Activation of Nrf2 by sulfuretin stimulates chondrocyte differentiation and increases bone lengths in zebrafish.

Elongation of most bones occur at the growth plate through endochondral ossification in postnatal mammals. The maturation of chondrocyte is a crucial factor in longitudinal bone growth, which is regulated by a complex network of paracrine and endocrine signaling pathways. Here, we show that a phytochemical sulfuretin can stimulate hypertrophic chondrocyte differentiation in vitro and in vivo. We found that sulfuretin stabilized nuclear factor (erythroid-derived 2)-like 2 (Nrf2), stimulated its transcriptional activity, and induced expression of its target genes. Sulfuretin treatment resulted in an increase in body length of zebrafish larvae and induced the expression of chondrocyte markers. Consistently, a clinically available Nrf2 activator, dimethyl fumarate (DMF), induced the expression of hypertrophic chondrocyte markers and increased the body length of zebrafish. Importantly, we found that chondrocyte gene expression in cell culture and skeletal growth in zebrafish stimulated by sulfuretin were significantly abrogated by Nrf2 depletion, suggesting that such stimulatory effects of sulfuretin were dependent on Nrf2, at least in part. Taken together, these data show that sulfuretin has a potential use as supporting ingredients for enhancing bone growth. [BMB Reports 2023; 56(9): 496-501].

Systematic expression profiling of neuropathy-related aminoacyl-tRNA synthetases in zebrafish during development.

Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.

Non-Rodent Genetic Animal Models for Studying Tauopathy: Review of Drosophila, Zebrafish, and C. elegans Models.

Tauopathy refers to a group of progressive neurodegenerative diseases, including frontotemporal lobar degeneration and Alzheimer's disease, which correlate with the malfunction of microtubule-associated protein Tau (MAPT) due to abnormal hyperphosphorylation, leading to the formation of intracellular aggregates in the brain. Despite extensive efforts to understand tauopathy and develop an efficient therapy, our knowledge is still far from complete. To find a solution for this group of devastating diseases, several animal models that mimic diverse disease phenotypes of tauopathy have been developed. Rodents are the dominating tauopathy models because of their similarity to humans and established disease lines, as well as experimental approaches. However, powerful genetic animal models using Drosophila, zebrafish, and C. elegans have also been developed for modeling tauopathy and have contributed to understanding the pathophysiology of tauopathy. The success of these models stems from the short lifespans, versatile genetic tools, real-time in-vivo imaging, low maintenance costs, and the capability for high-throughput screening. In this review, we summarize the main findings on mechanisms of tauopathy and discuss the current tauopathy models of these non-rodent genetic animals, highlighting their key advantages and limitations in tauopathy research.

Modeling Neurodevelopmental Disorders and Epilepsy Caused by Loss of Function of kif2a in Zebrafish.

In recent years there has been extensive research on malformations of cortical development (MCDs) that result in clinical features like developmental delay, intellectual disability, and drug-resistant epilepsy (DRE). Various studies highlighted the contribution of microtubule-associated genes (including tubulin and kinesin encoding genes) in MCD development. It has been reported that de novo mutations in KIF2A, a member of the kinesin-13 family, are linked to brain malformations and DRE. Although it is known that KIF2A functions by regulating microtubule depolymerization via an ATP-driven process, in vivo implications of KIF2A loss of function remain partly unclear. Here, we present a novel kif2a knock-out zebrafish model, showing hypoactivity, habituation deficits, pentylenetetrazole-induced seizure susceptibility and microcephaly, as well as neuronal cell proliferation defects and increased apoptosis. Interestingly, kif2a-/- larvae survived until adulthood and were fertile. Notably, our kif2a zebrafish knock-out model demonstrated many phenotypic similarities to KIF2A mouse models. This study provides valuable insights into the functional importance of kif2a in zebrafish and phenotypical hallmarks related to KIF2A mutations. Ultimately, this model could be used in a future search for more effective therapies that alleviate the clinical symptoms typically associated with MCDs.

A threonyl-tRNA synthetase-mediated translation initiation machinery.

