三七总皂苷对斑马鱼胚胎初级和次级造血的抑制作用

目的探讨三七总皂苷(Panax notoginsenosides,PNSs)对斑马鱼胚胎造血的作用,为三七的药理学应用提供实验依据。方法通过乙醇提取得到PNSs,用50、100μg/mL的PNSs从75%外包时期开始处理斑马鱼胚胎。收集发育至不同时期的胚胎,检测药物处理后,斑马鱼初级造血和次级造血的分子标记的变化。结果 PNSs处理后,初级造血的分子标记gata1、hbbe3明显下降,生成的红细胞显著减少;PNSs处理还可以抑制造血干细胞(hematopoietic stem cell, HSC)发育。次级造血的分子标记runx1,cmyb在PNSs处理下表达下调,由HSC分化生成的T淋巴细胞分子标记rag1表达也显著降低。有意思的是,PNSs对斑马鱼初级和次级造血的抑制作用均呈剂量依赖效应。结论孕期可能需要慎重使用三七总皂苷药物。     

Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis.

Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.     

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.     

Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila

Hedgehog (Hh) signaling is important for development and homeostasis in vertebrates and invertebrates. Ligand-independent, deregulated Hh signaling caused by loss of negative regulators such as Patched causes excessive cell proliferation, leading to overgrowth in Drosophila and tumors in humans, including basal-cell carcinoma and medulloblastoma. We show that in Drosophila deregulated Hh signaling also promotes cell survival by increasing the resistance to apoptosis. Surprisingly, cells with deregulated Hh activity do not protect themselves from apoptosis; instead, they promote cell survival of neighboring wild-type cells. This non-cell autonomous effect is mediated by Hh-induced Notch signaling, which elevates the protein levels of Drosophila inhibitor of apoptosis protein-1 (Diap-1), conferring resistance to apoptosis. In summary, we demonstrate that deregulated Hh signaling not only promotes proliferation but also cell survival of neighboring cells. This non-cell autonomous control of apoptosis highlights an underappreciated function of deregulated Hh signaling, which may help to generate a supportive micro-environment for tumor development.     

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.     

Non-cell autonomous expression of TNF-alpha-converting enzyme ADAM17 is required for normal lymphocyte development

TNF-alpha converting enzyme (TACE; ADAM17), a member of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, has been shown to cleave a wide variety of cell surface proteins of immunological importance. Due to the broad expression of TACE and the early postnatal lethality of TACE-deficient mice, it has been difficult to assess the role of TACE in lymphocyte development. Indeed, it is not known whether hemopoietic and/or nonhemopoietic expression of TACE is required for normal lymphocyte development. In the current study, we analyzed the lymphoid system of tace(DeltaZn/DeltaZn) mice and tace(DeltaZn/DeltaZn) bone marrow RAG1(-/-) recipients. Our results clearly show that nonlymphocyte expression of TACE is required for normal lymphocyte development and lymphoid organ structure. Lack of TACE function resulted in a partial block in T cell development at the double-negative 4:double-positive transition in the thymus, a loss of B cell development/maturation in the spleen, and a lack of B cell follicle and germinal center formation in the spleen. Thus, TACE serves as a lymphocyte extrinsic factor that is essential for normal T development and peripheral B cell maturation.     

Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila

ABSTRACT

Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.     

Dissecting hematopoiesis and disease using the zebrafish

The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.     

Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development

Limb development in the Drosophila embryo requires a pattern-forming system to organize positional information along the proximal–distal axis of the limb. This system must function in the context of the well characterized anterior–posterior and dorsal–ventral pattern-forming systems that are required to organize the body plan of the embryo. By genetic criteria the Distal-less gene appears to play a central role in limb development. Lack-of-function Distal-less mutations cause the deletion of a specific subset of embryonic peripheral sense organs that represent the evolutionary remnants of larval limbs. Distal-less activity is also required in the imaginal discs for the development of adult limbs. This requirement is cell autonomous and region specific within the developing limb primordium. Production of genetically mosaic imaginal discs, in which clones of cells lack Distal-less activity, indicates the existence of an organized proximal–distal positional information in very young imaginal disc primordia. We suggest that this graded positional information may depend on the activity of the Distal-less gene.     

A parental requirement for dual-specificity phosphatase 6 in zebrafish

Background

Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood.

Results

Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults.

Conclusions

The fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.

     

The role of stat1b in zebrafish hematopoiesis

STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by sub-functionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development.     

Erratum: Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development

[This corrects the article on p. 2045 in vol. 8.].     

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.