斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

Approaches to measuring calcium in zebrafish: focus on neuronal development

Calcium ions are known to act as important cellular signals during nervous system development. In vitro studies have provided significant information on the role of calcium signals during neuronal development; however, the function of this messenger in nervous system maturation in vivo remains to be established. The zebrafish has emerged as a valuable model for the study of vertebrate embryogenesis. Fertilisation is external and the rapid growth of the transparent embryo, including development of internal organs, can be observed easily making it well suited for imaging studies. The developing nervous system is relatively simple and has been well characterised, allowing individual neurons to be identified. Using the zebrafish model, both intracellular and intercellular calcium signals throughout embryonic development have been characterised. This review summarises technical approaches to measure calcium signals in developing embryonic and larval zebrafish, and includes recent developments that will facilitate the study of calcium signalling in vivo. The application of calcium imaging techniques to investigate the action of this messenger during embryogenesis in intact zebrafish is illustrated by discussion of their contribution to our understanding of neuronal development in vivo.     

Zebrafish as a model system to study the physiological function of telomeric protein TPP1

Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin) is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1), and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish.     

Developmental control of Presenilin1 expression,endoproteolysis, and interaction in zebrafish embryos

Dominant mutations in presenilin1 (PS1) and presenilin2 (PS2) are a major cause of early-onset Alzheimer's disease. In this report we analyze the expression of the zebrafish presenilin1 (Psen1) and presenilin2 (Psen2) proteins during embryogenesis. We demonstrate that Psen1 and Psen2 holoproteins are relatively abundant in zebrafish embryos and are proteolytically processed. Psen1 is maternally expressed, whereas Psen2 is expressed at later stages during development. The Psen1 C-terminal proteolytic fragment (CTF) is present at varying levels during embryogenesis, indicating the existence of developmental control mechanisms regulating its production. We examine the codependency of Psen1 and Psen2 expression during early embryogenesis. Forced overexpression of psen2 increases expression of Psen2 holoprotein, but not the N-terminal fragment (NTF), indicating that levels of Psen2 NTF are strictly controlled. Overexpression of psen2 did not alter levels of Psen1 holoprotein, CTF, or higher molecular weight complexes. Reduction of Psen1 activity in zebrafish embryos produces similar developmental defects to those seen for loss of PS1 activity in knockout mice. The relevance of these results to previous work on presenilin protein regulation and function are discussed. Our work shows that zebrafish embryos are a valid and valuable system in which to study presenilin interactions, regulation, and function.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

ADAM12‐deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects

ADAM (a d isintegrin a nd m etalloprotease) constitutes a family of multi‐domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino‐acid sequence. We examined adam12 expression in zebrafish embryos by whole mount in situ hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12‐knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.     

Characterization of a cell line derived from zebrafish (brachydanio rerio) embryos

Summary During the last decade, zebrafish (Brachydanio rerio) have emerged as a novel and attractive system to study embryogenesis and organogenesis in vertebrates. The main reason is that both extensive genetic studies and detailed embryologic analysis are possible using this small tropical fresh water teleost. However, in vitro analysis using cell culture or molecular genetics are still far less advanced than in other vertebrate systems. Here we report the generation and characterization of a fibroblast like cell line, ZF4, derived from 1-day-old zebrafish embryos. The hyperploid cell line has been stable in multiple passages for more than 2 yr now and is the first zebrafish cell line that can be maintained in conventional medium containing mammalian serum. Using a series of plasmids for expression of a marker gene, we evaluate in ZF4 cells the relative strength of expression from several different viral, fish, and mammalian promoters. Stable integration can be obtained by using G418 selection. We hope that our cell line will be a useful tool for the analysis of gene regulation in zebrafish.     

Adriamycin inhibits embryonic development in zebrafish through downregulation of Kruppel‐like factor4

Adriamycin is an effective anticancer drug used in a wide range of cancers. Anticancer drugs modulate oncogenes and nodal regulatory molecules that affect cell differentiation and organismal development. In this study, we explore the effect of adriamycin on Kruppel‐like factor4 (Klf4), an essential pluripotent factor by choosing zebrafish embryos as a model system. Klf4 is involved in the regulation of cellular growth, proliferation, and differentiation. In zebrafish embryogenesis, Klf4 is a major regulator of differentiation of polster in the anterior mesendoderm region of cells into hatching gland cells. The importance of this study is to check the effect of adriamycin on embryonic development. We found, adriamycin dose dependently altered the gene expression level of Klf4 that occurs in parallel to its detrimental effect on hatching. Supportively, cathepsin L and cyclase‐associated protein1 are the other two markers of hatching that are altered along with Klf4.     

There is more to life than death: a moonlighting function of a Bcl-2 member

Members of the Bcl-2 family proteins are best known for their roles in apoptosis regulation. In this issue of Developmental Cell, Popgeorgiev et al. (2011) have uncovered a new, nonapoptotic role for a Bcl-2 homolog during early embryogenesis in zebrafish.     

