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During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog–Vegf–Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.

Nascent hematopoietic stem and progenitor cells (HSPCs) acquire definitive hematopoietic characteristics only when they develop into fetal HSPCs; however, the mechanisms underlying fetal HSPC development are poorly understood. Here, we profiled the chromatin accessibility and transcriptional features of zebrafish nascent and fetal HSPCs using ATAC-seq and RNA-seq and revealed dynamic changes during HSPC transition. Functional assays demonstrated that chromatin remodeler-mediated epigenetic programming facilitates fetal HSPC development in vertebrates. Systematical screening of chromatin remodeler-related genes identified that smarca5 is responsible for the maintenance of chromatin accessibility at promoters of hematopoiesis-related genes in fetal HSPCs. Mechanistically, Smarca5 interacts with nucleolin to promote chromatin remodeling, thereby facilitating genomic binding of transcription factors to regulate expression of hematopoietic regulators such as bcl11ab. Our results unravel a new role of epigenetic regulation and reveal that Smarca5-mediated epigenetic programming is responsible for fetal HSPC development, which will provide new insights into the generation of functional HSPCs both in vivo and in vitro.

Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP ‘pocket-like’ units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage–HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell–cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

Summary Cell transplantation into immunodeficient recipients is a widely used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a foxn1/Casper mutant zebrafish that is transparent and exhibits T cell deficiency. By employing the line for hematopoietic stem cell (HSC) transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization zebrafish embryos produce a better transplant outcome in foxn1/Casper mutants, compared with adult HSCs. In addition to HSCT, the foxn1/Casper mutant is feasible for allografts of myelodysplastic syndrome-like and muscle cells, as well as xenografts of medaka muscle cells. In summary, foxn1/Casper mutants permit the nonconditioned engraftment of multiple cell types and visualized characterization of transplanted cells in vivo., Graphical Abstract, Highlights • foxn1/Casper mutant zebrafish permit unconditioned and visualized cell transplantation • Zebrafish fetal HSCs possess more robust engraftment ability than adult HSCs • foxn1/Casper mutant zebrafish permit allogeneic MDS-like cell transplantation • Allograft and xenograft of muscle cells can be monitored in foxn1/Casper mutant zebrafish, In this article, Liu and colleagues generated a foxn1/Casper mutant zebrafish line with immunodeficiency and transparency to permit allograft and xenograft of normal and malignant cells without preconditioning. In addition, they evaluated the engraftment efficiency of HSCs from different developmental stages in foxn1/Casper recipients. Additional applications included the engraftment of MDS-like cells and xenogenic medaka muscle cells.

Hematopoietic stem and progenitor cells (HSPCs) are capable of producing all mature blood lineages, as well as maintaining the self-renewal ability throughout life. The hairy-like organelle, cilium, is present in most types of vertebrate cells, and plays important roles in various biological processes. However, it is unclear whether and how cilia regulate HSPC development in vertebrates. Here, we show that cilia-specific genes, involved in primary cilia formation and function, are required for HSPC development, especially in hemogenic endothelium (HE) specification in zebrafish embryos. Blocking primary cilia formation or function by genetic or chemical manipulations impairs HSPC development. Mechanistically, we uncover that primary cilia in endothelial cells transduce Notch signal to the earliest HE for proper HSPC specification during embryogenesis. Altogether, our findings reveal a pivotal role of endothelial primary cilia in HSPC development, and may shed lights into in vitro directed differentiation of HSPCs., Haematopoietic stem and progenitor cells (HSPCs) produce all blood lineages and arise from the haemogenic endothelium (HE) during embryogenesis. Here the authors show that genes specific to cilia formation are required for HSPC development in the HE in zebrafish through transduction of Notch signal.

Significance The caudal hematopoietic tissue (CHT) is characterized as a hematopoietic organ for fetal hematopoietic stem and progenitor cell (HSPC) expansion in zebrafish. In this study, we used scRNA-seq combined with functional assays to decode the developing CHT. First, we resolved fetal HSPC heterogeneity, manifested as lineage priming and metabolic gene signatures. We further analyzed the cellular interactions among nonhematopoietic niche components and HSPCs and identified an endothelial cell-specific factor, Gpr182, followed by experimental validation of its role in promoting HSPC expansion. Finally, we uncovered the conservation and divergence of developmental hematopoiesis between human fetal liver and zebrafish CHT. Our study provides a valuable resource for fetal HSPC development and clues to establish a supportive niche for HSPC expansion in vitro., During vertebrate embryogenesis, fetal hematopoietic stem and progenitor cells (HSPCs) exhibit expansion and differentiation properties in a supportive hematopoietic niche. To profile the developmental landscape of fetal HSPCs and their local niche, here, using single-cell RNA-sequencing, we deciphered a dynamic atlas covering 28,777 cells and 9 major cell types (23 clusters) of zebrafish caudal hematopoietic tissue (CHT). We characterized four heterogeneous HSPCs with distinct lineage priming and metabolic gene signatures. Furthermore, we investigated the regulatory mechanism of CHT niche components for HSPC development, with a focus on the transcription factors and ligand–receptor networks involved in HSPC expansion. Importantly, we identified an endothelial cell-specific G protein–coupled receptor 182, followed by in vivo and in vitro functional validation of its evolutionally conserved role in supporting HSPC expansion in zebrafish and mice. Finally, comparison between zebrafish CHT and human fetal liver highlighted the conservation and divergence across evolution. These findings enhance our understanding of the regulatory mechanism underlying hematopoietic niche for HSPC expansion in vivo and provide insights into improving protocols for HSPC expansion in vitro.

