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Methods for introducing morpholinos into the chicken embryo
Developmental Dynamics
Abstract
The use of antisense morpholino oligos to inhibit the translation of a target transcript has been applied recently to studies of the chicken embryo. In contrast to other developmental systems such as in frog, sea urchin, and zebrafish that permit the direct microinjection of morpholinos into a blastomere, square pulse electroporation is used to introduce fluorescently tagged morpholinos into specific populations of chick embryo cells in ovo. This article reviews the methods that have proven successful, the types of controls that are necessary when performing knockdowns of gene expression in the chick embryo, and discusses the limitations of the current technique, as well as directions for further research.
... Consequently, the latter molecule will act as a competitive inhibitor of the wild-type form and perturb the associated regulatory pathway (Akamatsu et al. 1999; Bartkowska et al. 2007, Barembaum andBronner-Fraser 2007). Another common strategy consists in blocking the transcription of the gene of interest, which is classically achieved through morpholino electroporation (Mellitzer et al. 2002;Kos et al. 2003). These nucleic acids are in fact antisense oligonucleotides which are either directed against the proximal region of the translation initiation site, to sterically inhibit the initiation complex, or designed to encompass the intron/exon boundaries and thus interfere with RNA splicing. ...
... Again in the nervous system, one has relied on the central canal of the spinal cord in chick (Pekarik et al. 2003) and Xenopus . Other examples include, at earlier stages, the proamniotic (Soares et al. 2008), the amniotic (Mellitzer et al. 2002), and the yolk sac (Giroux et al. 2007) cavities in post-implantation mouse embryos as well as the space between the vitelline membrane and the epiblast (Cui et al. 2006, Iimura and Pourquié 2008, Hatakeyama and Shimamura 2008, Muramatsu et al. 1996 Fig. 2a), the lumen of the neural tube (Kos et al. 2003;Krull 2004), and the somitocoels (Scaal et al. 2004) in avian embryos. Likewise, utilization has been made of the subretinal space in mouse pup (Donovan and Dyer 2006), of the rays of the caudal fin in zebrafish (Tawk et al. 2002), and of the hemocoel (Thomas 2003) and of the body cavity (Ando and Fujiwara 2013) in silkworm. ...
... On one hand, manipulating the electrodes allows the experimenter to easily adapt the protocol to individuals, which can be important when considering the non-negligible size and shape variability encountered in embryologyespecially for higher organisms or when a lack of accessibility makes it difficult to synchronize the experimental timing with the developmental one. On the other hand, adjusting the position of the electrodes can be tricky and thus time consuming (Kos et al. 2003;Krull 2004;Scaal et al. 2004). Furthermore, relying on a well-defined experimental device may be preferable since it enables some standardization, synonym of a better reproducibilitythis is especially the case when using microsystems that had been fabricated with a precision close to the micrometer (Fox et al. 2006;Geng and Lu 2013;Wang and Lee 2013; chapters "▶ Description by Electroporation in Microfluidic Devices" and "▶ Nanochannel Electroporation: Delivery of Precise Amounts of Biomolecules into Living Cells"). ...
During the formation of a complex organism, cells divide, die, migrate, and differentiate. Biologists have established tools to observe those phenomena but also to change their course, which subsequently enables to infer causal relationships between various events occurring in different cell groups. More precisely, present approaches mostly rely on modifications of gene expression. For instance, cells are labeled with fluorescent proteins and tracked within the embryo, molecular signals are switched on and off to perturb regulatory pathways. Importantly, in all those experiments, the exogenous genetic material must be delivered at the right place and with the appropriate timing: requirements that can both be fulfilled by electroporation. After 15 years of constant refinement, this technique has now superseded methods like viral infection, microinjection, and lipofection. Applications encompass a large number of model organisms, targeted anatomical structures, and molecular biology techniques.
... Electroporation serves to enhance uptake of morpholinos into chick embryonic cells [there is evidence that morpholinos alone can penetrate the plasma membrane and enter the cytosol, (Kos et al., 2003)] and to spatially target their application to a desired tissue. As standard nonlabeled morpholinos have no charge, researchers should either use Gene Tools charged morpholinos, consisting of nonionic morpholino/DNA heteroduplexes electrostatically bound to ethoxylated polyethylenimine or fluorescently tagged morpholinos (fluorescein-or lissamine-tagged). ...
... In addition to facilitating visualization of morpholino uptake, tagging also fortuitously introduces a weak charge by virtue of the tag (negative or positive, respectively). Lightly charged morpholinos have been shown to be more amenable to electroporation, even though some success has been reported with the electroporation of the standard noncharged morpholino into trunk neuroepithelial cells (Kos et al., 2001(Kos et al., , 2003. Although the exact mechanism is unknown, it is thought that the application of electric pulses to the cells temporarily disrupts the stability of the plasma cell membrane creating the transient pores through which the nucleic acid molecules (DNA or RNA) to be transected are transported into the cytosol. ...
... When morpholinos are fluorescently tagged, they are also visible upon injection, so the use of solution dyes such as vegetable dye (2% FD&C Blue in Ringer's solution) or Fast Green is unnecessary. Moreover the use of Fast Green in morpholino solutions has been reported to inhibit the uptake of the morpholinos into the cells (Kos et al., 2003). A recent recommendation by GeneTools specialist is to reconstitute the morpholino in nuclease free-water to a desired stock concentration of 1-2 mM, but to keep both stock solution and individual aliquots in a dry and obscure place at room temperature. ...
The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re‐established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001).
... Consequently, the latter molecule will act as a competitive inhibitor of the wild-type form and perturb the associated regulatory pathway (Akamatsu et al. 1999; Bartkowska et al. 2007, Barembaum andBronner-Fraser 2007). Another common strategy consists in blocking the transcription of the gene of interest, which is classically achieved through morpholino electroporation (Mellitzer et al. 2002;Kos et al. 2003). These nucleic acids are in fact antisense oligonucleotides which are either directed against the proximal region of the translation initiation site, to sterically inhibit the initiation complex, or designed to encompass the intron/exon boundaries and thus interfere with RNA splicing. ...
... Again in the nervous system, one has relied on the central canal of the spinal cord in chick (Pekarik et al. 2003) and Xenopus . Other examples include, at earlier stages, the proamniotic (Soares et al. 2008), the amniotic (Mellitzer et al. 2002), and the yolk sac (Giroux et al. 2007) cavities in post-implantation mouse embryos as well as the space between the vitelline membrane and the epiblast (Cui et al. 2006, Iimura and Pourquié 2008, Hatakeyama and Shimamura 2008, Muramatsu et al. 1996 Fig. 2a), the lumen of the neural tube (Kos et al. 2003;Krull 2004), and the somitocoels (Scaal et al. 2004) in avian embryos. Likewise, utilization has been made of the subretinal space in mouse pup (Donovan and Dyer 2006), of the rays of the caudal fin in zebrafish (Tawk et al. 2002), and of the hemocoel (Thomas 2003) and of the body cavity (Ando and Fujiwara 2013) in silkworm. ...
... On one hand, manipulating the electrodes allows the experimenter to easily adapt the protocol to individuals, which can be important when considering the non-negligible size and shape variability encountered in embryologyespecially for higher organisms or when a lack of accessibility makes it difficult to synchronize the experimental timing with the developmental one. On the other hand, adjusting the position of the electrodes can be tricky and thus time consuming (Kos et al. 2003;Krull 2004;Scaal et al. 2004). Furthermore, relying on a well-defined experimental device may be preferable since it enables some standardization, synonym of a better reproducibilitythis is especially the case when using microsystems that had been fabricated with a precision close to the micrometer (Fox et al. 2006;Geng and Lu 2013;Wang and Lee 2013; chapters "▶ Description by Electroporation in Microfluidic Devices" and "▶ Nanochannel Electroporation: Delivery of Precise Amounts of Biomolecules into Living Cells"). ...