A fundamental question in biology is how vertebrates evolved and differ from invertebrates, and little is known about differences in the regulation of translation in the two systems. Herein, we identify a threonyl-tRNA synthetase (TRS)-mediated translation initiation machinery that specifically interacts with eIF4E homologous protein, and forms machinery that is structurally analogous to the eIF4F-mediated translation initiation machinery via the recruitment of other translation initiation components. Biochemical and RNA immunoprecipitation analyses coupled to sequencing suggest that this machinery emerged as a gain-of-function event in the vertebrate lineage, and it positively regulates the translation of mRNAs required for vertebrate development. Collectively, our findings demonstrate that TRS evolved to regulate vertebrate translation initiation via its dual role as a scaffold for the assembly of initiation components and as a selector of target mRNAs. This work highlights the functional significance of aminoacyl-tRNA synthetases in the emergence and control of higher order organisms.

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic 'naphthalene' moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

Genome-wide identification and functional analysis of genes expressed ubiquitously in rice.

Genes that are expressed ubiquitously throughout all developmental stages are thought to be necessary for basic biological or cellular functions. Therefore, determining their biological roles is a great challenge. We identified 4034 of these genes in rice after studying the results of Agilent 44K and Affymetrix meta-anatomical expression profiles. Among 105 genes that were characterized by loss-of-function analysis, 79 were classified as members of gene families, the majority of which were predominantly expressed. Using T-DNA insertional mutants, we examined 43 genes and found that loss of expression of six genes caused developing seed- or seedling-defective phenotypes. Of these, three are singletons without similar family members and defective phenotypes are expected from mutations. Phylogenomic analyses integrating genome-wide transcriptome data revealed the functional dominance of three ubiquitously expressed family genes. Among them, we investigated the function of Os03g19890, which is involved in ATP generation within the mitochondria during endosperm development. We also created and evaluated functional networks associated with this gene to understand the molecular mechanism. Our study provides a useful strategy for pheonome analysis of ubiquitously expressed genes in rice.

Genome-wide Identification and Functional Analysis of Genes Expressed Ubiquitously in Rice.

Genes that are expressed ubiquitously throughout all developmental stages are thought to be necessary for basic biological or cellular functions. Therefore, determining their biological roles is a great challenge. We identified 4,034 of these genes in rice after studying the results of Agilent 44K and Affymetrix meta-anatomical expression profiles. Among the 105 that were characterized by loss-of-function analysis, 79 were classified as members of gene families, with the majority of them being predominantly expressed. Using T-DNA insertional mutants, we examined 43 genes and found that loss-of-expression in six caused developing seed- or seedling-defective phenotypes. Of them, three are singletons without same family members and easily expect the defective phenotypes by the mutations. Phylogenomic analyses integrating genome-wide transcriptome data revealed the functional dominance of three ubiquitously expressed family genes. From them, we investigated the role of Os03g19890, which is involved in ATP generation within the mitochondria during endosperm development. We also created and evaluated functional networks associated with that gene to understand the molecular mechanism. Our study will provide useful strategy for pheonome analysis of ubiquitously expressed genes.

Genome-wide identification and analysis of Japonica and Indica cultivar-preferred transcripts in rice using 983 Affymetrix array data.

BACKGROUND: Accumulation of genome-wide transcriptome data provides new insight on a genomic scale which cannot be gained by analyses of individual data. The majority of rice (O. sativa) species are japonica and indica cultivars. Genome-wide identification of genes differentially expressed between japonica and indica cultivars will be very useful in understanding the domestication and evolution of rice species. RESULTS: In this study, we analyzed 983 of the 1866 entries in the Affymetrix array data in the public database: 595 generated from indica and 388 from japonica rice cultivars. To discover differentially expressed genes in each cultivar, we performed significance analysis of microarrays for normalized data, and identified 490 genes preferentially expressed in japonica and 104 genes in indica. Gene Ontology analyses revealed that defense response-related genes are significantly enriched in both cultivars, indicating that japonica and indica might be under strong selection pressure for these traits during domestication. In addition, 36 (34.6%) of 104 genes preferentially expressed in indica and 256 (52.2%) of 490 genes preferentially expressed in japonica were annotated as genes of unknown function. Biotic stress overview in the MapMan toolkit revealed key elements of the signaling pathway for defense response in japonica or indica eQTLs. CONCLUSIONS: The percentage of screened genes preferentially expressed in indica was 4-fold higher (34.6%) and that in japonica was 5-fold (52.2%) higher than expected (11.1%), suggesting that genes of unknown function are responsible for the novel traits that distinguish japonica and indica cultivars. The identification of 10 functionally characterized genes expressed preferentially in either japonica or indica highlights the significance of our candidate genes during the domestication of rice species. Functional analysis of the roles of individual components of stress-mediated signaling pathways will shed light on potential molecular mechanisms to improve disease resistance in rice.