An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Nonmuscle myosin II-B (myh10) expression analysis during zebrafish embryonic development

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes (myh9, myh10, and myh14) encoding different nonmuscle myosin heavy chains. The three NM II isoforms share a very similar molecular structure and play important roles in a variety of fundamental biological processes. NM II-B (myh10) has been shown to be essential for the formation of mouse neural system and heart. But so far the complete knowledge for its expression in developing zebrafish embryos is lacking. In current study, we proved the conservation of zebrafish NM II-B in vertebrate evolution by in silicon analysis. Afterwards the NM II-B (myh10) expression was demonstrated to initiate after gastrulation stage. At 20 hpf, the expression is mainly restricted in central nervous system (CNS). It was maintained and expanded to sensor organ including eye, otic vesicle, and olfactory bulb at 36 hpf and later. We also detected myh10 mRNA hybridization signal in 48 hpf zebrafish heart. In addition, we investigated myh9a and myh9b mRNA distribution in zebrafish developing embryos. It was shown that myh10 and myh9 have distinct expression pattern, with myh9s not in neural system but in epidermis, enveloping layer (EVL). Our study provides new insight into the NM II expression and the use of this model organism to tackle future studies on the role of NM II in embryo development.     

Clinically Approved Iron Chelators Influence Zebrafish Mortality,Hatching Morphology and Cardiac Function

Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.     

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens ¹. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates ². In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling ³. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.     

斑马鱼akt3 基因的克隆及其表达图谱与过表达分析

采用RT-PCR 和RACE 相结合的方法, 克隆得到了斑马鱼的akt3/pkb 基因, 其cDNA 全长为2874 bp,编码479 个氨基酸。斑马鱼akt3 具有akt 家族成员间保守的PH 结构域、催化活性结构域和调节结构域以及两个保守的磷酸化位点Thr305 和Ser472。与已发表的人、大鼠、小鼠的akt3 氨基酸序列比较, 相似性分别为95.8%、94.7%和95.4%。对斑马鱼早期胚胎进行RT-PCR 检测显示, akt3 在0-4hpf(hours post fertilization)含量水平较高, 6hpf 到12hpf 降低至较低水平, 16hpf 后表达量开始逐渐上升, 60hpf 至96hpf 则稳定在较高水平。原位杂交结果表明: akt3 在2hpf 至96hpf 的胚胎中整体都有表达, 没有组织特异性。在成鱼中, 除鳃部外, akt3 在所检测的其他各组织器官中均有表达, 在脑部和卵巢表达量较高; 利用显微注射持续表达myr-akt3 mRNA 研究其功能充分性时结果显示, 过量表达斑马鱼akt3 mRNA 能使斑马鱼胚胎发育滞后且伴随着尾部短粗、体节模糊、尾末端膨大甚至严重缩短等不同程度畸形。而在斑马鱼myr-akt3 注射组发育至24hpf 时观察(以排除akt3 造成的发育延迟的影响), 发现注射过akt3 的斑马鱼胚胎的脑部厚度较对照组显著增大, 表明akt3 对斑马鱼胚胎脑部尺寸发育有影响。       

Bioenergetic profiling of zebrafish embryonic development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.     

Proteome analysis of a single zebrafish embryo using three different digestion strategies coupled with liquid chromatography-tandem mass spectrometry

Zebrafish is a powerful model to analyze vertebrate embryogenesis and organ development. Although a number of genes have been identified to specify embryonic development processes, only a few large-scale proteomic analyses have been reported in regard to these events to date. Here the total proteins of a single embryo were analyzed by urea-, sodium deoxycholate (SDC)-, and performic acid (PA)-assisted trypsin digestion strategies coupled to capillary liquid chromatography-tandem mass spectrometry (CapLC-MS/MS) identification. In total, 509 and 210 proteins were detected from the embryos at 72 and 120 hours postfertilization (hpf), respectively, with a false identification rate of less than 1%. Approximately 95% of those proteins could be observed by combining the urea- and SDC-assisted digestion strategies, suggesting that these two methods are more effective than the PA-assisted method. Compared with 0.5% SDC, 1% SDC was more effective to identify proteins in zebrafish embryos. In addition, removal of the predominant yolk proteins could significantly improve protein identification efficiency. Our study represents the first overview of the protein expression profile of a single zebrafish embryo at 72 or 120 hpf. More important, this single individual proteome methodology could be applied to multiple development stages of wide-type or mutant embryos, providing a simple and powerful way to further our understanding of embryonic development.     

硫代硫酸钠干扰斑马鱼胚胎发育并致畸

硫的衍生物潜在的威胁着胚胎的发育过程.斑马鱼被用于研究不同浓度（1×10-6～1 mol/L）的硫代硫酸钠（sodium thiosulfate, STS）对胚胎发育的影响,在解剖显微镜下实时观察斑马鱼胚胎发育的全过程.采用Western 印迹法检测乙酰化的微管蛋白——α-微管蛋白（acetylated tubulin, α-tubulin）和神经元增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达,分别检测STS暴露后胚胎的运动神经元功能,神经元的增殖状态.发育中的斑马鱼胚胎暴露于0.1～1 mol/L STS,呈现出严重的发育迟缓,并且伴随多脏器畸形;暴露于10 μmol/L～10 mmol/L STS,胚胎呈现循环系统,神经系统以及颌面部畸形.胚胎在48 hpf (hours post fertilization)时,对STS的暴露敏感高于24 hpf和96 hpf.STS可能干扰细胞的增殖及运动神经元的正常分化.STS可能干扰正常的细胞骨架结构,并在胚胎发育晚期影响细胞增殖,对胚胎神经系统、循环系统及颌面部有致畸作用.     

