Normal Zebrafish Development Research Articles

Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

The Zebrafish (Danio rerio) Embryo Model as a Tool to Assess Drinking Water Treatment Efficacy for Freshwater Impacted by Crude Oil Spill.

Traditional approaches toward evaluating oil spill mitigation effectiveness in drinking water supplies using analytical chemistry can overlook residual hydrocarbons and treatment byproducts of unknown toxicity. Zebrafish (Danio rerio) were used to address this limitation by evaluating the reduction in toxicity to fish exposed to laboratory solutions of dissolved crude oil constituents treated with 3 mg/L ozone (O3 ) with or without a peroxone-based advanced oxidation process using 0.5 M H2 O2 /M O3 or 1 M H2 O2 /M O3 . Crude oil water mixtures (OWMs) were generated using three mixing protocols-orbital (OWM-Orb), rapid (OWM-Rap), and impeller (OWM-Imp) and contained dissolved total aromatic concentrations of 106-1019 µg/L. In a first experiment, embryos were exposed at 24 h post fertilization (hpf) to OWM-Orb or OWM-Rap diluted to 25%-50% of full-strength samples and in a second experiment, to untreated or treated OWM-Imp mixtures at 50% dilutions. Toxicity profiles included body length, pericardial area, and swim bladder inflation, and these varied depending on the OWM preparation, with OWM-Rap resulting in the most toxicity, followed by OWM-Imp and then OWM-Orb. Zebrafish exposed to a 50% dilution of OWM-Imp resulted in 6% shorter body length, 83% increased pericardial area, and no swim bladder inflation, but exposure to a 50% dilution of OWM-Imp treated with O3 alone or with 0.5 M H2 O2 /M O3 resulted in normal zebrafish development and average total aromatic destruction of 54%-57%. Additional aromatic removal occurred with O3 + 1 M H2 O2 /M O3 but without further attenuation of toxicity to zebrafish. This study demonstrates using zebrafish as an additional evaluation component for modeling the effectiveness of freshwater oil spill treatment methods. Environ Toxicol Chem 2022;41:2822-2834. © 2022 SETAC.

Lipid-related metabolism during zebrafish embryogenesis under unbalanced copper homeostasis.

Copper (Cu) is an essential trace element, playing an important role in lipid metabolism, and its transporters ATP7A and ATP7B, as Cu-transporting P-type ATPases, are involved in maintaining the Cu homeostasis in cells. Numerous studies in mammals have shown that Cu homeostasis and lipid metabolism are closely related, but studies on the link between the effects of excess Cu, ATP7A, and ATP7B on lipid metabolism during vertebrate embryogenesis are scarce. In this study, zebrafish disease models with Cu overload and ATP7A and ATP7B inactivation, respectively, were used to study the lipid metabolism-related differentially expressed genes (DEGs) which were enriched in the models. The dynamic and spatiotemporal expressions of the DEGs in WTs, atp7a-/-, and atp7b-/- mutants with or without Cu stress were unveiled in this study and they mostly distributed in brain at 24 hpf then in liver and intestine at 96 hpf, suggesting their potential roles in lipid and glycogen metabolism to apply energy for normal development in zebrafish. Meanwhile, the correlation analysis for the DEGs among the three groups unveiled that most of the DEGs were involved in the glyceride metabolism pathway. This is the first report to establish the relationship between atp7a and atp7b with Cu-stimulated intestinal and liver lipid metabolism during fish embryogenesis, and this study will provide a theoretical basis for fish embryonic development and lipid metabolism disorders under unbalanced copper homeostasis.

Patterns and Crucial Regulation of Alternative Splicing During Early Development in Zebrafish

Many vertebrate genes generate multiple transcript variants that may encode functionally distinct protein isoforms, but the transcriptomes of various developmental stages are poorly defined. Identifying the transcriptome and its regulation during the normal developmental process is the key to deciphering the developmental stage-specific functions of genes. Here we presented a systematic assessment of the temporal alternative splicing (AS) events during the critical development stages to capture the dynamic gene expression changes and AS in zebrafish. An unexpected transcriptome complexity generated by AS was observed during zebrafish development. The patterns of AS events varied substantially among developmental stages despite the similarities in the total proportion of AS genes. We further found that AS afforded substantial functional diversification of genes through the generation of stage-specific AS events from broadly protein-coding genes as an essential developmental regulatory mechanism. Skipped exon (SE) showed the strongest signals among developmental AS (devAS), suggesting that devAS events generated by SE may be necessary for the normal development of zebrafish. Most developmental genes regulated by AS mechanisms were not modulated in terms of their overall expression levels, indicating that AS shaped the transcriptome independently from transcriptional regulation during development. 128-cell stage was a critical stage for gene transcription during embryonic development. Splicing factors as an essential developmental regulator underwent AS in the potential autoregulatory feedback loop and expressed multiple isoforms. Thus, zebrafish development was shaped by an interplay of programs controlling gene expression levels and AS. Overall, we provided a global view of developmental patterns of AS during zebrafish development and revealed that AS transitions were the crucial regulatory component of zebrafish embryonic development.

