Transgenic technology for visualization and manipulation of the neural circuits controlling behavior in zebrafish

The vertebrate brain is innately equipped with neural circuits that make quick behavioral decisions possible. Elucidating these neural circuits, determining how their master plans are encoded in the genome, and revealing how they can be modified by postnatal experiences will facilitate our understanding of how nature and nurture interact to establish an animal's behavior. In this review, we explain how transgenic zebrafish can cast insights into the developmental mechanisms and functional roles of the neural circuits that directly and indirectly control visuomotor behavior, by taking as an example a transgenic line Tg( brn3a-hsp70:GFP ) enabling visualization of the tectobulbar and habenulo-interpeduncular tracts. These insights emphasize the benefits of applying advanced transgenic technology in zebrafish to future research into this area.     

利用Tol2转座子构建斑马鱼心脏组织特异表达转基因载体及其表达分析

为了制备用于在斑马鱼心脏中特异表达目的基因的转基因载体,通过分子克隆的方法对能够在斑马鱼心脏中特异表达EGFP报告基因的Tol2载体进行了改造,在原有的CMLC2启动子与EGFP编码区之间插入带有多克隆位点的IRES序列,获得pTol2-CMLC2-IRES-EGFP转基因表达载体,该载体可以实现在同一个启动子CMLC2的驱动下分别同时表达目的基因和EGFP;为了验证该表达载体的有效性,进一步在CMLC2启动子与IRES序列之间插入DsRed-Monome编码区,利用得到的pTol2-CMLC2-RED-IRES-EGFP转基因载体显微注射到斑马鱼单细胞期胚胎中进行表达分析,结果表明外源目的基因DsRed-Monome和报告基因EGFP均能以相同的表达模式在斑马鱼心脏组织中特异表达。pTol2-CMLC2-IRES-EGFP转基因表达载体的成功构建对于建立心脏发育候选基因的斑马鱼转基因实验模型具有重要意义。     

In vivo synthesis of meganuclease for generating transgenic zebrafish Danio rerio

The authors show that co-injection at the one-cell stage of mRNA encoding a nuclear-targeted meganuclease I- Sce I together with expression cassettes flanked by cognate restriction sites results in efficient stable transgenesis in zebrafish Danio rerio .     

Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

心脏特异表达绿色荧光斑马鱼模型的建立与评估

本课题组前期研究中, 利用斑马鱼cmlc2 (Cardiac myosin light chain 2)基因启动子构建了一个用于斑马鱼心脏组织特异表达外源基因的转基因表达载体pTol2-cmlc2-IRES-EGFP。文章利用该载体构建了一个稳定表达EGFP的转基因斑马鱼品系, 并初步分析了EGFP的表达对该转基因斑马鱼品系的心脏发育和功能的影响。结果表明, 在建立的转基因斑马鱼品系早期胚胎发育过程中, 绿色荧光信号在心脏中特异表达, 该表达模式与原位杂交分析的cmlc2的表达模式结果相同; 该转基因斑马鱼品系的心脏形态及发育生长正常; 进一步通过M-Mode分析心脏生理学功能的结果表明：该转基因品系心动周期、心率、收缩与舒张表面积及表面积缩短率等重要生理指标与正常野生型的斑马鱼对照组相比没有显著差别。以上结果表明该转基因品系中绿色荧光蛋白的表达对斑马鱼心脏的发育和功能没有影响。研究结果为进一步利用该载体建立外源目的基因转基因表达模型, 研究心脏表达基因的功能奠定了重要基础。     

The evolution of tenascins and fibronectin

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.     

Gene transfer into zebrafish by sperm nuclear transplantation

A technique for fertilizing zebrafish eggs by injection of sperm nuclei is described. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults. If sperm nuclei are preincubated for 20 min with DNA encoding the green fluorescent protein, transgene expression can be detected in all cells of the embryo. The use of condensed sperm nuclei allows injection with a small bore pipette, which is critical for successful injection of the relatively small zebrafish egg. This technique enables the generation of ubiquitously expressing transgenic zebrafish directly by microinjection. Hence, experiments involving transgenic fish can be completed in days, without the need for growing and breeding founders. This technique may also be used to generate transgenic lines, as transgene expression was visible in the offspring of transgenic founders. The method described here is likely to be applicable to other teleosts, such as medaka and salmon.     

Direct and indirect requirements of Shh/Gli signaling in early pituitary development

Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation.     

从小鼠11.5d胚胎cDNA文库中筛选Dishevelled2相互作用蛋白

Dishevelled (Dvl)是个多功能、进化上非常保守的蛋白,在Wnt信号传导通路中起着重要的作用。为了研究Dishevelled介导Wnt信号传递的分子机制,利用GAL4酵母双杂交系统筛选了小鼠11．5d胚胎cDNA库,发现了15个可与小鼠Dvl2 DEP结构域和羧基端相互作用的蛋白质。将阳性库质粒测序并对测序结果做BLAST分析,发现其中一个阳性克隆是编码Gli3蛋白氨基端(6—122aa)的cDNA片段,从而暗示Gli3蛋白可能与Dishevelled一起作用并参与某些生物学过程。     

Lamination of the cerebral cortex is disturbed in Gli3 mutant mice

The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt^J/Pdn which lacks the cortical hem, a major source of reelin⁺ Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt^J/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin⁺ cells which are probably derived from an expanded Dbx1⁺ progenitor pool in the mutant. However, while these reelin⁺ neurons and also Calretinin⁺ cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.