During the formation of a complex organism, cells divide, die, migrate, and differentiate. Biologists have established tools to observe those phenomena but also to change their course, which subsequently enables to infer causal relationships between various events occurring in different cell groups. More precisely, present approaches mostly rely on modifications of gene expression. For instance, cells are labeled with fluorescent proteins and tracked within the embryo, molecular signals are switched on and off to perturb regulatory pathways. Importantly, in all those experiments, the exogenous genetic material must be delivered at the right place and with the appropriate timing: requirements that can both be fulfilled by electroporation. After 15 years of constant refinement, this technique has now superseded methods like viral infection, microinjection, and lipofection. Applications encompass a large number of model organisms, targeted anatomical structures, and molecular biology techniques.
... The ADAM10 morpholinos (ADAM10-mo) with the targeting sequence of 59-ggattatcgtcctcgctagatccat-39 and the standard negative-control morpholinos (Fluo-mo) of 59-cctcttacctcagttacaatttata-39 were purchased from the Gene Tools (Philomath, USA). This ADAM10-mo can specifically repress ADAM10 protein translation in chicken embryos [12,25]. Both morpholinos were labeled with fluorescein at the 39-end of the oligonucleotides in order to trace transfected cells after in vivo electroporation. ...
... The fluorescein labeled in the 39-end of ADAM10-mo and the GFP protein were used as reporter markers to trace the ADAM10-mo and dn-ADAM10-me transfected cells, respectively. Both AD-AM10-mo and dn-ADAM10-me have been identified to efficiently and specifically repress ADAM10 protein translation in chicken embryos [12,25]. The construct of dn-ADAM10-me, in which the metalloprotease active-site glutamic acid (E385) is changed to an alanine (A), can efficiently block the ADAM10 metalloprotease function [12,38]. ...
... When the control Fluo-mo was transfected alone at E4, the expression of the ADAM10 protein (red, Fig. 1B, C) between the transfected side (green, Fig. 1A, C) and the untransfected side (left side in Fig. 1A-D) at E6 was similar, although the fluorescein-positive (transfected) cells were observed in both the ventricular zone (arrows in Fig. 1A-D) and the mantle layer. In contrast, when ADAM10-mo was transfected, the expression of ADAM10 protein (red, Fig. 1F, G) in the transfected region (green, Fig. 1E, G) was decreased (arrows in Fig. 1F, G), when compared to the untransfected side (left side in Fig. 1E-H), suggesting that ADAM10-mo can efficiently downregulate AD-AM10 protein expression in vivo, consistent with the previous studies [12,25]. Interestingly, the prospective ventricular zone (arrow in Fig. 1H) disappeared when transfected with ADAM10mo (arrows in Fig. 1E-H). ...
Members of the ADAM (a disintegrin and metalloprotease) family are involved in embryogenesis and tissue formation via their proteolytic function, cell-cell and cell-matrix interactions. ADAM10 is expressed temporally and spatially in the developing chicken spinal cord, but its function remains elusive. In the present study, we address this question by electroporating ADAM10 specific morpholino antisense oligonucleotides (ADAM10-mo) or dominant-negative ADAM10 (dn-ADAM10) plasmid into the developing chicken spinal cord as well as by in vitro cell culture investigation. Our results show that downregulation of ADAM10 drives precocious differentiation of neural progenitor cells and radial glial cells, resulting in an increase of neurons in the developing spinal cord, even in the prospective ventricular zone. Remarkably, overexpression of the dn-ADAM10 plasmid mutated in the metalloprotease domain (dn-ADAM10-me) mimics the phenotype as found by the ADAM10-mo transfection. Furthermore, in vitro experiments on cultured cells demonstrate that downregulation of ADAM10 decreases the amount of the cleaved intracellular part of Notch1 receptor and its target, and increases the number of βIII-tubulin-positive cells during neural progenitor cell differentiation. Taken together, our data suggest that ADAM10 negatively regulates neuronal differentiation, possibly via its proteolytic effect on the Notch signaling during development of the spinal cord.
... This adaptation still allows binding of the complementary nucleic sequences by traditional Watson-Crick base pairing. Importantly, it has one major advantage over conventional antisense oligonucleotides: this backbone makes MOs completely resistant to nucleases [12][13][14] and, unlike other knockdown strategies, MOs do not depend on harnessing the cellular machinery, like the RNA-induced silencing complex and RNAse-H activity. Furthermore, since the backbone does not carry a negative charge, MOs are less likely to interact non-specifically with cellular proteins, and may therefore be less toxic [14]. ...
... In the chick embryo, electroporation is widely used for MO delivery, although recently a few other methods have been described [33,34]. For successful electroporation, MOs must be fluorescein-labelled since the negatively charged fluorescein at the 3' end appears to enhance electroporation [4,13]. The major advantage of fluorescein-labelled MOs is that electroporated cells can be visualised immediately after electroporation and can easily be compared with their wild-type neighbours. ...
... Successful electroporation is easily detected by fluorescence microscopy. It has been reported that MOs remain in neural tube cells for at least 48 hours after electroporation, with the intensity of the fluorescence being reduced at later time points [13]. We have not systematically tested this, and therefore cannot comment. ...
... Morpholinos have been used in developmental biology research, and particularly in embryonic chick studies, with great success [86,[119][120][121]. The most commonly used method to deliver MOs in live chicks in ovo is through electroporation. ...
... The concentration of the MO working solution needs to be determined experimentally as it's efficiency will vary depending on several factors, including the abundance of the target mRNA. Also, the addition of fast green to this solution is not recommended since it has been reported to inhibit the uptake of MOs [120]; however, addition of a contrast dye is not necessary when using MOs carrying a fluorescent tag, as they are easily visible, especially under blue light. The protocol for injecting MOs in the neural tube (ED1.5) or the eye (ED3-4) is similar to that described for RCAS virus injections (see above). ...
... Moreover, MO incorporation (and potentially the extent of phenotypic effect) may vary from embryo to embryo. This variability can be due to many factors such as the volume and concentration of the injected solution, backflow from the neuropore or the injection site, placement of the electrodes, etc. [120]. Therefore, it is good practice to analyze a reasonably large number of embryos, and if possible consider only those in which adequate MO incorporation or efficacy can be verified. ...
The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. The present article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.
... Stage 22 (E3.5) embryos were injected subretinally with morpholino and electroporated in ovo (Kos et al., 2003), and retinas were analyzed after 72 hr, corresponding to st30 (E6.5). The relative density of Lim1 þ cells was twice as high in the anti-Nolz1 morpholino-treated retinas (2.1 6 0.02; 1,351 cells counted; n ¼ 4), compared with the control (1.0 6 0.01; 535 cells counted; n ¼ 4; Fig. 3H-J). ...
... Morpholinos are DNA analogues that can block translation by binding to the 5 0 -UTR, and are often used in zebrafish, and have also been used in chicken embryos (Kos et al., 2001). Although the morpholino in itself is not charged, fluorescein-tagged morpholinos may be electroporated owing to the negatively charged fluorescein tag (Kos et al., 2003). A total of 300 nmol morpholino oligo (Gene Tools, LLC, Philomath, OR) was diluted to 1 mM in distilled water. ...
Background:
The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1 and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We therefore studied Nolz1 during retinal development.
Results:
Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells.
Conclusions:
We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation. This article is protected by copyright. All rights reserved.
... However, nonspecific effects including activation and loss of unrelated transcripts have been reported in particular in young chick embryos (Mende et al., 2008) demonstrating the critical importance of appropriate controls (see below). Antisense morpholinos provide a good alternative, especially for early embryos, and result in reproducible and reliable gene inactivation (Basch et al., 2006; Christophorou et al., 2010; Kos et al., 2003; Mende et al., 2008; Sheng et al., 2003; Strobl-Mazzulla et al., 2010; Voiculescu et al., 2008). Antisense morpholinos target either the translation start site to interfere with the initiation complex or splice junctions resulting in exon deletion or intron inclusion. ...