Dicistronic Gene Expression in Developing Zebrafish

Internal ribosome entry sites (IRESs) allow ribosomal access to messenger RNA without a requirement for cap recognition and subsequent scanning to an initiator AUG. Hence, IRESs have been adapted into dicistronic vectors for the expression of more than one gene from a single mRNA. Dicistronic vectors have been used for many applications in mammalian tissue culture and transgenesis. However, whether the IRESs from mammalian viruses function without temporal or spatial restrictions in nonmammalian organisms like zebra fish (Danio rerio) is unknown. Therefore, we have examined the expression capabilities of the encephalomyocarditis virus (EMCV) IRES during zebrafish embryogenesis. We determined that the EMCV IRES was sufficient to permit detectable expression of several second cistron reporters during zebrafish embryogenesis, including luciferase and green fluorescent protein. This suggests that our dicistronic vectors are suitable for general use in any vertebrate system, from fish to humans. Received March 26, 1999; accepted June 14, 1999.     

Zebrafish as a model to understand autophagy and its role in neurological disease

In the past decade, the zebrafish (Danio rerio) has become a popular model system for the study of vertebrate development, since the embryos and larvae of this species are small, transparent and undergo rapid development ex utero, allowing in vivo analysis of embryogenesis and organogenesis. These characteristics can also be exploited by researchers interested in signaling pathways and disease processes and, accordingly, there is a growing literature on the use of zebrafish to model human disease. This model holds great potential for exploring how autophagy, an evolutionarily conserved mechanism for protein degradation, influences the pathogeneses of a range of different human diseases and for the evaluation of this pathway as a potential therapeutic strategy. Here we summarize what is known about the regulation of autophagy in eukaryotic cells and its role in neurodegenerative disease and highlight how research using zebrafish has helped further our understanding of these processes.     

“Casting” light on the role of glycosylation during embryonic development: Insights from zebrafish

Zebrafish (Danio rerio) remains a versatile model organism for the investigation of early development and organogenesis, and has emerged as a valuable platform for drug discovery and toxicity evaluation [1–6]. Harnessing the genetic power and experimental accessibility of this system, three decades of research have identified key genes and pathways that control the development of multiple organ systems and tissues, including the heart, kidney, and craniofacial cartilage, as well as the hematopoietic, vascular, and central and peripheral nervous systems [7–31]. In addition to their application in large mutagenic screens, zebrafish has been used to model a variety of diseases such as diabetes, polycystic kidney disease, muscular dystrophy and cancer [32–36]. As this work continues to intersect with cellular pathways and processes such as lipid metabolism, glycosylation and vesicle trafficking, investigators are often faced with the challenge of determining the degree to which these pathways are functionally conserved in zebrafish. While they share a high degree of genetic homology with mouse and human, the manner in which cellular pathways are regulated in zebrafish during early development, and the differences in the organ physiology, warrant consideration before functional studies can be effectively interpreted and compared with other vertebrate systems. This point is particularly relevant for glycosylation since an understanding of the glycan diversity and the mechanisms that control glycan biosynthesis during zebrafish embryogenesis (as in many organisms) is still developing. Nonetheless, a growing number of studies in zebrafish have begun to cast light on the functional roles of specific classes of glycans during organ and tissue development. While many of the initial efforts involved characterizing identified mutants in a number of glycosylation pathways, the use of reverse genetic approaches to directly model glycosylation-related disorders is now increasingly popular. In this review, the glycomics of zebrafish and the developmental expression of their glycans will be briefly summarized along with recent chemical biology approaches to visualize certain classes of glycans within developing embryos. Work regarding the role of protein-bound glycans and glycosaminoglycans (GAG) in zebrafish development and organogenesis will also be highlighted. Lastly, future opportunities and challenges in the expanding field of zebrafish glycobiology are discussed.     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. The effective concentration to cause 50% malformations (EC₅₀) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang- 1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.     

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.     

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.     

Zebrafish as a model system for biomedical studies

Zebrafish (Danio rerio) is now firmly recognized as a powerful research model for many areas of biology and medicine. Here, we review some achievements of zebrafish-based assays for modeling human diseases and for drug discovery and development. For drug discovery, zebrafish is especially valuable during the earlier stages of research as its represents a model organism to demonstrate a new treatment’s efficacy and toxicity before more costly mammalian models are used. This review considers some examples of known compounds which exhibit both physiological activity and toxicity in humans and zebrafish. The major advantages of zebrafish embryos consist in their permeability to small molecules added to their incubation medium and chorion transparency that enables the easy observation of the development. Assay of acute toxicity (LC₅₀ estimation) in embryos can also include the screening for developmental disorders as an indicator of teratogenic effects. We have used the zebrafish model for toxicity testing of new drugs based on phospholipid nanoparticles (e.g. doxorubicin). Genome organization and the pathways involved into control of signal transduction appear to be highly conserved between zebrafish and humans and therefore zebrafish may be used for modeling of human diseases. The review provides some examples of zebrafish application in this field.