ reported that Gadd45b was upregulated in glomeruli of mice with podocyte-specific deletion of Dicer, suggesting the involvement of Gadd45b in podocyte injury. However, no functional characterization of Gadd45 genes in podocytes has been conducted to date and the role of GADD45B in the context of podocyte injury remains unclear.Zebrafish has emerged as a new vertebrate model system for renal glomerular research. The podocytes and renal glomeruli in zebrafish kidney are structurally, molecularly and functionally conserved, rendering zebrafish a valuable and relevant model for podocyte studies. To characterize the role of GADD45b in podocyte injury, we therefore employed zebrafish as an in vivo model system and human podocytes as an in vitro model. We observed the upregulation of GADD45B on podocyte injury in zebrafish renal glomeruli as well as in cultured human podocytes treated with TGF-β and PAN. We further showed that podocyte-specific overexpression of zebrafish orthologs of gadd45b predisposed podocytes to injury, whereas inhibition of gadd45b expression in zebrafish larvae ameliorated podocyte injury and reduced proteinuria. Furthermore, we found that the ROS-GADD45B-p38 pathway was involved in the regulation of GADD45B expression and deleterious role in podocyte injury. Collectively, we have identified GADD45B as an important player in podocyte injury.     

The zebrafish as a tool to identify novel therapies for human cardiovascular disease

Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as silent heart and pickwick^m171, which mimic titin mutations observed in human dilated cardiomyopathy. Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease.KEY WORDS: Cardiovascular, Drug discovery, Zebrafish     

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9 ^{sa1022/sa1022} mutants have a defect in 28S rRNA processing. The nol9 ^{sa1022/sa1022} larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9 ^{sa1022/sa1022} larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9 ^{sa1022/sa1022} embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9 ^{sa1022/sa1022} mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.