... Identification of genes downstream of the Shh signalling in the developing chick wing and synexpressed with Hoxd13 using microarray and 3D computational analysis. Mech Dev 127:428–441.Table 1 Summary of Perturbation Experiments as Described in the Text Morpholino MOs complementary to the transcription start site block translation; MOs complementary to splice junctions result in exon deletion or intron inclusion Basch et al., 2006; Christophorou et al., 2010; Kos et al., 2003; Mende et al., 2008; Sheng et al., 2003; Strobl-Mazzulla et al., 2010; Voiculescu et al., 2008 Small ...
... However, nonspecific effects including activation and loss of unrelated transcripts have been reported in particular in young chick embryos (Mende et al., 2008) demonstrating the critical importance of appropriate controls (see below). Antisense morpholinos provide a good alternative, especially for early embryos, and result in reproducible and reliable gene inactivation (Basch et al., 2006; Christophorou et al., 2010; Kos et al., 2003; Mende et al., 2008; Sheng et al., 2003; Strobl-Mazzulla et al., 2010; Voiculescu et al., 2008). Antisense morpholinos target either the translation start site to interfere with the initiation complex or splice junctions resulting in exon deletion or intron inclusion. ...
... Identification of genes downstream of the Shh signalling in the developing chick wing and synexpressed with Hoxd13 using microarray and 3D computational analysis. Mech Dev 127:428–441.Table 1 Summary of Perturbation Experiments as Described in the Text Morpholino MOs complementary to the transcription start site block translation; MOs complementary to splice junctions result in exon deletion or intron inclusion Basch et al., 2006; Christophorou et al., 2010; Kos et al., 2003; Mende et al., 2008; Sheng et al., 2003; Strobl-Mazzulla et al., 2010; Voiculescu et al., 2008 Small ...
... Antisense morpholinos provide a good alternative, especially for early embryos, and result in reproducible and reliable gene inactivation (Basch et al., 2006;Christophorou et al., 2010;Kos et al., 2003;Mende et al., 2008;Sheng et al., 2003;Strobl-Mazzulla et al., 2010;Voiculescu et al., 2008). Antisense morpholinos target either the translation start site to interfere with the initiation complex or splice junctions resulting in exon deletion or intron inclusion. ...
... This work is funded by the BBSRC and Deafness Research UK. Basch et al., 2006;Christophorou et al., 2010;Kos et al., 2003;Mende et al., 2008;Sheng et al., 2003;Strobl-Mazzulla et al., 2010;Voiculescu et al., 2008 Small interfering RNA (siRNA) RNA degradation Bron et al., 2004;Das et al., 2006;Hu et al., 2002;Katahira and Nakamura, 2003 Misexpression Ectopic expression of the gene of interest by electroporation or transfection; grafting beads or transfected cells for signaling molecules Nakamura et al., 2004 Dominant negative forms of TFs, receptors ...
Setting up the body plan during embryonic development requires the coordinated action of many signals and transcriptional regulators in a precise temporal sequence and spatial pattern. The last decades have seen an explosion of information describing the molecular control of many developmental processes. The next challenge is to integrate this information into logic ‘wiring diagrams' that visualise gene actions and outputs, have predictive power and point to key control nodes. Here we provide an experimental workflow on how to construct gene regulatory networks using the chick as model system. © 2012 Wiley Periodicals, Inc.
... Antisense technology is a useful tool for specific gene silencing during development and has been applied in many species (Audic et al, 2001; Coonrod et al, 2001; Howard et al, 2001; Yang et al, 2001; Kos et al, 2003). Chemical modification of oligonucleotides has improved their stability and therefore increased their applicability in vivo. ...
... As an alternative , viral vector-based expression systems were developed to express dominant-negative proteins (Morgan and Fekete, 1996; Logan and Tabin, 1998). Morpholinos were also used successfully in chicken embryos (Kos et al, 2003; Tucker, 2004), although they may be more difficult to target to specific tissues than regular oligonucleotides (Krull, 2004). More recently, in ovo electroporation as an efficient method of gene transfer in chicken embryos for the temporally and spatially controlled ectopic expression of a gene of interest was established (Table 2; Muramatsu et al, 1997; Momose et al, 1999; Nakamura and Funahashi, 2001; reviewed in Bourikas and Stoeckli, 2003). ...
Loss-of-function approaches are important tools for functional gene analysis. Due to the availability of sophisticated methods to manipulate gene expression in embryonic stem cells that can be used to generate mutant mice, the mouse is by far the most important vertebrate model organism for basic and applied biomedical research. Unfortunately, the available methods do not allow for precise temporal and spatial control of gene silencing during embryonic development limiting the usefulness of the mouse for developmental studies. Due to their easy accessibility chicken embryos have been one of the preferred model organisms for developmental studies. Their disadvantage, the lack of genetic tools, could be overcome by the development of in ovo RNAi (in ovo RNA interference), a method that allows for temporal and spatial control of gene silencing in vivo.
... Morpholinos were first applied to chicken embryos by Erickson and colleagues and have now been extensively used to knockdown gene expression [12,13]. Morpholinos are short oligo chains of about 25 bp, with each subunit containing a morpholine ring with a nucleic acid base [14]. ...
The polarity of cellular components is essential for cellular shape changes, oriented cell migration, and modulating intra- and intercellular mechanical forces. However, many aspects of polarized cell behavior-especially dynamic cell shape changes during the process of morphogenesis-are almost impossible to study in cells cultured in plastic dishes. Avian embryos have always been a treasured model system to study vertebrate morphogenesis for developmental biologists. Avian embryos recapitulate human biology particularly well in the early stages due to their flat disc gastruloids. Since avian embryos can be manipulated in ovo they present paramount opportunities for highly localized targeting of genetic mechanisms during cellular and developmental processes. Here, we review the application of these methods for both gain of function and loss of function of a gene of interest at a specific developmental stage during left-right (LR) asymmetric gut morphogenesis. These tools present a powerful premise to investigate various polarized cellular activities and molecular processes in vivo in a reproducible manner.
... Several DNA constructs containing a robust chicken β-actin promoter, a CMV promoter, an internal ribosome entry site (IRES), and a bicistronic reporter with green fluorescent protein (GFP) have been widely adopted including pMES, pCIG, and pCAβ (Swartz et al., 2001;Megason and McMahon, 2002;McLarren et al., 2003;Sauka-Spengler and Barembaum, 2008;Jhingory et al., 2010;Hall et al., 2014;Yang et al., 2014;Gammill et al., 2019;Wu and Taneyhill, 2019). Electroporation can also efficiently enable gene repression using RNA interference (RNAi) and antisense morpholino oligonucleotides (Tucker, 2001;Kos et al., 2003;Chesnutt and Niswander, 2004;Krull, 2004;Nakamura et al., 2004;Rao et al., 2004;Das et al., 2006;Sauka-Spengler and Barembaum, 2008;Gammill et al., 2019). However, due to the extrachromosomal nature of these vectors such treatments are only transient since plasmids and short oligonucleotides degrade and dilute following the proliferation of transfected cells, and misexpression is almost entirely eliminated by 72 to 96 h (Sauka-Spengler and Barembaum, 2008;Wang et al., 2011;Hall et al., 2014;Bourgeois et al., 2015). ...
Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.
... Electroporation has become a standard method for delivery of Morpholinos into chick embryos (Kos et al., 2003), especially for studies of neural tube development (Tucker, 2004). Electroporation has also been used to deliver Morpholinos into other embryos including mice (Mellitzer et al., 2002), into brains of developing rats (Takahashi et al., 2002), into zebrafish (Cerda et al., 2006), into clipped fins of zebrafish (Thummel et al., 2006), and into cell cultures (Jubin, 2005). ...
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis—nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.
