Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

反义技术及其在胚胎异常发育机制研究中的应用

综述了反义研究中的几个关键问题,提出严格的ODNs设计,有效的ODNs转运和设计合理的对照等是获得良好实验结果的关键;介绍该技术在各种体外胚胎试验中的应用。     

Knocked down and out: PACAP in development, reproduction and feeding

One approach to understanding the role of PACAP in vivo is to knockdown the translation of PACAP mRNA to protein or to knock out the PACAP gene by targeted disruption. In this paper, we review the effect of PACAP knockdown with morpholinos on early brain development in zebrafish. Also reviewed is the role of PACAP at several stages of reproduction as assessed in mice with a disrupted PACAP gene. New data are presented to analyze PACAP's action in energy homeostasis (body mass, food intake, endocrine parameters) using female PACAP-null mice. The evidence suggests PACAP is important for brain development in zebrafish and is required for normal reproduction, but not for body mass or food intake in mice maintained near thermoneutrality.     

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.     

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.     

Antisense and ribozyme constructs in transgenic animals

Geneticists have long sought the ability to add or subtract individual genes from an organism's genome, or to be able to alter the level of expression of a gene in a targeted, developmentally and tissue-specific manner. The development of transgenic technology realized the possibilities of increasing the expression of a specific gene or the transfer of a new gene into an animal. Homologous recombination techniques allow the deletion or alteration of a genein vivo. The production of transgenic animals incorporating a gene construct that expresses a complimentary antisense RNA to a targeted gene, or an antisense RNA incorporating a catalytic, ribozyme sequence, have been suggested as a potential mechanism for obtaining the developmentally and tissue-specific down-regulation of expression of a targeted genein vivo. In this paper we review the current literature with respect to the application of antisense and ribozyme constructs in transgenic animals and conclude that such constructs can effectively down-regulate the level of mRNA from a target gene, the amount of protein produced in the cell, and result in phenotypic consequences.     

Peptide nucleic acid (PNA) antisense effects in Escherichia coli

Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as tools for microbial functional genomics.     

Use of fully modified 2'-O-methyl antisense oligos for loss-of-function studies in vertebrate embryos

Antisense oligonucleotides are commonly employed to study the roles of genes in development. Although morpholino phosphorodiamidate oligonucleotides (morpholinos) are widely used to block translation or splicing of target gene products' the usefulness of other modifications in mediating RNase-H independent inhibition of gene activity in embryos has not been investigated. In this study, we investigated the extent that fully modified 2'-O-methyl oligonucleotides (2'-OMe oligos) that can function as translation inhibiting reagents in vivo, using Xenopus and zebrafish embryos. We find that oligos against Xenopus β-catenin, wnt11, and bmp4 and against zebrafish chordin (chd), which can efficiently and specifically generate embryonic loss-of-function phenotypes comparable with morpholino injection and other methods. These results show that fully modified 2'-OMe oligos can function as RNase-H independent antisense reagents in vertebrate embryos and can thus serve as an alternative modification to morpholinos in some cases.     

Controlling morpholino experiments: don't stop making antisense

One of the most significant problems facing developmental biologists who do not work on an organism with well-developed genetics - and even for some who do - is how to inhibit the action of a gene of interest during development so as to learn about its normal biological function. A widely adopted approach is to use antisense technologies, and especially morpholino antisense oligonucleotides. In this article, we review the use of such reagents and present examples of how they have provided insights into developmental mechanisms. We also discuss how the use of morpholinos can lead to misleading results, including off-target effects, and we suggest controls that will allow researchers to interpret morpholino experiments correctly.     

Imprinting of the murine MAS protooncogene is restricted to its antisense RNA.

The Mas protooncogene encodes a G protein-coupled receptor with the common seven transmembrane domains and may be involved in the actions of angiotensins. The gene is located in close proximity to the paternally imprinted Igf2r gene and its maternal imprinting has been reported but remained controversial. We used mice carrying a targeted deletion of the Mas protooncogene on the maternal or paternal chromosome to clarify this issue. In all Mas-expressing organs of adult mice such as heart, kidney, testis or brain, no Mas mRNA was missing in heterozygous animals inheriting the deleted allele from the father excluding mono-allelic paternal expression. However, we show exclusive paternal expression of a Mas antisense RNA, confirming the maternal imprinting of this antisense RNA in all investigated adult tissues and in embryos. Our results strongly suggest that Mas is not imprinted in mice but that an antisense RNA probably starting in the neighboring Igf2r gene is maternally imprinted in both embryos and adult organs.     

Muscle development in Ciona intestinalis requires the b-HLH myogenic regulatory factor gene Ci-MRF

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.     

SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.     

PhotoMorphs™: A novel light‐activated reagent for controlling gene expression in zebrafish

Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly used antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light‐ activatable knockdown reagent called PhotoMorph?. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein, No tail, and E‐cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E‐cadherin knockdown. A splice‐blocking PhotoMorph directed to the rheb gene showed light‐dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. genesis 47:736–743, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of Müller glial cell division blocks regeneration of the light-damaged zebrafish retina

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.     

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, β-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.     

Antisense oligonucleotides as therapeutic agents.

Antisense oligonucleotides can block the expression of specific target genes involved in the development of human diseases. Therapeutic applications of antisense techniques are currently under investigation in many different fields. The use of antisense molecules to modify gene expression is variable in its efficacy and reliability, raising objections about their use as therapeutic agents. However, preliminary results of several clinical studies demonstrated the safety and to some extent the efficacy of antisense oligodeoxynucleotides (ODNs) in patients with malignant diseases. Clinical response was observed in some patients suffering from ovarian cancer who were treated with antisense targeted against the gene encoding for the protein kinase C-alpha. Some hematological diseases treated with antisense oligos targeted against the bcr/abl and the bcl2 mRNAs have shown promising clinical response. Antisense therapy has been useful in the treatment of cardiovascular disorders such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Antisense oligonucleotides also have shown promise as antiviral agents. Several investigators are performing trials with oligonucleotides targeted against the human immunodeficiency virus-1 (HIV-1) and hepatitis viruses. Phosphorothioate ODNs now have reached phase I and II in clinical trials for the treatment of cancer and viral infections, so far demonstrating an acceptable safety and pharmacokinetic profile for continuing their development. The new drug Vitravene, based on a phosphorothioate oligonucleotide designed to inhibit the human cytomegalovirus (CMV), promises that some substantial successes can be reached with the antisense technique.     

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.