Technical Advancement

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1 INVITED ARTICLE Acta Histochem. Cytochem. 35 (5): , 2002 Technical Advancement Using Antisense Morpholino Oligos to Knockdown Gene Expression in the Chicken Embryo Richard P. Tucker 1 1 Department of Cell Biology and Human Anatomy, University of California at Davis, School of Medicine, 1 Shields Avenue, Davis CA , USA Received September 20, 2002; accepted October 7, 2002 Morpholinos are synthetic DNA analogs that are not degraded by nucleases. A technique has been developed for the microinjection and square-pulse electroporation of antisense morpholinos into the precursors of neural crest cells in the chicken embryo in ovo. This paper reviews the methods used for the introduction of morpholinos into the chicken embryo as well as how to analyze their effects on gene expression and morphogenesis. Key words: Antisense, Morpholino, Electroporation, Chicken, Tenascin-C I. Introduction The knockout of specific genes in the mouse by homologous recombination is a powerful tool for studying the roles of specific gene products in development, homeostasis and disease. However, many mice with a gene disrupted by this method either develop normally or die prior to implantation, complicating the analysis of gene function during specific development events. In recent years, antisense strategies with DNA or RNA probes have proven useful in some systems as an alternative approach. RNAi, for example, can be used in Drosophila, C. elegans and other invertebrates to target specific genes [4]. In addition, antisense DNA oligonucleotides can be directly microinjected into Xenopus oocytes [11] or introduced via a slow-release polymer to chicken embryos in ovo [8]. Unfortunately, applications of RNAi to vertebrate models have met with mixed results, and issues of toxicity and in vivo degradation limit the use of antisense DNA oligonucleotides. Recently, synthetic DNA analogs made with phosphorodiamidate-linked morpholine rings replacing the sugar-phosphate backbone of nucleic acids have been used successfully to knockdown the translation of target mrnas in zebrafish, Xenopus, tunicates, sea urchin and mouse (see [6], for review). The principal advantage of using antisense morpholino oligos ( morpholinos ) over traditional or even modified oligonucleotides is their longevity: as they are not targeted by degradation by nucleases, they can remain functional in the cytoplasm for days. Morpholinos are also less toxic than other antisense oligos. During the 18 months since the first descriptions of their use in Xenopus [5] and zebrafish [9], over sixty genes have been reported to be successfully knocked down with morpholinos. Many of these reports can be found in Volume 30 (Issue 3) of the journal Genesis. Morpholinos are introduced into zebrafish and Xenopus embryos by microinjection into single-celled embryos or blastomeres. Though efficient for organisms that develop externally, this approach is not practical for studies of amniotes. To introduce morpholinos into chicken embryos we microinject labeled oligos into the lumen of the neural tube and then drive them into surrounding neuroepithelial cells using square-pulse electroporation. This paper will concentrate on the techniques used to introduce morpholinos into chicken embryos and analyze the embryos for the knockdown of the target transcript. To illustrate the method, we will show how this technique was used to knockdown the expression of the extracellular matrix glycoprotein tenascin-c in migrating neural crest cells [14]. Correspondence to: Richard P. Tucker, Department of Cell Biology and Human Anatomy, University of California at Davis, 1 Shields Avenue, Davis, CA , USA. 361 II. Morpholinos Custom morpholinos are designed by and purchased

2 362 Tucker from GeneTools, LLC. Typical morpholinos are bases long and are complimentary to sequences straddling the start codon of the target transcript or the untranslated leader sequences just 5' to the start codon. The uncharged morpholinos are labeled with either fluorescein (green) or lissamine (red) to permit monitoring of the microinjection and uptake following electroporation. Morpholinos are diluted in sterile water to make a stock solution of 1 2 mm. Further dilutions are made in sterile PBS. Aliquots of the stock solution are stored at 20 C, though morpholinos remain potent even after prolonged storage at 4 C. III. Microinjection and Electroporation Fertile chicken eggs are incubated on their sides for 42 to 48 hr, when most of the embryos are between Hamburger and Hamilton [3] stage Two to three ml of albumen is withdrawn from the narrow pole of the egg with a syringe, and the hole made by the needle is closed with transparent tape. This draws the embryo, which is resting on top of the yolk, away from the inner surface of the shell. Forceps are then used to make a small opening in the shell to expose the embryo, which is clearly visible after injecting a small volume of ink into the subblastodermic space (Fig. 1A). An opening is then made in the vitelline embryo using fine needles. Approximately nl of morpholino is micro- Fig. 1. A: A chicken embryo at stage 14 prepared for microinjection by subblastodermal injection of ink and removal of the vitelline envelope. The cut border of the vitelline envelope is indicated with arrows. B: The fluorescent morpholino (arrow) is readily detectable with fiber optics following microinjection into the lumen of the neural tube. C: Gold-plated electrodes placed on either side of the embryo. Note the bubbles that have formed around the electrode to the right following the administration of the current. D: The fluorescent morpholino can be seen in half of the cervical neural tube 24 hr after microinjection and electroporation. E: A section through a control embryo stained with an antibody to tenascin-c (adapted from [14]). The neural crest staging area is bounded by the neural tube (nt), somite (s) and ectoderm (e). F: A section through an embryo with tenascin-c morpholino stained with anti-tenascin-c. Note the reduction of immunostaining in the vicinity of the morpholino. Cells in the staging area are indicated by the arrow. G: A similarly treated embryo stained with an antibody to cellular fibronectin. The tenascin-c morpholino does not affect the anti-fibronectin immunostaining pattern. H: The same section shown in G showing the morpholino in the neural tube as well as in cells in the staging area (arrow).