... Disaturated phospholipids and vascular endothelial growth factor were used as parameters of lung function at different stages of chicken embryo development, and a critical window of vulnerability to oxygen tension was established (Been et al., 2010). Another relevant example is surfactant protein B, which is expressed in chicken lungs from Nunoya et al., 1983 Evaluation of anticonvulsant drugs (Loscher, 1984;Johnson et al., 1985) and how seizures alter the development of the brain (Gong et al., 2003) Obese strain of chicken Studies of autoimmune thyroiditis as described in Wick, Sundick et al., 1974 andDietrich et al., 1999 Neuroendocrine alterations caused by thyroiditis (Schauenstein et al., 1987) Gain and loss of function Sauka-Spengler and Barembaum, 2008Krull, 2004 Gain of function In ovo electroporation Nakamura et al., 2004 Overexpression of genes in Purkinje cells (Luo and Redies, 2004) Tbx4 and Fgf10 in lung bud formation (Sakiyama et al., 2003) Retroviral vector Sato, 2004 Wnt5a in pulmonary development (Loscertales et al., 2008) Loss of function RNAi gene silencing Andermatt and Stoeckli, 2014 Axonin/TAG-1 in cerebellar development (Baeriswyl and Stoeckli, 2008) Morpholinos Kos et al., 2003 Transcription factor FoxD3 in the development of the neural crest (Kos et al., 2001) MRI, magnetic resonance imaging; PET, positron emission tomography; RNAi, RNA interference. (Bjornstad et al., 2014). ...
The advance of perinatal medicine has improved the survival of extremely premature babies, thereby creating a new and heterogeneous patient group with limited information on appropriate treatment regimens. The developing fetus and neonate have traditionally been ignored populations with regard to safety studies of drugs, making medication during pregnancy and of newborns a significant safety concern. Recent initiatives of the FDA and EMA have been passed with the objective of expanding the safe pharmacological treatment options in these patients. There is a consensus that neonates should be included in clinical trials. Prior to these trials, drug leads are tested in toxicity and pharmacology studies as governed by several guidelines summarized in the multidisciplinary ICH M3 (R2). Pharmacology studies must be performed in the major organ systems: cardiovascular, the respiratory and the CNS. The chicken embryo and fetus have features that make it a convenient animal model for non-clinical safety studies where effects on all these organ systems can be tested. The developing chicken is inexpensive, accessible and nutritionally self-sufficient with a short incubation time and is ideal for drug screening purposes. Other high throughput models have been implemented. However, many of these have limitations, including difficulty in mimicking natural tissue architecture and function (human stem cells) and obvious differences from mammals in the respiratory organ system and certain aspects of CNS development (C. elegans, zebrafish).This minireview outlines the potential and limitations of the developing chicken as an additional model for the early exploratory phase of development of new pharmaceuticals.
... There have been a raft of articles discussing in ovo electroporation and the best parameters to use to enhance survival and increase transfection efficiency (Muramatsu et al., 1997;Itasaki et al., 1999;Nakamura et al., 2000;Yasuda et al., 2000;Yasugi et al., 2000;Nakamura et al., 2001;Swartz et al., 2001;Katahira et al., 2003;Chen et al., 2004;Krull, 2004;Nakamura et al., 2004;Uchikawa et al., 2004;Sato et al., 2007). Several investigators have also determined how to introduce various types of interfering molecules into the developing chick using electroporation or viruses, making knock-down experiments feasible (Hu et al., 2002;Katahira et al., 2003;Kos et al., 2003;Pekarik et al., 2003;Chesnutt et al., 2004;Rao et al., 2004;Hernandez et al., 2005;Canto-Soler and Adler, 2006;Harpavat and Cepko, 2006;Watanabe et al., 2007). The use of electroporation has several advantages over the use of viruses to introduce DNA, such as there is no longer a need to clone sequences into the retroviral plasmid, no size restriction on insertions to the expression vector, no need to expend the effort and funds in making a viral stock with which to infect tissues, and no need to purchase the more expensive virus-free eggs. ...
The chick provides an excellent system to explore cell and molecular events during retina development and regeneration. Chick embryos are wonderful to work with in a variety of aspects. The retina can regenerate during early development. The embryonic chick can regenerate its retina via two modes. One requires the activation of stem/progenitor cells present in the ciliary margin, while the other involves the use of the classic process of transdifferentiation. One of the great advantages of working with the embryonic chick eye is that the retina can be repaired or replaced if damaged or removed. The accessibility of the embryo for microsurgery combined with the availability of molecular tools in the chick has made this a great system to study and dissect the early molecular events that take place during retina regeneration. The chick genome was also recently sequenced, and this provides a vast range of possibilities to study the early stages of retina regeneration, including the use of gene array technology to identify critical genes regulated during chick retina regeneration.
... Despite these limitations continued advances in mosaic transgenesis are beginning to establish the avian as a bonafide model in which genetic gain and loss of functions can be routinely preformed. Gain and loss of function studies in the avian embryo mirror techniques used in other model systems, with examples including construct based over expression of functional proteins, expression of dominant negative proteins, and introduction of morpholinos or shRNA for gene knockdown (Chen et al., 2004;Chesnutt and Niswander, 2004;Das et al., 2006;Itasaki et al., 1999;Katahira and Nakamura, 2003;Kos et al., 2003;Krull, 2004;Luo and Redies, 2004;Nakamura et al., 2004;Ogura, 2002;Swartz et al., 2001). What is unique in the avian system, however, is the ability to focally introduce genetic manipulations only at specific regions of the embryo during very precise developmental stages. ...
For more than 2,000 years, philosophers and scientists have turned to the avian embryo with questions of how life begins (Aristotle and Peck Generations of Animals. Loeb Classics, vol. XIII. Harvard University Press, Cambridge, 1943; Needham, A history of embryology. Abelard-Schuman, New York, 1959). Then, as now, the unique accessibility of the embryo both in terms of acquisition of eggs from domesticated fowl and ease at which the embryo can be visualized by simply opening the shell has made avians an appealing and powerful model system for the study of development. Thus, as the field of embryology has evolved through observational, comparative, and experimental embryology into its current iteration as the cellular and molecular biology of development, avians have remained a useful and practical system of study.
... Morpholino injection methods were first described by transfecting DNA [16][17][18][19][20] and then used in chick embryo with square pulse electroporation [21][22][23]. This method was adopted by regeneration research for adult axolotl [24] and adult zebrafish in fin regeneration studies [25,26]. ...
Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and other in vitro systems ultimately need to demonstrate proof in an in vivo model. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe an in vivo zebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4-6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.
... Electroporation has been applied to fish (Chen et al. 2009) and ascidian embryos (Corbo et al. 1997), and to the regenerating tissue (Thummel et al. 2006;Mochii et al. 2007). antisense oligonucleotide was adopted (Kos et al. 2003;Sugiyama & Nakamura 2003). It must be noted that morpholino antisense oligonucleotide interferes with translation, and consequently we need an antibody against the molecule to evaluate the results precisely. ...
Gene transfer by electroporation has become an indispensable method for the study of developmental biology. The technique is applied not only in chick embryos but also in mice and other organisms. Here, a short history and perspectives of electroporation for gene transfer in vertebrates are described.
... Expression of transgenes for avian vascular studies has been successfully achieved by direct DNA electroporation[11], replication incompetent retrovirus or adenovirus[23, 24], replication competent virus[25], lipofection[26, 27] and misexpression from implanted mammalian cell aggregates [11, 28] although the ability to directly apply peptides provides a simpler method than gene-based misexpression293031. Whereas gene-specific attenuation at the RNA level can be achieved through avian misexpression methods using RNAi or electroporation of modified oligonucleotides (morpholinos) [32, 33], these methods are less effective and reliable as mouse gene deletion technology. The most notable advance in avian vascular biology has been the development of live-embryo videography in conjunction with vascularspecific Tie1-GFP transgenic quail lines to directly observe amniote vascular cell movements[34, 35]. ...
Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog.
... Electroporation. Electroporation has become a standard method for delivery of Morpholinos into chick embryos (Kos et al., 2003), especially for studies of neural tube development (Tucker, 2004). Electroporation has also been used to deliver Morpholinos into other embryos including mice (Mellitzer et al., 2002), into brains of developing rats (Takahashi et al., 2002), into zebrafish (Cerda et al., 2006), into clipped fins of zebrafish (Thummel et al., 2005), and into cell cultures (Jubin, 2005). ...
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and maturation of miRNA, although they can also inhibit other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.
... While these techniques are powerful and useful for certain types of experiments (i.e., whole-embryo treatment with inhibitors, or targeted temporary inhibition or misexpression of ligands) the ability to perform more refined molecular manipulations-such as molecular misexpression using constitutively active promoters, molecular misexpression of dominant negatives, RNAi and morpholino-mediated translation inhibition-would revolutionize oviparous chondrichthyans as an evolutionary developmental model system. All of these molecular manipulations have been successfully performed in chicken-another oviparous, nongenetic model system-using microinjection and in ovo electroporation to transfect cells with DNA constructs (Eberhart et al., 2004), dsRNA (Pekarik et al., 2003), and morpholinos (Kos et al., 2003). Electroporation of EGFP reporter constructs has also been used to test tissue specificity of enhancers and noncoding regions in the chick (Uchikawa, 2008). ...
Alcian blue skeletal preparations of wild-type (front) and retinoic acid-treated (back) embryos of the little skate, Leucoraja erinacea. As in paired fins, the cartilaginous gill rays of L. erinacea are patterned by a retinoic acid-regulated Sonic hedghog (Shh)-Fibroblast growth factor 8 (Fgf8) feedback loop, and exposure to exogenous retinoic acid induces ectopic Shh expression and mirror-image gill ray duplications. (Cover design by Kalliopi Monoyios and Randy Dahn). See the review by Gillis and Shubin in this issue.
... Alternatively, candidate gene expression could theoretically be down-regulated, using RNA antisense technology in ovo (Kos et al., 2003) or in isolated gonads grown in vitro. RNA interference is now being widely used for specific gene knockdown in vertebrate cells and tissues, and in whole embryos (Pekarik et al., 2003). ...
... Fertile White Leghorn chicken eggs from the UC Davis Animal Science Department were incubated in a humidified 37°C incubator until they reached Hamburger and Hamilton (HH) stages 14-16 (Hamburger and Hamilton, 1992). mem-EGFP DNA (1-3 μg/μl) was injected into the lumen of the neural tube, and embryos were electroporated at the axial level of the segmental plate using a Gentronics BTX-T820 square wave generator with five 50-millisecond square pulses at 30 V (Itasaki et al., 1999;Kos et al., 2003). The other half of the neural tube was labeled by reversing the position of the electrodes and electroporating with one 50-millisecond pulse at 30 V. For co-electroporation studies, mCherry-tubulin (0.35 μg/μl) was coelectroporated with either GFP-actin (0.12 μg/μl), GFP-α-catenin (0.35 μg/μl) or γ-tubulin-GFP (0.2 μg/μl). ...
An epithelial-mesenchymal transition (EMT) is the process whereby epithelial cells become mesenchymal cells, and is typified by the generation of neural crest cells from the neuroepithelium of the dorsal neural tube. To investigate the neural crest EMT, we performed live cell confocal time-lapse imaging to determine the sequence of cellular events and the role of cell division in the EMT. It was observed that in most EMTs, the apical cell tail is retracted cleanly from the lumen of the neuroepithelium, followed by movement of the cell body out of the neural tube. However, exceptions to this sequence include the rupture of the neural crest cell tail during retraction (junctional complexes not completely downregulated), or translocation of the cell body away from the apical surface while morphologically rounded up in M phase (no cell tail retraction event). We also noted that cell tail retraction can occur either before or after the redistribution of apical-basolateral epithelial polarity markers. Surprisingly, we discovered that when an EMT was preceded by a mitotic event, the plane of cytokinesis does not predict neural crest cell fate. Moreover, when daughter cells are separated from the adherens junctions by a parallel mitotic cleavage furrow, most re-establish contact with the apical surface. The diversity of cellular mechanisms by which neural crest cells can separate from the neural tube suggests that the EMT program is a complex network of non-linear mechanisms that can occur in multiple orders and combinations to allow neural crest cells to escape from the neuroepithelium.
... Currently, many molecular manipulation techniques are available for the mis-expression of genes in avian systems. These include the use of replication-competent avian sarcoma (RCAS) retroviruses (Hughes, 2004), electroporation (Itasaki et al., 1999;Krull, 2004;Nakamura et al., 2004), and siRNA and antisense oligonucleotides for gene repression (Kos et al., 2003;Rao et al., 2004). A fortuitous characteristic of RCAS vectors is their apparent inability to spread across basement membranes, thereby confining mis-expression of genes specifically to mesenchyme or epithelium . ...
Avian embryos, which have been studied scientifically since Aristotle, continue to persevere as invaluable research tools, especially for our understanding of the development and evolution of the craniofacial skeleton. Whether the topic is beak shape in Darwin's finches or signaling interactions that underlie bone and tooth formation, birds offer advantages for craniofacial biology that uniquely complement the strengths of other vertebrate model systems, such as fish, frogs, and mice. Several papers published during the past few years have helped pinpoint molecular and cellular mechanisms that pattern the face and jaws through experiments that could only have been done together with our feathered friends. Ultimately, such knowledge will be essential for devising novel clinical approaches to treat and/or prevent diseases, injuries, and birth defects that affect the human craniofacial skeleton. Here we review recent insights plucked from avians on key developmental processes that generate craniofacial diversity.
... Trunk neuroepithelium was transiently transfected with experimental or control vectors (Table 2) by electroporation (Kos et al., 2003) and incubated for 18-72 hours. Expression vectors producing c-KIT, EDNRB2, EphB2 and control short hairpin RNA (shRNA,Table 1) were constructed using the pSilencer 1.0-U6 siRNA Expression Vector (Ambion) or the siSTRIKEhMGFP U6 Hairpin Cloning System (Promega). ...
Neural crest cells that become pigment cells migrate along a dorsolateral route between the ectoderm and the somite, whereas most other neural crest cells are inhibited from entering this space. This pathway choice has been attributed to unique, cell-autonomous migratory properties acquired by neural crest cells when they become specified as melanoblasts. By shRNA knockdown and overexpression experiments, we investigated the roles of three transmembrane receptors in regulating dorsolateral pathfinding in the chick trunk. We show that Endothelin receptor B2 (EDNRB2) and EphB2 are both determinants in this process, and that, unlike in other species, c-KIT is not. We demonstrate that the overexpression of EDNRB2 can maintain normal dorsolateral migration of melanoblasts in the absence of EphB2, and vice versa, suggesting that changes in receptor expression levels regulate the invasion of this pathway. Furthermore, by heterotopic grafting, we show that neural crest cell populations that do not rely on the activation of these receptors can migrate dorsolaterally only if this path is free of inhibitory molecules. We conclude that the requirement for EDNRB2 and EphB2 expression by melanoblasts is to support their migration by helping them to overcome repulsive or non-permissive cues in the dorsolateral environment.
... For loss-of-function, electroporation of morpholinos (Kos et al., 2003;Papanayotou et al., 2008;Sheng et al., 2003;Tucker, 2001;Voiculescu et al., 2007) or expression of dominant negative DNA constructs (Becker et al., 2001;Bel-Vialar et al., 2002;Chen and Cepko, 2002) have both been successful. Recently, methods for vector-based RNA interference have been described, requiring small amounts of DNA to be introduced into tissues, while allowing long lasting and stable expression 0925-4773/$ -see front matter Ó 2008 Elsevier Ireland Ltd. ...
