BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Simultaneous Imaging of Multiple miRNAs in Mitochondria Controlled by Fluorescently Encoded Upconversion Optical Switches for Drug Resistance Studies.

Mitochondrial miRNAs (mitomiRs) are essential regulators of biological processes by influencing mitochondrial gene expression and function. To comprehensively understand related pathological processes and treatments, simultaneous imaging of multiple mitomiRs is crucial. In this study, we present a technique that enables simultaneous monitoring of multiple mitomiRs in living cells using a near-infrared (NIR) photoactivated controlled detection probe (PD-mFleU) with a fluorescence-encoded error correction module and a nonsupervised machine learning data-processing algorithm. This method allows controlled sensing imaging of mitomiRs with a DNA reporter probe that can be activated by NIR light after targeted mitochondrial localization. Multilayer upconversion nanoparticles (UCNPs) are used for encoding probes and error correction. Additionally, the density-based spatial clustering of applications with the noise (DBSCAN) algorithm is used to process and analyze the image. Using this technique, we achieved rapid in situ imaging of the abnormal expression of three mitomiRs (miR-149, miR-590, and miR-671) related to mt-ND1 in drug-resistant cells. Furthermore, upregulating the three mitomiRs simultaneously efficiently reverted drug-resistant cells to sensitive cells. Our study provides an analytical strategy for multiplex imaging of mitomiRs in living cells with potential clinical applications.

Dual Functional Full-Color Carbon Dot-Based Organelle Biosensor Array for Visualization of Lipid Droplet Subgroups with Varying Lipid Composition in Living Cells.

In situ visualization of lipid composition diversity in lipid droplets (LDs) is essential for decoding lipid metabolism and function. However, effective probes for simultaneously localizing and reflecting the lipid composition of LDs are currently lacking. Here, we synthesized full-color bifunctional carbon dots (CDs) that can target LDs as well as respond to the nuance in internal lipid compositions with highly sensitive fluorescence signals, due to lipophilicity and surface state luminescence. Combined with microscopic imaging, uniform manifold approximation and projection, and sensor array concept, the capacity of cells to produce and maintain LD subgroups with varying lipid composition was clarified. Moreover, in oxidative stress cells, LDs with characteristic lipid compositions were deployed around mitochondria, and the proportion of LD subgroups changed, which gradually disappeared when treated with oxidative stress therapeutics. The CDs demonstrate great potential for in situ investigation of the LD subgroups and metabolic regulations.

Zebrafish cox17 modulates primitive erythropoiesis via regulation of mitochondrial metabolism to facilitate hypoxia tolerance.

Cox17 is required in the assembly of mitochondrial intermembrane space (IMS) and Cu metallization of cytochrome C oxidase (CcO) in mitochondria as well as Cu homeostasis in cells. Cox deficiency is associated with hematopoietic diseases such as tubulopathy and leukodystrophy, but whether and how cox17 functions in hematopoiesis are still unknown. Here, we report the effects of zebrafish cox17 deficiency on primitive erythropoiesis, mitochondrial metabolism, and hypoxia tolerance. Cox17-/- larvae were sensitive to hypoxia stress, with reduced primitive erythropoiesis. Meanwhile, cox17-/- mutants showed a significant reduction in the expression of pivotal transcriptional regulators in erythropoiesis, such as scl, lmo2, and gata1a at 14 h post fertilization (hpf), with expression remaining downregulated for scl but upregulated for lmo2 and gata1a at 24 hpf. Mechanistically, cox17-/- mutants showed impaired mitochondrial metabolism, coupled with a significant decrease in the mitochondrial membrane potential, ATP and SAM content, and the ratio of SAM and SAH. Additionally, disrupting mitochondrial metabolism in wild type (WT) larvae treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) could mimic the primitive erythropoiesis defects observed in cox17-/- mutants. Moreover, cox17-/- mutants exhibited significantly downregulated WNT signaling and upregulated ER stress, with a significant reduction of beta-Catenin in gata1a+ cells and of binding enrichment in both scl and lmo2 promoters of the WNT transcriptional factor TCF4. This is the first report on the novel linkage of cox17 deficiency with defective primitive erythropoiesis and reduced hypoxia tolerance. This study has shed light on the potential mechanism by which Cox deficiency, especially cox17 deficiency, induces Cu homeostasis imbalance, leading to hematopoietic diseases.

Seed color in lettuce is determined by the LsTT2, LsCHS, and Ls2OGD genes from the flavonoid biosynthesis pathway.

KEY MESSAGE: The mutated LsTT2 and Ls2OGD genes are responsible for white seeds and yellow seeds in lettuce, respectively. Three LsCHS genes are involved in the biosynthesis of flavonoid in seed coats. Lettuce seeds have several different colors, including black, yellow, and white. The genetic mechanisms underlying color variations of lettuce seeds remain unknown. We used genome-wide association studies (GWAS) and map-based cloning approaches to clone genes controlling the color of lettuce seeds. LsTT2, which encodes an R2R3-MYB transcription factor and is homologous to the TT2 gene in Arabidopsis, was shown to be the causal gene for the variation of black and white seeds in lettuce. A point mutation leads to the lack of stop codon in the LsTT2 transcript, resulting in white seeds. Knockout of the LsTT2 gene converted black seeds to white seeds. The locus controlling yellow seeds was mapped to Chromosome 2. Knockout of two 2-oxoglutarate-dependent dioxygenases (2OGD) genes from the candidate region converted black seeds to yellow seeds, suggesting that these two 2OGD proteins catalyze the conversion of yellow metabolites to black metabolites. We also showed that three LsCHS genes from the candidate region are associated with flavonoid biosynthesis in seeds. Knockout mutants of the three LsCHS genes decreased color intensity. This study provides new insights into the regulation of flavonoid biosynthesis in plants.

Copper ions impair zebrafish skeletal myofibrillogenesis via epigenetic regulation.

Unbalanced copper (Cu2+ ) homeostasis is associated with the developmental defects of vertebrate myogenesis, but the underlying molecular mechanisms remain elusive. In this study, it was found that Cu2+ stressed zebrafish embryos and larvae showed reduced locomotor speed as well as loose and decreased myofibrils in skeletal muscle, coupled with the downregulated expression of muscle fiber markers mylpfa and smyhc1l and the irregular arrangement of myofibril and sarcomere. Meanwhile, the Cu2+ stressed zebrafish embryos and larvae also showed significant reduction in the expression of H3K4 methyltransferase smyd1b transcripts and H3K4me3 protein as well as in the binding enrichment of H3K4me3 on gene mylpfa promoter in skeletal muscle cells, suggesting that smyd1b-H3K4me3 axis mediates the Cu2+ -induced myofibrils specification defects. Additionally, whole genome DNA methylation sequencing unveiled that the gene smyd5 exhibited significant promoter hyper-methylation and increased expression in Cu2+ stressed embryos, and the ectopic expression of smyd5 in zebrafish embryos also induced the myofibrils specification defects as those observed in Cu2+ stressed embryos. Moreover, Cu2+ was shown to suppress myofibrils specification and smyd1b promoter transcriptional activity directly independent of the integral function of copper transporter cox17 and atp7b. All these data may shed light on the linkage of unbalanced copper homeostasis with specific gene promoter methylation and epigenetic histone protein modification as well as the resultant signaling transduction and the myofibrillogenesis defects.

Characterization of four polymorphic genes controlling red leaf colour in lettuce that have undergone disruptive selection since domestication.

Anthocyanins protect plants from biotic and abiotic stressors and provide great health benefits to consumers. In this study, we cloned four genes (Red Lettuce Leaves 1 to 4: RLL1 to RLL4) that contribute to colour variations in lettuce. The RLL1 gene encodes a bHLH transcription factor, and a 5-bp deletion in some cultivars abolishes its function to activate the anthocyanin biosynthesis pathway. The RLL2 gene encodes an R2R3-MYB transcription factor, which was derived from a duplication followed by mutations in its promoter region. The RLL3 gene encodes an R2-MYB transcription factor, which down-regulates anthocyanin biosynthesis through competing with RLL2 for interaction with RLL1; a mis-sense mutation compromises the capacity of RLL3 to bind RLL1. The RLL4 gene encodes a WD-40 transcription factor, homologous to the RUP genes suppressing the UV-B signal transduction pathway in Arabidopsis; a mis-sense mutation in rll4 attenuates its suppressing function, leading to a high concentration of anthocyanins. Sequence analysis of the RLL1-RLL4 genes from wild and cultivated lettuce showed that their function-changing mutations occurred after domestication. The mutations in rll1 disrupt anthocyanin biosynthesis, while the mutations in RLL2, rll3 and rll4 activate anthocyanin biosynthesis, showing disruptive selection for leaf colour during domestication of lettuce. The characterization of multiple polymorphic genes in this study provides the necessary molecular resources for the rational breeding of lettuce cultivars with distinct levels of red pigments and green cultivars with high levels of health-promoting flavonoids.