3 Antisense Morpholinos in the Chicken Embryo 363 injected into the lumen of the neural tube just rostral to the level of the segmental plate using a fine glass micropipette (Fig. 1B). The amount of morpholino that remains in the lumen of the neural tube varies from embryo to embryo depending on the amount of reflux through the hole made in the roof plate. This variability necessitates both the analysis of many embryos (at least 10) as well as the microinjection of a range of concentrations of morpholinos. Up to 1 mm of control morpholino (e.g. sense sequence) has no effect on normal development, and the fluorescent tag of morpholinos diluted to 0.01 mm can still be detected in tissue sections. The best results have been obtained using morpholinos in the 1 mm to 0.5 mm range. Following the microinjection, gold-plated electrodes spaced approximately 1 mm from each other are gently lowered onto the surface of the embryo on either side of the neural tube (Fig. 1C). We typically use three 100 msec square pulses of 9 12 V to deliver the morpholino into the neuroepithelium. The electrodes are then lifted away, the hole in the shell is sealed with transparent tape, and the egg is returned to the incubator for 18 to 48 hr. IV. Histochemistry After an appropriate period of incubation the embryo is removed from the egg and fixed in cold 4% paraformaldehyde in PBS for 3 hr to overnight. The success of the uptake of the morpholino can be determined in the fixed embryos using either a 4 objective on an upright fluorescence microscope or by using a dissecting microscope fitted with epiillumination optics (Fig. 1D). Uptake of the morpholino is usually limited to the side of the embryo where the positive electrode is placed. If this is not desired, the poles of the electrodes can be alternated during the electroporation. Fixed embryos can then be processed either for routine paraffin embedding and sectioning or frozen for cryosectioning. The fluorescent tag on the morpholino is still present after sectioning, and appropriate sections can be selected and processed for immunohistochemistry. Primary antibodies used to generate the images in this review were an anti-tenascin- C monoclonal antibody (M1 [2]), anti-cellular fibronectin (Sigma, product number F-6140), and the monoclonal antibody neural crest cell marker HNK-1 [12]. Since we used fluorescein-labeled morpholinos, a red secondary antibody was used for the immunohistochemistry (goat anti-mouse TRITC, Accurate Chemical and Scientific). V. Discussion Figure 1E shows a cross section through a stage-18 control (electroporation only) embryo after immunostaining with a tenascin-c monoclonal antibody. Tenascin-C immunoreactivity is seen in the neural crest staging area bounded dorsally by the ectoderm, laterally by the somites and medially by the neural tube. In a similar section through a stage- 18 embryo 24 hours after electroporation of the tenascin-c antisense morpholino, tenascin-c immunoreactivity is reduced in the staging area (Fig. 1F). Immunostaining with an antibody to a second extracellular matrix glycoprotein known to interact with tenascin-c, fibronectin, is unchanged (Fig. 1G, H). These results demonstrate that the morpholinos are readily detectable 24 hr following electroporation and that the morpholinos specifically reduce the immunostaining of the target protein. Neural crest cells make tenascin-c shortly after leaving the dorsal neural tube and as they migrate ventrally through the staging area and into the anterior half of each somite [13]. To determine if the synthesis of tenascin-c by the neural crest cells is necessary for their dispersal from the neural tube, sections of embryos electroporated with either 1 mm of control morpholino or with 0.5 mm of tenascin-c morpholino were immunostained with the neural crest cell marker HNK-1. HNK-1-positive cells are seen streaming from the neural tube in control embryos (Fig. 2A). These cells also contain the fluorescent control morpholino (Fig. 2B, C). In contrast, the neural crest cells of 14 out of 19 embryos with tenascin-c morpholino migrated abnormally (Fig. 2D). In most of the embryos with abnormally migrating neural crest cells, HNK-1-positive cells are seen in the staging area, but are not seen migrating ventrally into the somites. In some embryos HNK-1 positive cells were seen in the lumen of the neural tube, and in others HNK-1 cells were seen in both the lumen and within the neuroepithelium. These phenotypes resemble those reported by others [1] who microinjected in ovo a tenascin-c monoclonal antibody into the pathway followed by cranial neural crest cells. The effect of the morpholino on migration was also seen after 48 hr (Fig. 2E, F). The neural crest-derived dorsal root ganglia are smaller on the side of the embryo where the tenascin-c morpholinos (only barely visible at this time point) were taken up. These results indicate that control morpholinos do not effect neural crest cell migration, that neural crest cells must make tenascin-c to migrate normally, and that morpholinos can still be detected 48 hr after electroporation. Note that in addition to our study with tenascin-c morpholinos [14], others have shown that neural crest cells migrate precociously into the space between the ectoderm and somites following knockdown of the transcription factor FoxD3 with morpholinos [7]. When homologous recombination was used to knockout the tenascin-c gene from a mouse, the animals developed without any characteristic neural crest-related defects [10]. Why do we see abnormally migrating neural crest cells in chicken embryos following knockdown of tenascin-c expression? One likely explanation is the spatiotemporal specificity of the knockdown with morpholinos. When a gene is functionally removed by homologous recombination, the embryo may respond by altering the levels of expression of compensatory genes. In the mouse, this could mean the expression of the closely related gene tenascin-w, or it could mean the downregulation of genes that work with tenascin-c like fibronectin. By introducing morpholinos into a specific population of cells just before they make the target transcript, the embryo may be less likely to compensate for the knockdown and the function of the target