In the chick embryo, two methods are now used for studying the developmental role of genes by loss-of-function approaches: vector-based shRNA and morpholino oligonucleotides. Both have the advantage that loss-of-function can be conducted in a spatially and temporally controlled way by focal electroporation. Here, we compare these two methods. We find that the shRNA expressing vectors pRFPRNAi, even when targeting a non-expressed protein like GFP, cause morphological phenotypes, mis-regulation of non-targeted genes and activation of the p53 pathway. These effects are highly reproducible, appear to be independent of the targeting sequence and are particularly severe at primitive streak and early somite stages. By contrast, morpholinos do not cause these effects. We propose that pRFPRNAi should only be used with considerable caution and that morpholinos are a preferable approach for gene knock-down during early chick development.
Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.
Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.
Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-β) in chicken embryo fibroblast (DF-1) cells.
Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-β proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-β PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.
Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log 10 increase in viral production was observed in anti-IFN-α and IFN-β PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log 10 increase in virus titre when treated with anti-IFN-α and IFN-β PPMO compared to virus titre when treated with JAK/STAT inhibitors.
Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.
The chicken embryo has historically been a key animal model to investigate the mechanisms of inner ear development. With the ongoing progress in methods for genetic manipulation of avian embryos, and in particular the advent of CRISPR/Cas9 technology, a wide range of approaches are now available to investigate gene function in the chicken inner ear. In this chapter, we provide a standard protocol for in ovo electroporation of the inner ear and discuss the advantages and limitations of the genetic methods available for gain- and loss-of-function studies in the embryonic chicken inner ear.
This book provides a comprehensive overview of the development of therapeutic oligonucleotides for therapeutic applications, touching on a number of additional oligonucleotides including a number of small interfering RNAs currently in various phases of clinical development. Written by leading expert scientists from both academia and leading biotechnical companies, the authors provide a compelling update on current status of RNA interference with emphasis on fascinating topics including oligonucleotides: antisense oligonucleotides, ribozymes, siRNAs, decoy oligonucleotides and aptamers. This exceptional work will be a valid resource for researchers and students as well as academia, consultants and scientists.
The paper reports the synthesis of a series of antisense oligonucleotides (aONs) directed against different segments of the influenza A virus genome (H1N1) and screening of their antiviral activity in the influenza A virus (H1N1)/MDCK cells and influenza A virus (H1N1)/A549 cells systems. The results of screening have shown that PB1-2AUG-R aON targeted towards the AUG codon region of the segment 2 of virus genome possessed the highest antiviral activity. The synthesized morpholino analog (PMO) and the new phosphoryl guanidine oligodeoxyribonucleotide (PGO) with the sequence of oligonucleotide PB1- 2AUG-R provided a comparable biological effects: the influenza virus titer in MDCK cell culture was
reduced by 15 times compared to the control when PGO was used in the concentration of 10 μM and by 40 times when PMO was used in the concentration of 20 μM. For the delivery of electrically neutral analogs of oligonucleotides (PGO and PMO), the perforation method proposed for PMO was used.
Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.
To study gene function in vivo during development of the central nervous system (CNS) efficient model systems that allow for temporal and spatial control of gene expression are required. In our lab, we have used the chicken embryo in combination with in ovo electroporation and RNA interference (RNAi) for gene silencing during early stages of nervous system development. In particular, we used dorsal commissural neurons to study axonal pathfinding. These neurons extend their axons toward the floor plate, the ventral midline of the spinal cord. Guidance cues derived from the floor plate, the intermediate target of these axons, are important for axon growth toward and across the midline. Using in ovo RNAi, we could show that interference with the function of the transmembrane glycoprotein Endoglycan (PODXL2) resulted in the failure to turn or in erroneous caudal turns after midline crossing. Furthermore, the morphology of the floor plate was severely disturbed in the absence of Endoglycan. During later stages of neural development, Endoglycan is expressed by Purkinje cells in the cerebellum. For the functional characterization of Endoglycan during cerebellar development, we extended the experimental accessibility of chicken embryos to much older stages. We established the procedure of ex ovo electroporation and RNAi to manipulate gene expression in the developing cerebellum. The cerebellum represents a well characterized neuronal structure, and therefore, an ideal system to study CNS development, including neurogenesis, differentiation, migration, axon guidance, as well as synapse formation. To demonstrate the applicability and the efficiency of ex ovo RNAi, we analyzed the function of the cell adhesion molecule Axonin-1 in cerebellar development. Axonin-1 is expressed by postmitotic granule cells at the time when they extend their axons, the parallel fibers. In the absence of Axonin-1 the arrangement of granule cell axons within the molecular layer was aberrant and fibers no longer extended parallel but towards the cerebellar surface. The effect of Axonin-1 was not on parallel fiber elongation but affected specifically parallel fiber navigation. The same effects on parallel fiber development observed after ex ovo RNAi were reproduced in embryos treated with function-blocking anti-Axonin-1 antibodies, indicating that ex ovo RNAi efficiently and reproducibly silenced axonin-1 in the developing cerebellum. Thus, we used ex ovo RNAi to study the function of Endoglycan during cerebellar development. Endoglycan is expressed by Purkinje cells at the time when they migrate towards the pial surface to establish the Purkinje cell monolayer. Interference with Endoglycan function caused a severe migration defect of Purkinje cells. Moreover, due to the aberrant formation of the Purkinje cell monolayer, the thickness of the external germinal layer and the proliferation rate of granule cells were significantly reduced causing disturbed foliation and reduced size of the cerebellum. Für die Analyse von Genfunktionen während der Entwicklung des zentralen Nervensystems (ZNS) werden effiziente Systeme benötigt, welche es ermöglichen, die Genexpression zeitlich und räumlich zu kontrollieren. In unserem Labor benutzen wir in ovo RNAi (RNA interference), um im Hühnerembryo während früher neuraler Entwicklungsstadien Gene gezielt auszuschalten. Anhand dorsaler Kommissuralneurone studieren wir Wegweisermoleküle, welche Axone zu ihren Zielzellen dirigieren. Axone der Kommissuralneurone wachsen gegen die ventrale Mittelline des Rückenmarks, die Bodenplatte, überqueren diese und wachsen anschliessend in rostraler Richtung weiter. Wegweisermoleküle, welche direkt auf der Bodenplatte lokalisiert sind oder von dieser sezerniert werden, sind wichtig für die korrekte Wegfindung dieser Axone. Mittels in ovo RNAi konnten wir zeigen, dass das Transmembran-Glykoprotein Endoglycan (PODXL2) nach dem Überqueren der Bodenplatte für die anschliessende rostrale Drehung von Kommissuralaxonen wichtig ist. Der Verlust von Endoglycan-Aktivität führte zu Stillstehen am Ausgang der Bodenplatte und fehlerhaftem caudalen Wachsen von Kommissuralaxonen. Das Blockieren von Endoglycan führte ausserdem zu einer Veränderung der Morphologie der Bodenplatte. Während späterer Entwicklungsstadien wird Endoglycan von migrierenden Purkinje- Zellen des Kleinhirns (Cerebellum) exprimiert. Um die Funktion von Endoglycan im Kleinhirn zu untersuchen, haben wir mit ex ovo RNAi eine Methode entwickelt, welche das gezielte Hemmen einzelner Gene während fortgeschrittener Stadien der Entwicklung des Nervensystems ermöglicht. Die neuronale Struktur des Kleinhirns ist gut charakterisiert und daher ein ideales System um verschiedene Entwicklungsschritte des Nervensystems zu studieren. Um die Anwendbarkeit und Effizienz von ex ovo RNAi auf unsere Fragestellung zu testen, haben wir zunächst die Funktion des Zelladhäsionsmoleküls Axonin-1 im Kleinhirn untersucht. Während des Auswachsens der Axone, den so genannten Parallelfasern, exprimieren Körnerzellen Axonin-1. Der Verlust von Axonin-1-Aktivität hat zur Folge, dass die Organisation der Parallelfasern in der Molekularschicht des Kleinhirns defekt ist. Dieser Defekt beruht auf der fehleraften Navigation von Parallelfasern. Den gleichen Effekt auf die Entwicklung der Parallelfasern konnten wir mittels Injektionen von funktionsblockierenden Antikörpern erzielen. Dies deutet darauf hin, dass ex ovo RNAi effizient und reproduzierbar Axonin-1-Aktivität im Kleinhirn hemmen kann. Daher verwendeten wir diese Methode für die Untersuchung von Endoglycan im Kleinhirn. Dieses Gen wird von Purkinje-Zellen während ihrer radialen Migration in Richtung Oberfläche des Kleinhirns exprimiert. Das Fehlen von Endoglycan verursachte einen Migrationsdefekt der Purkinje-Zellen und eine anormale Ausbildung der Purkinje-Zellschicht. Dies wiederum führte zu einer verminderten Dicke der Körnerzellschicht bedingt durch eine Reduktion der Zellteilungsrate.