Macrophages play pivotal roles in immunity, hematopoiesis, and tissue homeostasis. In mammals, macrophages have been shown to originate from yolk-sac-derived erythro-myeloid progenitors and aorta-gonad-mesonephros (AGM)-derived hematopoietic stem cells. However, whether macrophages can arise from other embryonic sites remains unclear. Here, using single-cell RNA sequencing, we profile the transcriptional landscape of mouse fetal placental hematopoiesis. We uncover and experimentally validate that a CD44+ subpopulation of placental endothelial cells (ECs) exhibits hemogenic potential. Importantly, lineage tracing using the newly generated Hoxa13 reporter line shows that Hoxa13-labeled ECs can produce placental macrophages, named Hofbauer cell (HBC)-like cells. Furthermore, we identify two subtypes of HBC-like cells, and cell-cell interaction analysis identifies their potential roles in angiogenesis and antigen presentation, separately. Our study provides a comprehensive understanding of placental hematopoiesis and highlights the placenta as a source of macrophages, which has important implications for both basic and translational research.

It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition., Cell-autonomous Notch signaling regulated by the membrane trafficking protein Rab5c plays an instructive role in hematopoietic stem and progenitor cell specification, while the AKT signaling seems to provide a permissive signal to maintain hemogenic endothelium survival.

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 ( dnmt1 ) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates.

Cell transplantation into immunodeficient recipients is a widely-used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a zebrafish foxn1/Casper mutant that is transparent and immunodeficient with deficient T cells. By employing the mutant for hematopoietic stem cell transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization produce a better transplant outcome in adult recipients, compared to adult HSCs. Furthermore, adult HSCs from the newly generated Tg (runx1:en-GFP) line have higher engraftment efficiency than that from the Tg (CD41:GFP) line. In addition to HSCT, foxn1/Casper mutant is feasible to allogeneic leukemia and muscle cell transplantation, as well as xenogenic medaka HSCT. Collectively, foxn1/Casper mutant permits the nonconditioned engraftment of multiple cell types with superiority in imaging and characterization of transplanted cells at single cell resolution in vivo.

Technological advances are important for innovative biological research. Development of molecular tools for DNA manipulation, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas), has revolutionized genome editing. These approaches can be used to develop potential therapeutic strategies to effectively treat heritable diseases. In the last few years, substantial progress has been made in CRISPR/Cas technology, including technical improvements and wide application in many model systems. This review describes recent advancements in genome editing with a particular focus on CRISPR/Cas, covering the underlying principles, technological optimization, and its application in zebrafish and other model organisms, disease modeling, and gene therapy used for personalized medicine.

Programming bugs are quite normal and usual for novice programmers. This article reports the findings of our research investigating disciplinary variations in the computer programming field from three perspectives, which are (i) the knowledge structure implied by the performance of each disciplinary group of students in the 26 question categories covering different knowledge points, (ii) the programming ability illustrated by the common bugs made by them respectively, and (iii) the knowledge acquisition ability suggested by the changes happened to the common bugs over a one-year period. The results support the hypothesis that the thinking pattern and the knowledge structure of students is shaped by their disciplinary context, and call into question the extent to which the knowledge acquisition process differs between different disciplinary groups. Overall, Students may use our result to look up the most common bugs in attempt to avoid them in the future. While instructors can use this result to continuously improve their curriculum and teaching practices for different disciplinary group of students respectively.

With the widespread use of online learning platforms, learning outside of class plays an increasingly important role in student learning. The impact of learners' learning behavior on their academic performance in online learning platform becomes an increasingly important but neglected research perspective. Thus, this article will focus on: Study the relevance of learning behavior indexes in the online learning platform and course scores. The experimental data in this article comes from the database of Beihang online judge platform (www.buaacoding.cn), and the Programming course scores of the students who are studying in Information College of Beihang university (There are more than 500 students one year in this college). We use Difference analysis, correlation analysis, and contrastive analysis to analyze the experimental data. The experimental results show that the learning behavior outside the classroom, such as the online learning platform, has an impact on the students' course scores.