Maintenance and Breeding of Zebrafish under Laboratory Conditions for Animal Research

Background: Zebrafish is widely used in biomedical research to explore several diseases and abnormalities. A thorough understanding of husbandry is required to maintain effectively, breed and produce healthy and diversified colonies. Diet, age, size, light exposure, mating behaviour, tank temperature and time used for egg formation are all factors that influence the breeding process. Methods: The fishes were kept in the zebrafish housing system with frequent water quality checks and a breeding duration of 7 days. Developing eggs and larvae were photographed and further processed with image view software. The normal development of zebrafish goes through seven stages: zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching stage. Result: The average number of eggs deposited per female in the experiment was 341±4.21, with 318±4.81 viable eggs. 298±4.99 eggs hatched after 72 hours and 270±4.34 fingerlings survival was recorded after a week. The present paper discusses the care of zebrafish, setting up and maintenance in laboratory conditions. Once the system is standardized, it is easy to access healthy eggs, larvae and adults throughout the year for research and teaching purposes.

Effect of Water-Pipe Smoking on the Normal Development of Zebrafish.

Background: Among all types of tobacco consumption, Water-Pipe Smoking (WPS) is the most widely used in the Middle East and second-most in several other countries. The effect of WPS on normal development is not yet fully understood, thus the aim of this study is to explore the acute toxicity effects of WPS extract on zebrafish larvae. Methods: In this study, we compared the effects of WPS smoke condensates at concentrations varying from 50 to 200 µg/mL on developmental, cardiac, and behavioural (neurotoxicity) functions. Gene expression patterns of cardiac biomarkers were also evaluated by RT-qPCR. Results: Exposing zebrafish embryos to 50, 100, 150 and 200 µg/mL WPS for three days did not affect the normal morphology of Zebrafish embryos, as the tail flicking, behavioural and locomotion assays did not show any change. However, WPS deregulated cardiac markers including atrial natriuretic peptide (ANP/NPPA) and brain natriuretic peptide (BNP/NPPB). Furthermore, it induced apoptosis in a dose-dependent manner. Conclusion: Our data demonstrate that WPS can significantly affect specific cardiac parameters during the normal development of zebrafish. Further investigations are necessary to elucidate the pathogenic outcome of WPS on different aspects of human life, including pregnancy.

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3'UTR intron-containing NMD targets.

Many post-transcriptional mechanisms operate via mRNA 3′UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3′UTR introns (3′UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3′UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3′UI in zebrafish, mouse and humans. foxo3b is one such proximal 3′UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3′UIs may serve gene regulatory function in vertebrates.

Molecular insights and cell cycle assessment upon exposure to Chaga (Inonotus obliquus) mushroom polysaccharides in zebrafish (Danio rerio)

Chaga (Inonotus obliquus) mushroom is considered as one of the most powerful antioxidants across the world. Though the therapeutic effects of Chaga components are well characterized in vitro, the in vivo developmental effects are not elucidated in detail. In this study, we assessed the in vivo developmental effects of Chaga polysaccharides in zebrafish, along with revealing the effects on cell cycle and apoptosis. Chaga mushroom polysaccharides comprised xylulose, rhamnose, mannose, glucose, inositol, and galactose, in addition to phenolic compounds; zebrafish embryos exhibited normal embryonic development upon transient exposure to Chaga extract (24 hours). Most embryos (>90%) were found to be healthy even at high concentrations (5 mg/mL). In addition, staining with the DNA binding dye, acridine orange showed that Chaga polysaccharides alleviated oxidative stress. Flow cytometric analysis using H2DCFDA that specifically binds to cells with fragmented DNA showed significantly reduced levels of intracellular reactive oxygen species (ROS) (p < 0.05), which in turn reduced apoptosis in the developing embryos. Cell cycle analysis by measuring the DNA content using flow cytometry revealed that Chaga polysaccharides moderately arrested the cells at G1 stage, thereby inhibiting cell proliferation that can be further explored in cancer studies. Overall, transient exposure of Chaga polysaccharide extract reduced intracellular ROS and assisted in the normal development of zebrafish.