Phosphorescence approach based on silica protected carbon dots for autofluorescence interference-free and highly selective detection of fluoride.

BACKGROUND: Fluoride (F-), an anion with the smallest ionic radius and highest charge density, plays an important role in biomedical and environmental processes, making the development of accurate F- detection methods of great importance. Fluorometric methods with simplicity and sensitivity have gained considerable attention in F- detection. However, their accuracy faces challenges due to issues like autofluorescence interference during real-time light excitation and limited selectivity. Therefore, it is important to establish a simple, real-time light excitation-free, and highly selective method for the accurate determination of F- in complicated samples. RESULTS: Herein, a novel phosphorescent approach is developed for the selective and accurate detection of F- in complex samples. Phosphorescence emission CDs@SiO2 is fabricated by confining CDs in a silica protective layer. This design retains the favorable water solubility of silica while benefitting from its inertness, making it resistant to most substances. Furthermore, phosphorescent analysis without real-time light excitation eliminates autofluorescence interference, significantly improving the signal-to-noise ratio (SNR) and simplifying sample pretreatment. The specific interaction between F- and the Si-O bond can lead to the degradation of the silica protective layer, exposing the CDs to the solution, resulting in phosphorescence quenching, achieving the highly accurate and sensitive detection of F- with a linear range of 0.001-4 mM and a limit of detection (LOD) of 1 µM. SIGNIFICANCE: This novel F- phosphorescence method based on the metal-free phosphorescent nanomaterial CDs@SiO2 integrates the benefits of no autofluorescence interference, high selectivity, and full aqueous compatibility, and its combination with a smartphone provides a simple, portable, and cost-effective detection platform for accurate and highly sensitive determination of F- in complex samples.

Copper overload induces apoptosis and impaired proliferation of T cell in zebrafish.

Copper is an essential biometal for cell development and function, however, unbalanced copper homeostasis in T cell development and the underlying mechanisms are largely unexplored. Here, we use a zebrafish model to investigate the effect of copper overload in T cell development. We show that copper stressed zebrafish larvae exhibit a significant reduction in T cells with increased cell apoptosis and impaired cell proliferation. T cell progenitors, hematopoietic stem and progenitor cells, also exhibit increased cell apoptosis. Copper overload induces production of ROS and the down-regulations of its resistance genes foxos, and ectopic expression of foxo3a, ROS scavenger GSH, could both effectively rescue the reduction of T cells in copper overload larvae. Moreover, foxm1-cytoskeleton axis, parallel to ROS-foxo axis, also mediates the copper overload induced T cell developmental defects. Meanwhile, ROS destroys expression of cytoskeleton rather than of foxm1 in the cells to induce cell apoptosis and the impaired proliferation. The functional integrity of copper transporters cox17 and atp7b are required for copper stress in inducing T cell apoptosis and proliferation impairment. Our findings demonstrate that the down-stream ROS-foxo/cytoskeleton and foxm1-cytoskeleton signaling pathways contribute jointly to copper overload induced T cell apoptosis and proliferation defects, which are depend on the integral function of Cox17 and Atp7b, and provide new insight into the copper homeostasis in T lymphocyte development.

EPC1/2 regulate hematopoietic stem and progenitor cell proliferation by modulating H3 acetylation and DLST.

Enhancers of polycomb 1 (EPC1) and 2 (EPC2) are involved in multiple biological processes as components of histone acetyltransferases/deacetylase complexes and transcriptional cofactors, and their dysfunction was associated with developmental defects and diseases. However, it remains unknown how their dysfunction induces hematopoietic stem and progenitor cell (HSPC) defects. Here, we show that depletion of EPC1/2 significantly reduced the number of hematopoietic stem and progenitor cells (HSPCs) in the aorta-gonad mesonephros and caudal hematopoietic tissue regions by impairing HSPC proliferation, and consistently downregulated the expression of HSPC genes in K562 cells. This study demonstrates the functions of EPC1/2 in regulating histone H3 acetylation, and in regulating DLST (dihydrolipoamide S-succinyltransferase) via H3 acetylation and cooperating with transcription factors serum response factor and FOXR2 together, and in the subsequent HSPC emergence and proliferation. Our results demonstrate the essential roles of EPC1/2 in regulating H3 acetylation, and DLST as a linkage between EPC1 and EPC2 with mitochondria metabolism, in HSPC emergence and proliferation.

Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance.

EAF1 and EAF2, the eleven-nineteen lysine-rich leukemia (ELL)-associated factors which can assemble to the super elongation complex (AFF1/4, AF9/ENL, ELL, and P-TEFb), are reported to participate in RNA polymerase II to actively regulate a variety of biological processes, including leukemia and embryogenesis, but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear. Here, we unveiled that deletion of EAF1/2 (eaf1-/- and eaf2-/-) caused reduction in hypoxia tolerance in zebrafish, leading to reduced erythropoiesis during hematopoietic processes. Meanwhile, eaf1-/- and eaf2-/- mutants showed significant reduction in the expression of key transcriptional regulators scl, lmo2, and gata1a in erythropoiesis at both 24 h post fertilization (hpf) and 72 hpf, with gata1a downregulated while scl and lmo2 upregulated at 14 hpf. Mechanistically, eaf1-/- and eaf2-/- mutants exhibited significant changes in the expression of epigenetic modified histones, with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters. Additionally, eaf1-/- and eaf2-/- mutants exhibited a dynamic expression of canonical WNT/ß-catenin signaling during erythropoiesis, with significant reduction in p-ß-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf. These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits, such as growth, disease resistance, hypoxia tolerance, and so on.

Phosphorescence, fluorescence, and colorimetric triple-mode sensor for the detection of acid phosphatase and corresponding inhibitor.

Acid phosphatase (ACP) as a clinical diagnostic biomarker for several pathophysiological diseases has aroused widespread interest. Compared to commonly developed single-mode ACP detection technology, the multi-mode detection method with self-validation can provide more reliable results. Herein, we proposed a triple-mode phosphorescence, fluorescence, and colorimetric method for ACP detection in combination with CDs@SiO2. HAuCl4 with oxidase-like activity can catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the blue oxide TMB (TMBox), offering absorption signals and quenching the phosphorescence and fluorescence of CDs@SiO2 based on the internal filtration effect (IFE). ACP can hydrolyze ascorbic acid 2-phosphate (AAP) to yield ascorbic acid (AA), thereby reducing TMBox to TMB, triggering solution fading and restoring phosphorescence and fluorescence signals. When the ACP inhibitor malathion is present, the reduction of TMBox is hindered, which successively led to the suppression of CDs@SiO2 phosphorescence and fluorescence signal recovery. According to these principles, triple-mode ACP (LOD = 0.0026 mU mL-1) and malathion detections (LOD = 0.039 µg mL-1) with favorable accuracy and sensitivity are realized. With simplicity, robustness, and versatility, the triple-mode sensor can be extended to the detection of the AAP hydrolase family and the screening of corresponding inhibitors.

Atp7b deficiency induces zebrafish eye developmental defects.

As a copper (Cu) transport ATPase, ATP7B plays an important role in maintaining Cu homeostasis in the body and its dysfunction is associated with retinal disease. How ATP7B dysfunction and the subsequent Cu overload induce retinal damage, however, are unknown. Here, we show that atp7b-/- homozygous zebrafish larvae are insensitive to light stimulation, with a reduction in retinal cells but normal like morphological phenotypes. Additionally, a series of differentially expressed genes are unveiled in atp7b-/- mutated larvae, which enrich in photo-transduction, structural constituent of eye lens, sensory perception of light stimulus, oxidative phosphorylation, and ATPase activity. Moreover, we show the Cu accumulation in retinal cells in atp7b-/- mutated larvae, which results in endoplasmic reticulum (ER) stress and retinal cell apoptosis and subsequent retinal defects. The integral data in this study demonstrate that atp7b mutation leads to Cu accumulation in zebrafish retinal cells and the consequence ER stress and retinal cell death. These data may give some possible hints to explain retinal disease occurred in the Cu dysregulation syndromes Wilson's disease with ATP7B mutation.

Copper overload impairs hematopoietic stem and progenitor cell proliferation via prompting HSF1/SP1 aggregation and the subsequently downregulating FOXM1-Cytoskeleton axis.

Unbalanced Cu homeostasis has been suggested to be associated with hematopoietic disease, but the roles of Cu overload in the hematopoietic system and the potential mechanisms are obscure. Here, we report a novel association and the novel potential pathways for Cu overload to induce proliferation defects in zebrafish embryonic hematopoietic stem and progenitor cells (HSPCs) via down-regulating expression of foxm1-cytoskeleton axis, which is conserved from fish to mammals. Mechanistically, we show the direct binding of Cu to transcriptional factors HSF1 and SP1 and that Cu overload induces the cytoplasmic aggregation of proteins HSF1 and SP1. These result in the reduced transcriptional activities of HSF1 and SP1 on their downstream FOXM1 as well as the FOXM1 transcriptional activities on cytoskeletons in HSPCs, which leads to ultimately cell proliferation impairment. These findings unveil the novel linkage of Cu overload with specific signaling transduction as well as the subsequent HSPC proliferation defects.

Effects of parental environmental copper stress on offspring development: DNA methylation modification and responses of differentially methylated region-related genes in transcriptional expression.

Parental environmental copper (Cu) exposure is widespread, causing problems for sustainability of fish populations, and epigenetics is suggested to be fundamental during the process, but the mechanism is scarcely reported. Here, we describe the effects of parental environmental Cu exposure on zebrafish developmental abnormality in subsequent generation. This study demonstrated for the first time that: 1. offspring from Cu-stressed paternal adult zebrafish showed developmental defects in the nervous and digestive system and changes in transcriptome; 2. Cu-induced alterations in sperm methylome and transcriptome could induce loci-specific alterations in DNA methylome and corresponding changes in the related gene transcription in offspring; 3. differentially methylated regions in pmpcb, crebl2 and tab2 promoters acted pivotally in their transcription; 4. pmpcb, crebl2 and tab2 are key individual contributors to parental Cu exposure-induced developmental defects in the nervous system, retina and digestive system of the offspring. Those data revealed that Cu-induced alterations in sperm methylome and transcriptome can be passed down to their fertilized offspring, reprogramming the epigenetic and transcriptional regulation of embryogenesis and causing embryonic developmental defects, suggesting that environmental Cu might pose a huge threat to the sustainability of fish populations.

Copper stress induces zebrafish central neural system myelin defects via WNT/NOTCH-hoxb5b signaling and pou3f1/fam168a/fam168b DNA methylation.

Unbalanced copper (Cu) homeostasis is associated with neurological development defects and diseases. However, the molecular mechanisms remain elusive. Here, central neural system (CNS) myelin defects and the down-regulated expression of WNT/NOTCH signaling and its down-stream mediator hoxb5b were observed in Cu2+ stressed zebrafish larvae. The loss/knockdown-of-function of hoxb5b phenocopied the myelin and axon defects observed in Cu2+ stressed embryos. Meanwhile, the activation of WNT/NOTCH signaling and ectopic expression of hoxb5b could rescue Cu induced myelin defects. Additionally, fam168b, similar to pou3f1/2, exhibited significant promoter hypermethylation and reduced expression in Cu2+ stressed embryos. The hypermethylated locus in fam168b promoter acted pivotally in its transcription, and the loss/knockdown of fam168b/pou3f1 also induced myelin defects. This study also demonstrated that fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis: Cu induced the down-regulated expression of the WNT&NOTCH-hoxb5b axis through the function of copper transporter cox17, coupled with the promoter methylation of genes fam168b/pou3f1, and its subsequent down-regulated expression through the function of another transporter atp7b, making joint contributions to myelin defects in embryos.

Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm formation via suppressing TGF-ß signaling.

Eaf family genes act in multiple cellular responses such as tumor suppression and embryonic development. In our previous work, Eaf1/2 was found to modulate convergence and extension (C&E) movements and pattern the embryonic anterior-posterior axis during zebrafish embryogenesis. Here, we found that loss-of-function of eaf1/2 caused expanded mesoderm and endoderm in zebrafish embryos and led to the recovery of endoderm specification in TGF-ß factor-mzoeptz257 mutants, while gain-of-function of eaf1/2 induced reduced mesoderm and endoderm. Analyses of gene expression profiles in Eaf deleted or over-expressed mammalian cells indicated that the roles of Eaf1 and Eaf2 in inhibiting TGF-ß signals were conserved from fish to mammals. By taking advantages of TGF-ß reporters, eaf1/2-fused engrailed proteins, and P53M214K mutant, we revealed that Eaf1 and Eaf2 might suppress TGF-ß transduction by synergistically inhibiting none-P53 and P53-required TGF-ß signaling. Furthermore, Eaf1/2 might co-localize and interact with TGF-ß transcriptional factors in the transcriptional complex as repressors to target and suppress TGF-ß signaling activity. Our study unveiled a previously unrecognized link of Eaf1/2 genes with TGF-ß and P53 in vertebrates and demonstrated a conservation of TGF-ß suppression activity for Eaf1/2 family genes from fish to mammals, which might shed some light on the molecular mechanistic basis of Eaf1 and Eaf2 in tumor suppression.