4 364 Tucker Fig. 2. A: A section through an embryo following electroporation of a control morpholino immunostained with HNK-1, a neural crest cell marker. Note the neural crest cells streaming through the staging area and into the somite. nt, neural tube; s, somite; e, ectoderm. B: The same section shown in A viewed to reveal the fluorescent control morpholino. C: Merged images showing the morpholino in neural crest cells in the somite (arrows). D: After 24 hr the neural crest cells migrate normally from the left-side of the neural tube, but not on the right where the tenascin- C morpholino was taken up. nt, neural tube; s, somite; e, ectoderm. E: After 48 hr the dorsal root ganglia form normally on the control side of the embryo. F: On the experimental side, the fluorescent morpholino is barely visible after 48 hr. The dorsal root ganglion (drg) on the experimental side is smaller than on the control side, indicating that fewer neural crest cells migrated from the neural tube following uptake of the tenascin-c morpholino. gene can be analyzed. Microinjection and square-pulse electroporation of morpholinos may prove to be a useful technique for those wishing to knockdown gene expression in tissues bordering canals or vesicles where the morpholinos can be introduced. In addition to the central nervous system, future targets could include the lens, otic vesical, branchial arches, heart and blood vessel walls. VI. References 1. Bronner-Fraser, M. (1988) Distribution and function of tenascin during cranial neural crest development in the chick. J. Neurosci. Res. 21; Chiquet, M. and Fambrough, D. M. (1984) Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis. J. Cell Biol. 98; Hamburger, V. and Hamilton, H. L. (1992) A series of normal stages in the development of the chick embryo Dev. Dyn. 195; Hannon, G. J. (2002) RNA interference. Nature 418; Heasman, J., Kofron, M. and Wylie, C. (2000) Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222; Heasman, J. (2002) Morpholino oligos: making sense of antisense? Dev. Biol. 243; Kos, R., Reedy, M. V., Johnson, R. L. and Erickson, C. A. (2001) The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 128; Morales, A. V. and de Pablo, F. (1992) Inhibition of gene expression by antisense oligonucleotides in chick embryos in vitro and in vivo. Curr. Top. Dev. Biol. 36; Nasevicius, A. and Ekker, S. C. (2000) Effective targeted gene knockdown in zebrafish. Nat. Genet. 26; Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. and Aizawa, S. (1992) Mice develop normally without tenascin. Genes Dev. 6;

5 Antisense Morpholinos in the Chicken Embryo Soreq, H. and Seidman, S. (1992) Xenopus oocyte microinjection: from gene to protein. Methods Enzymol. 207; Tucker, G. C., Aoyama, H., Lipinski, M., Tursz, T. and Thiery, J. P. (1984) Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes. Cell Differ. 14; Tucker, R. P. and McKay, S. E. (1991) The expression of tenascin by neural crest cells and glia. Development 112; Tucker, R. P. (2001) Abnormal neural crest cell migration after the in vivo knockdown of tenascin-c expression with morpholino antisense oligonucleotides. Dev. Dyn. 222;