In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships.

Inflammatory signaling has been shown to be essential for stress hematopoiesis in adult bone marrow, either through increasing proliferation or by directing differentiation of hematopoietic stem and progenitor cells (HSPCs) toward myeloid or lymphoid lineages. However, its role in embryonic normal hematopoiesis has been unknown. Here, we demonstrate that in both zebrafish and mouse embryos, inflammatory signaling is necessary and sufficient for HSPC emergence, in the absence of infection or pathological inflammation. Mechanistically, inflammatory signaling regulates hemogenic endothelium-derived HSPC development through a conserved Toll-like receptor 4 (TLR4)-nuclear factor κ-light-chain enhancer of activated B core (NF-κB) signaling, which then promotes Notch activity, a well-known signal required for HSPC specification in vertebrates. Our findings establish a previously unrecognized link between inflammatory signaling and HSPC emergence, and provide new insights into regenerative medicine and novel therapies to treat innate immune-related diseases.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5−/− mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.

Massive traffic scene data for algorithm research and model training is the fundamental for self-driving car technology development. In the procedure of scene image labeling, the most accurate method is manual annotation, but with the increasing of the amount of image data, artificial annotation method becomes infeasible due to its disadvantages of vast cost, inefficiency and subjective deviation. In order to reduce the time and cost of annotation processing while ensuring the accuracy, this paper proposed a semi-automatic annotation procedure. The core idea is using an automatic preprocessing method developed with convolution neural network to roughly annotate the image, just before the human review and revision. The innovation of the proposed automatic annotation method includes: 1.) the results of CNN processing are improved by combining with the object detection outcome; 2.) a variable parameter outlier-merging algorithm based on the sliding window is provided to deal with the large number of outliers. It shows about 5 percentage improvement in class average accuracy and 4/5 decrease of time to process one picture by using our processing method.

Using deep learning for visual scene parsing will satisfy the demand of the next generation of automatic driving technology. However, current parsing algorithms are not mature enough for practical applications unless higher accuracy and efficiency are obtained. We propose a novel scene parsing algorithm framework which integrates the object detection technologies into convolution neural network to improve the overall effectiveness. The framework consists of three components: i) a scene parsing network presenting primary semantic segmentation result. ii)an object detection network calculating the location and confidence of the targets in images. iii) an integration and filter module that cascades previous two results. Extensive experiments suggest that our model is capable of practical use and achieving more favorable scene parsing performance of mIoU score as 69.4% on CamVid dataset.

Nuclear receptor corepressors (Ncors) are important for developmental and homeostatic processes in vertebrates, which exert transcriptional repression by coordinating with histone deacetylases. However, little is known about their roles in definitive hematopoiesis. In this study, we show that in zebrafish, ncor2 is required for hematopoietic stem cell (HSC) development by repressing fos-vegfd signaling. ncor2 is specifically expressed in the aorta-gonad-mesonephros (AGM) region in zebrafish embryos. ncor2 deficiency reduced the population of HSCs in both the AGM region and T cells in the thymus. Mechanistically, ncor2 knockdown upregulated fos transcription by modulating the acetylation level in the fos promoter region, which then enhanced Vegfd signaling. Consequently, the augmented Vegfd signaling induced Notch signaling to promote the arterial endothelial fate, therefore, possibly repressing the hemogenic endothelial specification, which is a prerequisite for HSC emergence. Thus, our findings identify a novel regulatory mechanism for Ncor2 through Fos-Vegfd-Notch signaling cascade during HSC development in zebrafish embryos.

Summary In mammals, hematopoietic stem and progenitor cells (HSPCs) rapidly expand in the fetal liver (FL), but the underlying mechanism remains unclear. Here, we characterize zebrafish caudal hematopoietic tissue (CHT) and identify an important cellular and molecular mechanism of HSPC expansion. Time-lapse imaging showed that HSPCs localize adjacent to vascular endothelial cells (ECs), and their migration and expansion display caudal vein-specific orientation in the CHT. RNA sequencing and functional analysis identified that an EC-expressed transcription factor, Kruppel-like factor 6a (Klf6a), is essential for the CHT niche. We further demonstrated that Klf6a directly regulates the expression of the chemokine (C-C motif) ligand 25b to modulate HSPC lodgment and proliferation. Ex vivo culture results support the conserved role of Ccl21/Ccr7 signaling in promoting HSPC expansion in mammals. Together, we identify the Klf6a-Ccl25b/Ccr7 axis in controlling the complex HSPC-CHT niche interaction, which may be applicable to in vitro expansion or engraftment of HSPCs after transplantation.