In ovo‐electroporation is a well‐established method to introduce transgenes into a number of tissues in chicken embryos, e.g. neural tissue, limb mesenchyme and somites. This method has been widely used to investigate cell lineage, cell morphology and molecular pathways by localized expression of fluorescent reporter constructs. Furthermore gain‐ and loss‐of‐function experiments can be performed by electroporating transgenes or gene silencing constructs.
We have developed a new technique to electroporate tissues positioned oppositely to each other with different plasmids using an electroporation chamber. As proof of principle, we electroporated the dorsal surface ectoderm with a reporter construct expressing mCherry and the subjacent somites with a reporter construct expressing EGFP. This double electroporation technique allows investigating the localization of two different proteins of interest in two adjacent tissues and will be useful to examine the cellular and molecular interaction of neighboring structures during embryonic development.
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The origin of animals or metazoans from their unicellular ancestors is one of the most important evolutionary transitions in the history of life. To decipher the molecular mechanisms involved in this transition, it is crucial to understand both the early evolution of animals and their unicellular prehistory. Recent phylogenomic analyses have shown that there are at least three distinct unicellular or colonial lineages closely related to metazoans: choanoflagellates, ichthyosporeans and filastereans. However, until recently, choanoflagellates had been the only lineage for which an entire genome sequence was available. Moreover, the lack of transgenesis tools in any of these unicellular lineages had precluded the possibility of performing functional analyses. To better understand the unicellular prehistory of animals, we have recently obtained the genome sequences of both filastereans and ichthyosporeans. Analyses of their genomes identified many important genes for metazoan multicellularity and development, some of which are absent from the choanoflagellate genomes and thus were thought to be metazoan-specific. We have also established methods for transgenesis and gene silencing in ichthyosporeans. The combination of genomic information and molecular tools in filastereans and ichthyosporeans facilitate efficient functional analyses to understand how the key genes in the evolution of multicellularity were co-opted during the unicellular-tomulticellular transition that gave rise to metazoans. We propose that filastereans and ichthyosporeans are ideal model organisms for investigating the origin of metazoan multicellularity.
Purpose: The chicken embryo lens is a classical model system for developmental and cell biology studies. To understand the molecular mechanisms that underlie the morphological changes that occur during lens development, it is important to develop an effective gene transfer method that permits the analysis of gene functions in vivo. In ovo electroporation has been successfully used for introducing DNA into neural and mesenchymal tissues of chicken embryos. In this study, we explored the possibility of using this technique to manipulate gene expression in lens epithelial and fiber cells, as well as in other cells of the chicken eye. Methods: Two DNA constructs were used in this study. pCAX contains a chicken beta-actin promoter fused to the CMV IE enhancer to drive enhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses the same chimeric promoter to drive the expression of the chicken neurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. Plasmid DNA was injected into the lumen of the lens vesicle in chicken embryos at stage 15. For corneal epithelial and retinal cell electroporation, DNA was placed near the surface ectoderm in the eye region or injected into the vitreous cavity, respectively. Electroporation was performed with one electrode above the eye and the other underneath the head of the embryo. Chicken embryos were harvested at different time points for EGFP expression analysis by immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used to evaluate the effects of cNf2 on lens epithelial cell proliferation. Results: A strong EGFP signal can be detected in lens cells 4 h after electroporation. The transfected cells maintain high levels of EGFP expression for at least 5 days. Overexpressing cNf2 in lens epithelial cells significantly inhibits cell proliferation. Ectopic expression of EGFP in corneal epithelial and retinal cells was also achieved by in ovo electroporation. Conclusions: We have demonstrated that exogenous DNA can be effectively introduced into lens, corneal and retinal cells in the living embryo by in ovo electroporation. In comparison to viral infection and transgenic mouse approaches, in ovo electroporation offers an easier and quicker way to manipulate gene expression during embryonic development. This technique will be a useful tool for exploring the molecular mechanisms of lens and eye development.
This chapter describes the use of avian species as model organisms for research in telomere biology and aging. Cellular or replicative senescence is often utilized as a model for the aging process because of the hypothesis that cellular aging recapitulates organismal aging. A genomic alteration associated with cellular or replicative senescence in a variety of organisms, including the chicken, is the shortening of telomeres. Telomere arrays have been examined in a wide sampling of avian species, including chicken, using fluorescence in situ hybridization (FISH). A PCR-based technique that can be used to compare the abundance of telomere repeats is quantitative real-time PCR (Q-PCR). This technique quantifies the fold difference between telomere-repeat copy number in an experimental sample compared to a reference DNA sample. Real-time fluorescence-based PCR and RT-PCR have emerged as powerful methods for examining gene expression patterns in many contexts. In traditional PCR, an amplicon, which accumulates after a predetermined number of cycles, is analyzed by gel electrophoresis.
Transcriptome analysis by RNA-seq has emerged as a high-throughput, cost-effective means to evaluate the expression pattern of genes in organisms. Unlike other methods, such as microarrays or quantitative PCR, RNAseq is a target-free method that permits analysis of essentially any RNA that can be amplified from a cell or tissue. At its most basic, RNA-seq can determine individual gene expression levels by counting the number of times a particular transcript was found in the sequence data. Transcript levels can be compared across multiple samples to identify differentially expressed genes and infer differences in biological states between the samples. We have used this approach to examine gene expression patterns in chicken and human cells, with particular interest in determining response to heat stress.
High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species...
Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them.
Contents
79 The Chicken Leads the Way in Avian Genomics.
Prepared by J. Smith.
80 The Chicken Genome: Current Status of Genome Assembly and Annotations.
Prepared by D.W. Burt, L. Eöry, A.L. Archibald, B.L. Aken, P. Flicek, K. Howe, W. Chow, M. Dunn, J.M.D. Wood, R. Nag, and W.C. Warren.
83 The Avian RNAseq Consortium: A Community Effort to Annotate the Chicken Genome.
Prepared by J. Smith, D.W. Burt, and the Avian RNAseq Consortium.
89 Noncoding RNAs in the Chicken Genome.
Prepared by J. Hertel, M. Fasold, A. Nitsche, I. Erb, P. Prieto, D. Kedra, C. Notredame, T.E. Steeves, P.P. Gardner, and P.F. Stadler.
91 Genome Sequencing in Birds and Evolutionary Inferences from Avian Genome Sequences.
Prepared by H. Ellegren.
94 The Use of Avian BAC Libraries and Clones.
Prepared by M.N. Romanov and D.K. Griffin.
96 Comparative Genomics.
Prepared by D.M. Larkin, M. Farré, and J. Damas.
100 Avian Cytogenetics Goes Functional.
Prepared by D.K. Griffin, M.N. Romanov, R. O’Connor, K.E. Fowler, and D.M. Larkin.
105 Hypermethylated Chromosome Regions in Chicken and Other Birds.
Prepared by M. Schmid, C. Steinlein, A.-S. Schneider, I. Nanda, and T. Haaf.