Two paralogous znf143 genes in zebrafish encode transcriptional activator proteins with similar functions but expressed at different levels during early development

BackgroundZNF143 is an important transcriptional regulator protein conserved in metazoans and estimated to bind over 2000 promoter regions of both messenger RNA and small nuclear RNA genes. The use of zebrafish is a useful model system to study vertebrate gene expression and development. Here we characterize znf143a, a novel paralog of znf143b, previously known simply as znf143 in zebrafish. This study reveals a comparison of quantitative and spatial expression patterns, transcriptional activity, and a knockdown analysis of both ZNF143 proteins.ResultsZNF143a and ZNF143b have a fairly strong conservation with 65% amino acid sequence identity, and both are potent activators in transient transfection experiments. In situ hybridization analyses of both znf143 mRNAs show that these genes are expressed strongly in regions of the brain at 24 h post fertilization in zebrafish development. A transient knockdown analysis of znf143 expression from either gene using CRISPR interference revealed similar morphological defects in brain development, and caused brain abnormalities in up to 50% of injected embryos. Although present in the same tissues, znf143a is expressed at a higher level in early development which might confer an evolutionary benefit for the maintenance of two paralogs in zebrafish.Conclusionsznf143a encodes a strong activator protein with high expression in neural tissues during early embryogenesis in zebrafish. Similar to its paralogous gene, znf143b, both znf143 genes are required for normal development in zebrafish.

Synergistic toxicity of microcystin-LR and Cu to zebrafish (Danio rerio)

Toxic cyanobacterial blooms often coincide with metal pollution in a freshwater environment because of surface run-off enriched with nutrients and metals. However, the joint toxic effects of cyanobacterial toxins and metals on aquatic animals remain unknown. In this study, single and joint toxic effects and mechanisms of microcystin-LR (MCLR) and copper (Cu) were investigated in the early development of zebrafish (Danio rerio). The LC5072-h values were 2.79 mg/L for MCLR and 3.23 mg/L for Cu. The sublethal concentrations of MCLR (≤600 μg/L) did not affect the normal development of zebrafish but increased its hatchability. Strong synergistic toxic effects were observed after co-exposure to MCLR and Cu at environmental concentrations (≤60 μg/L). The synergistic toxic effects of these two compounds could be attributed to the increased bioaccumulation of MCLR and Cu, which was mediated by MCLR transporters (e.g., oatp1d1 and oatp2b1) and Cu transporters (e.g., ctr1 and atp7a), in zebrafish. Such bioaccumulation caused oxidative stress, as suggested by the disrupted gene expression of anti-oxidative enzymes (e.g., Cu/Zn-SOD, Mn-SOD, and CAT). Our results revealed for the first time the synergistic toxic effects and potential toxic mechanism of MCLR–Cu in aquatic animals. These synergistic effects should be considered when assessing the ecological risk of toxic cyanobacterial blooms.

Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome.

Two Dynamin-2 Genes Are Required for Normal Zebrafish Development

Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.

The transcriptional activator ZNF143 is essential for normal development in zebrafish

BackgroundZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA.ResultsThe loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein.ConclusionsNormal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.

Two Myristoylated Alanine‐Rich C‐Kinase Substrate (MARCKS) Paralogs are Required for Normal Development in Zebrafish

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC) and critical for mouse and Xenopus development. Herein two MARCKS paralogs, marcksa and marcksb, are identified in zebrafish and the role of these genes in zebrafish development is evaluated. Morpholino-based targeting of either MARCKS protein resulted in increased mortality and a range of gross phenotypic abnormalities. Phenotypic abnormalities were classified as mild, moderate or severe, which is characterized by a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail, respectively. All three phenotypes displayed abnormal neural architecture. Histopathology of Marcks targeted embryos revealed abnormalities in retinal layering, gill formation and skeletal muscle morphology. These results demonstrate that Marcksa and Marcksb are required for normal zebrafish development and suggest that zebrafish are a suitable model to further study MARCKS function.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal)—Experimental assessment using a zebrafish embryo test

Fish embryos are a particularly vulnerable stage of development, so they represent optimal targets for screening toxicological effects of waterborne xenobiotics. Herein, the toxicity potential of two mixtures of pharmaceuticals was evaluated using a zebrafish embryo test. One of the mixtures corresponds to an environmentally realistic scenario and both have carbamazepine, fenofibric acid, propranolol, trimethoprim and sulfamethoxazole. The results evidenced morphological alterations, such as spinal deformities and yolk-sac oedemas. Moreover, heart rates decreased after both mixture exposures, e.g., at 48 hpf, highest mixture versus blank control (47.8 ± 4.9 and 55.8 ± 3.7 beats/30 s, respectively). The tail lengths also diminished significantly from 3208 ± 145 μm in blank control to 3130 ± 126 μm in highest mixture. The toxicological effects were concentration dependent. Mortality, hatching rate and the number of spontaneous movements were not affected. However, the low levels of pharmaceuticals did interfere with the normal development of zebrafish, which indicates risks for wild organisms.