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The thymus is a central hematopoietic organ which produces mature T lymphocytes with diverse antigen specificity. During development, the thymus primordium is derived from the third pharyngeal endodermal pouch, and then differentiates into cortical and medullary thymic epithelial cells (TECs). TECs represent the primary functional cell type that forms the unique thymic epithelial microenvironment which is essential for intrathymic T-cell development, including positive selection, negative selection and emigration out of the thymus. Our understanding of thymopoiesis has been greatly advanced by using several important animal models. This review will describe progress on the molecular mechanisms involved in thymus and T cell development with particular focus on the signaling and transcription factors involved in this process in mouse and zebrafish.

The thymus is mainly comprised of thymic epithelial cells (TECs), which form the unique thymic epithelial microenvironment essential for intrathymic T-cell development. Foxn1, a member of the forkhead transcription factor family, is required for establishing a functional thymic rudiment. However, the molecular mechanisms underlying the function of Foxn1 are still largely unclear. Here, we show that Foxn1 functions in thymus development through Mcm2 in the zebrafish. We demonstrate that, in foxn1 knockdown embryos, the thymic rudiment is reduced and T-cell development is impaired. Genome-wide expression profiling shows that a number of genes, including some known thymopoiesis genes, are dysregulated during the initiation of the thymus primordium and immigration of T-cell progenitors to the thymus. Functional and epistatic studies show that mcm2 and cdca7 are downstream of Foxn1, and mcm2 is a direct target gene of Foxn1 in TECs. Finally, we find that the thymus defects in foxn1 and mcm2 morphants might be attributed to reduced cell proliferation rather than apoptosis. Our results reveal that the foxn1 - mcm2 axis plays a central role in the genetic regulatory network controlling thymus development in zebrafish.

The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600 μmol m−2 s−1. Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1β-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn’t interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1β-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions.

Potato virus Y (PVY) infections often lead to altered numbers of host plant chloroplasts, as well as changes in morphology and inhibited photosynthesis. The multifunctional protein helper component-proteinase, HC-Pro, has been identified in PVY-infected leaf chloroplasts. We used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that HC-Pro can interact with the chloroplast division-related factor NtMinD in yeast and tobacco cells, respectively. In addition, we confirmed that residues 271 to 314 in NtMinD are necessary for its interaction with PVY HC-Pro in a yeast two-hybrid analysis using four NtMinD deletion mutants. These residues are necessary for the dimerization of NtMinD, which plays a vital role in chloroplast division. Thus, PVY HC-Pro may affect NtMinD activity by inhibiting the formation of NtMinD homodimers, and this may interfere with chloroplast division and contribute to changes in the numbers of chloroplast per cell observed in PVY-infected plants.

The multifunctional protein helper component proteinase (HC-Pro) is thought to interfere with the activity of the 20S proteasome; however, no sites of interaction have been identified for either protein. Here, we first show that the Potato virus Y (PVY) HC-Pro protein can interact with three Arabidopsis 20S proteasome subunits (PAA, PBB, and PBE), using a yeast two-hybrid system and the bimolecular fluorescence complement assay. In addition, yeast two-hybrid analysis of the interaction between several mutant subunits of the 20S proteasome and PVY HC-Pro confirmed that residues 81 to 140 of PAA, 1 to 80 of PBB, and 160 to 274 of PBE are necessary for binding PAA, PBB, and PBE to PVY HC-Pro, respectively. Deletion mutant analysis of PVY HC-Pro showed that the N terminus (residues 1 to 97) is necessary for its interaction with three Arabidopsis 20S proteasome subunits. The ability of HC-Pro to interact and interfere with the activity of the 20S proteasome may help explain the molecular basis of its multifunctional character.

SummaryT lymphoid-primed progenitors are hematopoietic progenitors destined to enter the thymus. The in vivo characterization of these embryonic progenitors is challenging, however, due to the intrauterine development of mouse embryos. Thus, how the fate of these cells is determined has not been fully defined in mammals. Here we use zebrafish embryos to show that the homing of T lymphoid-primed progenitors to the thymus is impaired, concomitant with a decrease in ccr9a expression, in the absence of irf4a. Strikingly, fate mapping assays at the single-cell level showed a fate change of irf4a-deficient T lymphoid-primed progenitors to myeloid cells, accompanied by an increase in Pu.1 expression. These data indicate that in addition to regulating ccr9a expression, Irf4a is essential in T lymphoid-primed progenitors for repressing Pu.1 expression to prevent an alternate fate. Our findings provide insight into the fate determination mechanism of T lymphoid-primed progenitors.

Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships.