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The 5' UTRs of mRNAs are critical for translation regulation, but their in vivo regulatory features are poorly characterized. Here, we report the regulatory landscape of 5' UTRs during early zebrafish embryogenesis using a massively parallel reporter assay of 18,154 sequences coupled to polysome profiling. We found that the 5' UTR is sufficient to confer temporal dynamics to translation initiation, and identified 86 motifs enriched in 5' UTRs with distinct ribosome recruitment capabilities. A quantitative deep learning model, DaniO5P, revealed a combined role for 5' UTR length, translation initiation site context, upstream AUGs and sequence motifs on in vivo ribosome recruitment. DaniO5P predicts the activities of 5' UTR isoforms and indicates that modulating 5' UTR length and motif grammar contributes to translation initiation dynamics. This study provides a first quantitative model of 5' UTR-based translation regulation in early vertebrate development and lays the foundation for identifying the underlying molecular effectors.

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The link between oxidative stress and environmental factors plays an important role in chronic degenerative diseases; therefore, exogenous antioxidants could be an effective alternative to combat disease progression and/or most significant symptoms. Curcuma longa L. (CL), commonly known as turmeric, is mostly composed of curcumin, a multivalent molecule described as having antioxidant, anti-inflammatory and neuroprotective properties. Poor chemical stability and low oral bioavailability and, consequently, poor absorption, rapid metabolism, and limited tissue distribution are major restrictions to its applicability. The advent of nanotechnology, by combining nanosacale with multi-functionality and bioavailability improvement, offers an opportunity to overcome these limitations. Therefore, in this work, poly-Ɛ-caprolactone (PCL) nanoparticles were developed to incorporate the methanolic extract of CL, and their bioactivity was assessed in comparison to free or encapsulated curcumin. Their toxicity was evaluated using zebrafish embryos by applying the Fish Embryo Acute Toxicity test, following recommended OECD guidelines. The protective effect against paraquat-induced oxidative damage of CL extract, free or encapsulated in PCL nanoparticles, was evaluated. This herbicide is known to cause oxidative damage and greatly affect neuromotor functions. The overall results indicate that CL-loaded PCL nanoparticles have an interesting protective capacity against paraquat-induced damage, particularly in neuromuscular development that goes well beyond that of CL extract itself and other known antioxidants.

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The stability of RNA transcripts is regulated by signals within their sequences, but the identity of those signals still remain elusive in many biological systems. Recently introduced massively parallel tools for the analysis of regulatory RNA sequences provide the ability to detect functional cis-regulatory sequences of post-transcriptional RNA regulation at a much larger scale and resolution than before. Their application formulates the underlying sequence-based rules and predicts the impact of genetic variations. Here, we describe the application of UTR-Seq, as a strategy to uncover cis-regulatory signals of RNA stability during early zebrafish embryogenesis. The method combines massively parallel reporter assays (MPRA) with computational regression models. It surveys the effect of tens of thousands of regulatory sequences on RNA stability and analyzes the results via regression models to identify sequence signals that impact RNA stability and to predict the in vivo effect of sequence variations.

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High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction of tfam using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 ± 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring.

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Lipids exert substantial influences on vertebrate embryogenesis, but their metabolic dynamics at detailed structural levels remains elusive, primarily owing to the lack of a tool capable of resolving their huge structural diversity. Herein, we present the first large-scale and spatiotemporal monitoring of unsaturated lipids with C=C specificity in single developing zebrafish embryos enabled by photochemical derivatization and tandem mass spectrometry (MS). The lipid isomer composition was found extremely stable in yolk throughout embryogenesis, while notable differences in ratios of C=C location (e.g., PC 16:0_16:1 (7) vs. 16:0_16:1 (9)) and fatty acyl composition isomers (e.g., PC 16:1_18:1 vs. 16:0_18:2) were unveiled between blastomeres and yolk from zygote to 4 h post fertilization (hpf). From 24 hpf onwards, lipid isomer compositions in embryo head and tail evolved distinctively with development, suggesting a meticulously regulated lipid remodeling essential for cell division and differentiation. This work has laid the foundation for functional studies of structurally defined lipids in vertebrate embryology.

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All living systems function out of equilibrium and exchange energy in the form of heat with their environment. Thus, heat flow can inform on the energetic costs of cellular processes, which are largely unknown. Here, we have repurposed an isothermal calorimeter to measure heat flow between developing zebrafish embryos and the surrounding medium. Heat flow increased over time with cell number. Unexpectedly, a prominent oscillatory component of the heat flow, with periods matching the synchronous early reductive cleavage divisions, persisted even when DNA synthesis and mitosis were blocked by inhibitors. Instead, the heat flow oscillations were driven by the phosphorylation and dephosphorylation reactions catalyzed by the cell-cycle oscillator, the biochemical network controlling mitotic entry and exit. We propose that the high energetic cost of cell-cycle signaling reflects the significant thermodynamic burden of imposing accurate and robust timing on cell proliferation during development.

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The molecular genetic basis of teleost phenotypic diversity remains poorly understood, despite the increasing availability of genome and transcriptome data. Changes in gene expression, resulting from regulatory mutations, contribute to many or even most phenotypic innovations. In this study, we performed comparative genomic analyses to identify 44 Conserved Teleost-Specific MicroRNAs (CTSMs). An analysis of the sequences of CTSMs demonstrated that their sequence variability is significantly higher than that of Evolutionarily Conserved microRNAs (ECs - microRNAs conserved throughout vertebrates), and that this difference primarily results from variation in the loop regions. We computationally predicted target genes for CTSMs and found that the targets of CTSMs show significantly lower SNP density compared with targets of ECs as well as non-targets of CTSMs. The temporal expression profile of targets of CTSMs obtained from RNA-seq data revealed that the targets of CTSMs are specifically enriched in early-stage embryos and exhibit a broader expression spectrum. Finally, functional enrichment analysis (GO and KEGG) indicated that the targets of CTSMs play diverse functional roles, with a significantly enriched category being the "immune response". Our study provides the first systematic identification and characterization of conserved teleost-specific microRNAs and their targets and presents valuable foundation for a better understanding the genomic basis for the diversification of teleosts.

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