RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos

Short, hairpin RNA (shRNA) directed against bone morphogenetic protein 4 (Bmp-4) was delivered to early postimplantation staged mouse embryos via tail vein injection of pregnant dams. As early as 24 h postinjection, embryos expressed a DsRed marker and later exhibited defects of neural fold elevation and closure and of cardiac morphogenesis. Immunohistochemical analysis of sectioned embryos indicated that Bmp-4 protein was depleted and gene expression analysis indicated there was a reduction in Bmp-4 mRNA and an upregulation of the Bmp-4 antagonists, noggin and chordin, in embryos exposed to the shRNA, but not in control embryos. There was no change in the expression of Gata4, brachyury, or claudin6 in RNAi exposed embryos, indicating that RNA silencing was specific to Bmp-4 rather than producing widespread gene inhibition. Delivery of shRNA to embryos has the potential to specifically knockdown the expression of developmentally essential genes and to rescue gene mutations, significantly decreasing the time required to analyze the function(s) of individual genes in development.     

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.     

Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation

Mouse preimplantation development is characterized by three major transitions and two lineage segregations. Each transition or lineage segregation entails pronounced changes in the pattern of gene expression. Thus, research into the function of genes with obvious changes in expression pattern will shed light on the molecular basis of preimplantation development. We have described a simplified and effective method-electroporation-of introducing plasmid DNA and morpholinos into mouse preimplantation embryos and verified effectiveness of this approach by testing the procedure on the endogenous gene Oct4. Before electroporation, the zona pellucida was weakened by the treatment of acid Tyrode's solution. Then we optimized the parameters such as voltage, pulse duration, number of pulses and repeats, and applied these parameters to subsequent experiments. Compared with the control groups, the number of apoptotic cells and the expression and localization of OCT3/4 or CDX2 was not significantly changed in blastocysts developed from 1-cell embryos, which were electroporated with pIRES2-AcGFP1-Nuc eukaryotic expression vector or mismatched morpholino oligonucleotides. Furthermore, electroporated plasmid DNA and morpholinos targeting the endogenous gene Oct4 were able to sharply down regulate expression of OCT4 protein and actually cause expected phenotypes in mouse preimplantation embryos. In conclusion, plasmid DNA and morpholinos could be efficient delivered into mouse preimplantation embryos by electroporation and exert their functions, and normal development of preimplantation embryos was not affected.     

raldh2基因阻抑导致斑马鱼心脏发育畸形的机制

目的通过显微注射吗啡啉修饰的反义寡核苷酸(MO)阻抑视黄醛脱氢酶2(raldh2)基因表达,探讨raldh2基因阻抑对斑马鱼胚胎心脏发育的影响及可能的分子机制。方法根据斑马鱼raldh2基因起始密码区域序列设计合成吗啡啉修饰的反义寡核苷酸,采用显微注射方法阻抑斑马鱼胚胎raldh2基因表达。构建raldh2-EG-FP重组质粒进一步验证MO的特异性和有效性。分析raldh2基因阻抑后对胚胎发育,尤其心脏表型和功能的影响。通过胚胎整体原位杂交,分析心脏相关nppa和tbx20基因表达模式以及raldh2阻抑后对其表达的影响。结果显微注射raldh2-MO能有效地特异地阻抑斑马鱼胚胎raldh2基因表达,raldh2-MO对胚胎发育影响呈剂量依赖性。raldh2基因阻抑可导致胚胎心脏发育畸形,干扰正常的房室分化和向右环化,导致房室瓣血液反流。与野生型胚胎比较,raldh2基因阻抑组胚胎心率和心室收缩分数降低(P<0.05),心功能受损。整体原位杂交结果显示raldh2基因阻抑后nppa基因表达改变,心室部位nppa表达清晰,而心房部位表达减弱。tbx20基因在心脏、运动神经元、顶盖及视网膜表达,raldh2基因阻抑后,tbx20表达下调,在心脏表达减弱,以心房和流出道部位更显著。结论 raldh2基因在心脏早期发育的多个环节发挥重要作用,影响房室分化、心管环化和心肌收缩等。在心脏发育过程中nppa和tbx20基因表达受到raldh2基因调控,可能参与RA信号缺乏导致心脏畸形的潜在分子机制。     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos

Microtubules are the primary cytoskeletal constituent of extending neurites. We used antisense morpholinos to knock down expression of neuron-specific Class III beta tubulin in the right half of the neural tube of chicken embryos in ovo. There was a significant (p < 0.01) reduction in the number of Class III beta tubulin immunostained interneurons 24 h following electroporation of the morpholinos when compared with the contralateral side of the neural tube. However, neural crest-derived sensory neurons labeled with the fluorescently tagged morpholinos developed distinct processes. Moreover, there was no significant difference in the number of interneurons labeled on either side of the neural tube with a second marker of developing neurons, anti-microtubule associated protein (MAP) 1b. Neural tubes were also excised and dissociated following antisense or control morpholino electroporation. The resulting neurons were cultured for 48 h and immunostained with anti-Class III beta tubulin and anti-MAP 1b. Neurons that had taken up the antisense morpholino had significantly shorter neurites (p < 0.01) than neurons from the same neural tubes that did not; they also had significantly shorter neurites (p < 0.05) than labeled neurons from neural tubes electroporated with a control morpholino. Thus, normal expression of Class III beta tubulin may not be necessary for neurogenesis in the early avian spinal cord in situ, but is required for neurite outgrowth in vitro.     

Action of morpholinos in Ciona embryos

First, using morpholino against lacZ, we demonstrate that the morpholino specifically suppresses the translation of the gene introduced exogenously into Ciona eggs. Second, using morpholino against an alkaline phosphatase gene, we show that the morpholino suppresses the translation of the endogenous gene as well. Third, using morpholino against beta-catenin gene, we confirm that the suppression by the morpholino can be rescued by injection of beta-catenin mRNA. All of these results indicate that morpholino act in Ciona embryos to specifically block the function of endogenous genes as well as exogenously introduced genes. genesis 30: 103--106, 2001.     

昆虫RNAi技术及其应用

RNAi是近几年发展起来的抑制基因表达的新技术。部分昆虫存在RNAi信号的系统性传播现象,可以将dsRNA直接注射进昆虫的卵、血腔或局部组织,引发远距离靶基因的特异性沉默,建立起了Embryo RNAi,Larval RNAi,Adult RNAi,Parental RNAi,Feeding RNAi和基于转基因技术的可遗传RNAi等昆虫RNAi技术,使RNAi迅速成为了研究昆虫尤其是非模式昆虫基因功能的主要方法。文章拟就RNAi的系统性、昆虫RNAi技术及其应用进行综述。     

Zebrafish Bmp4 regulates left-right asymmetry at two distinct developmental time points

Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.     

DNA methylation,retroviruses, and embryogenesis

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or postimplantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfcctious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.     

Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.     

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.     

ankAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo

In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.     

Manipulation of gene expression in zebrafish using caged circular morpholino oligomers

Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their experimental applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.