Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Parkin Is Protective against Proteotoxic Stress in a Transgenic Zebrafish Model

Background

Mutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system.

Methodology/Principal Findings

We first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress.

Conclusions/Significance

Similarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo.     

A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique

Background

Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.

Methodology/Principal Findings

We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.

Conclusions/Significance

Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.     

Cross-species toxicogenomic analyses and phenotypic anchoring in response to groundwater low-level pollution

Background

Comparison of toxicogenomic data facilitates the identification of deregulated gene patterns and maximizes health risk prediction in human.

Results

Here, we performed phenotypic anchoring on the effects of acute exposure to low-grade polluted groundwater using mouse and zebrafish. Also, we evaluated two windows of chronic exposure in mouse, starting in utero and at the end of lactation. Bioinformatic analysis of livers microarray data showed that the number of deregulated biofunctions and pathways is higher after acute exposure, compared to the chronic one. It also revealed specific profiles of altered gene expression in all treatments, pointing to stress response/mitochondrial pathways as major players of environmental toxicity. Of note, dysfunction of steroid hormones was also predicted by bioinformatic analysis and verified in both models by traditional approaches, serum estrogens measurement and vitellogenin mRNA determination in mice and zebrafish, respectively.

Conclusions

In our report, phenotypic anchoring in two vertebrate model organisms highlights the toxicity of low-grade pollution, with varying susceptibility based on exposure window. The overlay of zebrafish and mice deregulated pathways, more than single genes, is useful in risk identification from chemicals implicated in the observed effects.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1067) contains supplementary material, which is available to authorized users.     

The mych gene is required for neural crest survival during zebrafish development

Background

Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes.

Principal Findings

We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain.

Significance

Mych is a novel factor required for neural crest cell survival in zebrafish.     

Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish

Background

Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.

Methodology/Principal Findings

We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.

Conclusions/Significance

These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.     

Alpha2 Macroglobulin-Like Is Essential for Liver Development in Zebrafish

Background

Alpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer''s disease, but little is known about a possible role in liver development loss-of-function in model systems.

Principal Findings

We report the isolation of the zebrafish α2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation.

Significance

This report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate.     

Integrated analyses of zebrafish miRNA and mRNA expression profiles identify miR-29b and miR-223 as potential regulators of optic nerve regeneration

Background

Unlike mammals, zebrafish have the ability to regenerate damaged parts of their central nervous system (CNS) and regain functionality of the affected area. A better understanding of the molecular mechanisms involved in zebrafish regeneration may therefore provide insight into how CNS repair might be induced in mammals. Although many studies have described differences in gene expression in zebrafish during CNS regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined.

Results

We used microarrays to analyse and integrate the mRNA and microRNA (miRNA) expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. Bioinformatic analysis identified 3 miRNAs and 657 mRNAs that were differentially expressed after injury. We then combined inverse correlations between our miRNA expression and mRNA expression, and integrated these findings with target predictions from TargetScan Fish to identify putative miRNA-gene target pairs. We focused on two over-expressed miRNAs (miR-29b and miR-223), and functionally validated seven of their predicted gene targets using RT-qPCR and luciferase assays to confirm miRNA-mRNA binding. Gene ontology analysis placed the miRNA-regulated genes (eva1a, layna, nefmb, ina, si:ch211-51a6.2, smoc1, sb:cb252) in key biological processes that included cell survival/apoptosis, ECM-cytoskeleton signaling, and heparan sulfate proteoglycan binding,

Conclusion

Our results suggest a key role for miR-29b and miR-223 in zebrafish regeneration. The identification of miRNA regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for CNS regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1772-1) contains supplementary material, which is available to authorized users.     

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.     

Clustered gene expression changes flank targeted gene loci in knockout mice

Background

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

Methodology/Principle Findings

Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

Conclusions/Significance

The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice     

Highly Conserved Non-Coding Sequences and the 18q Critical Region for Short Stature: A Common Mechanism of Disease?

Background

Isolated growth hormone deficiency (IGHD) and multiple pituitary hormone deficiency (MPHD) are heterogeneous disorders with several different etiologies and they are responsible for most cases of short stature. Mutations in different genes have been identified but still many patients did not present mutations in any of the known genes. Chromosomal rearrangements may also be involved in short stature and, among others, deletions of 18q23 defined a critical region for the disorder. No gene was yet identified.

Methodology/Principal Findings

We now report a balanced translocation X;18 in a patient presenting a breakpoint in 18q23 that was surprisingly mapped about 500 Kb distal from the short stature critical region. It separated from the flanking SALL3 gene a region enriched in highly conserved non-coding elements (HCNE) that appeared to be regulatory sequences, active as enhancers or silencers during embryonic development.

Conclusion

We propose that, during pituitary development, the 18q rearrangement may alter expression of 18q genes or of X chromosome genes that are translocated next to the HCNEs. Alteration of expression of developmentally regulated genes by translocation of HCNEs may represent a common mechanism for disorders associated to isolated chromosomal rearrangements.     

Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development

Background

The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.

Methodology/Principal Findings

We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.

Conclusions/Significance

Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.     

prox1b Activity is essential in zebrafish lymphangiogenesis

Background

The lymphatic vascular system, draining interstitial fluids from most tissues and organs, exerts crucial functions in several physiological and pathological processes. Lymphatic system development depends on Prox1, the first marker to be expressed in the endothelial cells of the cardinal vein from where lymph vessels originate. Prox1 ortholog in the optically clear, easily manipulated zebrafish model has been previously isolated and its contribution to lymphangiogenesis has been clarified. Because of a round of genome duplication occurred at the base of teleosts radiation, several zebrafish genes have been retained in duplicate through evolution. We investigated for the presence of additional prox1 genes and determined their role in zebrafish lymphangiogenesis.

Methodology/Principal Findings

We isolated a second ortholog, named prox1b, and analyzed its expression during development by whole mount in situ hybridization (WISH). We detected strong prox1b expression in the endothelium of the posterior cardinal vein (PCV) from where lymphatic precursors originate. To analyze prox1b involvement in lymphangiogenesis we utilized the fli1:GFP transgenics and followed the formation of the toracic duct (TD), the primary lymph vessel in fish, after prox1b knockdown. Our findings clearly demonstrated that the absence of prox1b activity severely hampers the formation of the TD.

Conclusions/Significance

This work provides substantial progress toward the understanding of zebrafish lymphangiogenesis. In light of the features shared by the lymphatic systems of zebrafish and higher vertebrates, the establishment of such lymphatic model will provide a powerful tool to study, for instance, disorders of body fluid homeostasis, inflammation and cancer metastasis, and may ultimately contribute to novel therapies.