Nesprin-3 augments peripheral nuclear localization of intermediate filaments in zebrafish

The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3β. Nesprin-3β is only expressed during early development and lacks seven amino acids in its first spectrin repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin β4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin β4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.

The Wnt/Planar Cell Polarity Protein-tyrosine Kinase-7 (PTK7) Is a Highly Efficient Proteolytic Target of Membrane Type-1 Matrix Metalloproteinase: IMPLICATIONS IN CANCER AND EMBRYOGENESIS

PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.

The abcc6a Gene Expression Is Required for Normal Zebrafish Development

Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene, which encodes a putative efflux transporter, ABCC6. The zebrafish (Danio rerio) has two ABCC6-related sequences. To study the function of abcc6 during zebrafish development, the mRNA expression levels were measured using RT-PCR and in situ hybridization. The abcc6a showed a relatively high level of expression at 5 days post-fertilization (d.p.f.) and the expression was specific to the Kupffer's vesicles. The abcc6b expression was evident at 6 hours post-fertilization (h.p.f.) and remained high up to 8 d.p.f., corresponding to embryonic kidney proximal tubules. Morpholinos were designed to both genes to prevent pre-mRNA splicing and block translation. Injection of the abcc6a morpholinos into 1-4 cell zebrafish embryos decreased gene expression by 54-81%, and induced a phenotype, pericardial edema and curled tail associated with death at around 8 d.p.f. Microinjecting zebrafish embryos with full-length mouse Abcc6 mRNA together with the morpholino completely rescued this phenotype. No phenotypic changes were observed when the abcc6b gene morpholino was injected into embryos with knock-down efficiency of 100%. These results suggest that abcc6a is an essential gene for normal zebrafish development and provide insight into the function of ABCC6, the gene mutated in PXE.

Small ubiquitin-related modifier paralogs are indispensable but functionally redundant during early development of zebrafish

The Small ubiquitin-related modifier (SUMO) conjugation to a variety of proteins regulates diverse cellular processes, including transcription, cell cycle regulation and maintenance of genome integrity. To investigate in vivo biological function of SUMO paralogs, we inactivated them in the early development of zebrafish. While zebrafish embryos deficient for all three SUMO paralogs, as Ubc9-deficient ones, displayed severe defects, loss of individual SUMO paralog was compatible with a normal development. SUMO-deficient embryos can be rescued by a single human or zebrafish SUMO. While key structural basic lysine residues and N-terminal unstructured stretch of SUMO are critical for in vivo rescue, the consensus K11 sumoylation site of SUMO2 is dispensable, implying that chain formation on this potential site is unessential for normal development. Inactivation of all three SUMOs triggered p53-dependent apoptosis and further inactivation of p53 restored normal zebrafish development. Interestingly, we also demonstrate that the dominant negative truncated form of p53, Delta113p53, significantly blunts SUMO depletion-induced p53 activity in vivo. Taken together, our results suggest that SUMO paralogs are indispensable, but redundant, in the early development of zebrafish.

Dispatched Homolog 2 is targeted by miR-214 through a combination of three weak microRNA recognition sites

MicroRNAs (miRNAs) regulate gene expression by inhibiting translation of target mRNAs through pairing with miRNA recognition elements (MREs), usually in 3′ UTRs. Because pairing is imperfect, identification of bona fide mRNA targets presents a challenge. Most target recognition algorithms strongly emphasize pairing between nucleotides 2–8 of the miRNA (the ‘seed’ sequence) and the mRNA but adjacent sequences and the local context of the 3′ UTR also affect targeting. Here, we show that dispatched 2 is a target of miR-214. In zebrafish, dispatched 2 is expressed in the telencephalon and ventral hindbrain and is essential for normal zebrafish development. Regulation of dispatched 2 by miR-214 is via pairing with three, noncanonical, weak MREs. By comparing the repression capacity of GFP reporters containing different dispatched 2 sequences, we found that a combination of weak sites, which lack canonical seed pairing, can effectively target an mRNA for silencing. This finding underscores the challenge that prediction algorithms face and emphasizes the need to experimentally validate predicted MREs.