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the “naked” embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24 h post-fertilization (hpf) with survival rates of ≥ 90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24 h of development. Dechorionation of younger stages (< 24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~ 400,000 Dalton, but becomes strongly toxic after dechorionation.     

1-苯基-2-硫脲对斑马鱼胚胎发育与黑色素生成的影响

目的1-苯基-2-硫脲（PTU）可抑制斑马鱼胚胎黑色素的产生,保持斑马鱼透明,便于形态观察和信号检测。本文研究了PTU对斑马鱼胚胎发育的影响和抑制斑马鱼胚胎黑色素生成,保持斑马鱼透明性的最佳浓度。方法用不同浓度PTU处理23hpf（受精后,hourspostfertilization,hpf）斑马鱼胚胎,作用57h后观察80hpf斑马鱼的形态学、生理学改变,计算死亡率和孵化率,测量心率和静脉窦-动脉球之间的距离。结果浓度为0．197mmo]／L、0．296mmol／LPTU可以有效抑制黑色素生成,保持斑马鱼整体透明,对斑马鱼心血管系统结构和生理功能无影响,且不影响斑马鱼正常孵化过程。随着PTU浓度的增加,斑马鱼死亡率增加,孵化率下降,出现心包水肿,心脏畸形等改变,心率下降,静脉窦-动脉球之间的距离增大。结论浓度不高于0．296mmol／L的PTU溶液能有效抑制斑马鱼黑色素生成,对斑马鱼心血管毒性研究无影响。     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。     

Tbx1基因功能下调影响斑马鱼Tbx20和Tbx2表达

目的采用吗啡啉修饰反义寡核苷酸显微注射方法下调斑马鱼Tbx1基因表达,研究斑马鱼Tbx1基因功能下调对其他两个T盒基因Tbx20和Tbx2表达的影响。方法采用吗啡啉修饰的反义寡核苷酸显微注射方法抑制斑马鱼Tbx1基因表达,分别将2.5、5、8、10 ng吗啡啉反义寡核苷酸在斑马鱼0-4细胞期注入胚胎,并构建Tbx20,骨形成蛋白2b（Bmp2b）和Tbx2反义RNA探针,进行整体原位杂交,观察Tbx1基因下调对Tbx20、Bmp2b及Tbx2表达的影响。结果Tbx1吗啡啉寡核苷酸显微注射组胚胎表现出鳃弓、耳囊、心血管系统和胸腺的发育异常。Tbx1基因下调导致Tbx20的表达出现改变,Tbx20在心脏的表达与对照组相比明显下调,神经元的表达范围明显缩小;Tbx1基因功能下调会导致Bmp2b在心脏和咽囊的表达减低,Bmp2b在后部咽囊的表达较前部咽囊减低得更为明显;Tbx1基因功能下调胚胎,Tbx2在鳃弓的表达模式发生改变,48 hpf,Tbx2在鳃弓的表达出现从后向前逐渐减低,鳃弓的表达范围较对照组明显缩小。结论Tbx1在发育过程中,会对其他T盒基因,如Tbx20和Tbx2具有激活或抑制的调控作用。Tbx1对Tbx20的作用可能是通过影响Bmp2b的途径,继发地影响Tbx20的表达。Tbx1基因功能下调,会改变Tbx2在鳃弓的表达模式。     

Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate intestine

The target of rapamycin (TOR) signaling pathway regulates cell growth and proliferation, however the extent to which TOR signaling mediates particular organogenesis programs remains to be determined. Here we report an examination of TOR signaling during zebrafish development, using a combination of small molecule treatment and morpholino-mediated gene knockdown. First, we amplified and sequenced the full-length cDNA for the zebrafish TOR ortholog (ztor). By in situ hybridization, we found that ztor is expressed ubiquitously in the early embryo, but displays a dynamic pattern in the gut between 48 and 72 h post-fertilization (hpf). Treatment of zebrafish embryos with rapamycin induced only a mild general developmental delay up to 72 hpf, but digestive tract development became arrested at the primitive gut tube stage. Rapamycin inhibited intestinal epithelial growth, morphogenesis and differentiation. Using morpholino-mediated gene knockdown of TOR pathway components, we show that this effect is mediated specifically by the rapamycin-sensitive TOR complex 1 (TORC1). Thus, in addition to regulating cell growth and proliferation, TOR signaling controls the developmental program guiding epithelial morphogenesis in the vertebrate intestine.     

Molecular cloning and expression of a second zebrafish aldehyde dehydrogenase 2 gene (aldh2b).

Aldehyde dehydrogenase 2 (ALDH2) is primarily responsible for detoxification of short-chain aldehydes in vivo. Previously it was reported that zebrafish has an aldh2 gene. Here we report the presence of a second aldh2 gene (aldh2b) in zebrafish. Zebrafish aldh2b locates adjacently to aldh2 on Chromosome 5 and the two genes share the same genomic organizations. aldh2b was predicted to encode a protein comprising 516 amino acids. The protein exhibits 95% amino acid identity with zebrafish ALDH2 and more than 76% identity with other vertebrate ALDH2s, respectively. Employing RT-PCR analysis, we demonstrated that both aldh2 and aldh2b mRNAs were present in embryos at cleavage stage (2 hpf: hour post fertilization) throughout protruding-mouth stage (72 hpf) and in different adult tissues of zebrafish. Taken together, our results reveal that zebrafish has two orthologues of aldh2 gene and the two genes share similar expression patterns during early development and in adult tissues.     

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 80-100 μm.Open in a separate windowClick here to view.^{(44M, flv)}