109 An Overview of Avian Evolution.
Prepared by S.B. Hedges.
114 An Update on Chicken Sex Determination and Gonadal Sex Differentiation.
Prepared by C.A. Smith.
119 Avian Epigenetics.
Prepared by H. Zhou.
122 Structural Variation and Copy Number Variation in Poultry.
Prepared by R.P.M.A. Crooijmans and M.A.M. Groenen.
124 SNPs and InDels – The Most Abundant Sources of Genetic Variations.
Prepared by A.A. Gheyas, C. Boschiero, and D.W. Burt.
130 Genetic Diversity of Village Chickens.
Prepared by T.T. Desta, R.A. Lawal, and O. Hanotte.
133 Mendelian Traits.
Prepared by D. Wragg.
137 Treasure the Exceptions: Utilizing Chicken Mutant Lines and Advanced Genetic Technologies to Uncover Genes Involved in Developmental Processes.
Prepared by E.A. O’Hare and M.E. Delany.
141 Genomic Landscape of the Chicken DT40 Cell Line.
Prepared by A. Motegi and M. Takata.
145 RNA-seq: Primary Cells, Cell Lines and Heat Stress.
Prepared by C.J. Schmidt, E.M. Pritchett, L. Sun, R.V.N. Davis, A. Hubbard, K.E. Kniel, S.M. Markland, Q. Wang, C. Ashwell, M. Persia, M.F. Rothschild, and S.J. Lamont.
148 Host-Viral Genome Interactions in Marek’s Disease.
Prepared by M.C. McPherson, C.M. Robinson, and M.E. Delany.
154 Transcriptome Variation in Response to Marek’s Disease Virus Acute Infection.
Prepared by L. Preeyanon, C.T. Brown, and H.H. Cheng.
163 The National Avian Research Facility.
Prepared by A. Hart, R. Kuo, L. Eöry, P. Kaiser, and D.W. Burt.
] The chick embryo has prevailed as one of the major models to study developmental biology, cell biology and regeneration. From all the anatomical features of the chick embryo, the eye is one of the most studied. In the chick embryo, the eye develops between 26 and 33 h after incubation (Stages 8-9, Hamburger and Hamilton, 1951). It originates from the posterior region of the forebrain, called the diencephalon. However, the vertebrate eye includes tissues from different origins including surface ectoderm (lens and cornea), anterior neural plate (retina, iris, ciliary body and retinal pigmented epithelium) and neural crest/head mesoderm (stroma of the iris and of the ciliary body as well as choroid, sclera and part of the cornea). After gastrulation, a single eye field originates from the anterior neural plate and is characterized by the expression of eye field transcriptional factors (EFTFs) that orchestrate the program for eye development. Later in development, the eye field separates in two and the optic vesicles form. After several inductive interactions with the lens placode, the optic cup forms. At Stages 14-15, the outer layer of the optic cup becomes the retinal pigmented epithelium (RPE) while the inner layer forms the neuroepithelium that eventually differentiates into the retina. One main advantage of the chick embryo, is the possibility to perform experiments to over-express or to down-regulate gene expression in a place and time specific manner to explore gene function and regulation. The aim of this protocol is to describe the electroporation techniques at Stages 8-12 (anterior neural fold and optic vesicle stages) and Stages 19-26 (eye cup, RPE and neuroepithelium). We provide a full description of the equipment, materials and electrode set up as well as a detailed description of the highly reproducible protocol including some representative results. This protocol has been adapted from our previous publications Luz-Madrigal et al. (2014) and Zhu et al. (2014).
The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos(1). Different structures such as DNA plasmids encoding genes(2-4), small interfering RNA (siRNA) plasmids(5), small synthetic RNA oligos(6), and morpholino antisense oligonucleotides(7) can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20--according to Hamburg and Hamilton)(8) and there are some disadvantages for its application in embryos at later stages (older than stage HH22--approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell. In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes(9) and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos(10-12).
Antisense morpholino oligonucleotides (MOs) have become a valuable method to knock down protein levels, to block mRNA splicing, and to interfere with miRNA function. MOs are widely used to alter gene expression during development of Xenopus and zebra fish, where they are typically injected into the fertilized egg or blastomeres. Here, we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells in the brain with current passing between two platinum plate electrodes, positioned on either side of the target brain area. The method is straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell-type specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.
The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Birth Defects Research (Part C) 99:121-133, 2013. © 2013 Wiley Periodicals, Inc.
General IntroductionNovel Techniques to Investigate NC Development and FateMechanisms of Specification and Emigration of NC CellsPeripheral Neuronal Lineages:Pluripotentiality and Early Restrictions of Migratory NCMolecular Control of Neuron Development in Peripheral GangliaGrowth Cone Navigation and Innervation of Peripheral TargetsFactors Controlling Neuronal Survival in the PNSGlial Cell Development from the NC-Peripheral Glial Lineages: Fate Choice and Early Developmental EventsSignals that Control Schwann Cell-precursor Survival and Schwann Cell Generation
Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.
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.
We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X–XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321–337; stages 1–10, Hamburger and Hamilton [1951] J Morphol 88:49–92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320–331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures. © 2001 Wiley-Liss, Inc.
We describe a simple method of chick whole-embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar-albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre-primitive streak stages to stage 10 (stages X–XIV, Eyal-Giladi and Kochav [1976] Dev Biol 49:321–337; stages 1–10, Hamburger and Hamilton [1951] J Morphol 88:49–92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320–331). Our modification of New's method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal-side up (in contrast to New's method) or ventral-side up, as desired; embryos younger than stage 8 can be culture only ventral-side up (as with New's method). We also discuss some alternative methods for setting up these cultures. © 2001 Wiley-Liss, Inc.
Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.
The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.
The winged-helix or forkhead class of transcription factors has been shown to play important roles in cell specification and lineage segregation. We have cloned the chicken homolog of FoxD3, a member of the winged-helix class of transcription factors, and analyzed its expression. Based on its expression in the dorsal neural tube and in all neural crest lineages except the late-emigrating melanoblasts, we predicted that FoxD3 might be important in the segregation of the neural crest lineage from the neural epithelium, and for repressing melanogenesis in early-migrating neural crest cells. Misexpression of FoxD3 by electroporation in the lateral neural epithelium early in neural crest development produced an expansion of HNK1 immunoreactivity throughout the neural epithelium, although these cells did not undergo an epithelial/mesenchymal transformation. To test whether FoxD3 represses melanogenesis in early migrating neural crest cells, we knocked down expression in cultured neural crest with antisense oligonucleotides and in vivo by treatment with morpholino antisense oligonucleotides. Both experimental approaches resulted in an expansion of the melanoblast lineage, probably at the expense of neuronal and glial lineages. Conversely, persistent expression of FoxD3 in late-migrating neural crest cells using RCAS viruses resulted in the failure of melanoblasts to develop. We suggest that FoxD3 plays two important roles in neural crest development. First, it is involved in the segregation of the neural crest lineage from the neuroepithelium. Second, it represses melanogenesis, thereby allowing other neural crest derivatives to differentiate during the early stages of neural crest patterning.
The winged-helix or forkhead class of transcription factors has been shown to play important roles in cell specification and lineage segregation. We have cloned the chicken homolog of FoxD3, a member of the winged-helix class of transcription factors, and analyzed its expression. Based on its expression in the dorsal neural tube and in all neural crest lineages except the late-emigrating melanoblasts, we predicted that FoxD3 might be important in the segregation of the neural crest lineage from the neural epithelium, and for repressing melanogenesis in early-migrating neural crest cells.
Misexpression of FoxD3 by electroporation in the lateral neural epithelium early in neural crest development produced an expansion of HNK1 immunoreactivity throughout the neural epithelium, although these cells did not undergo an epithelial/mesenchymal transformation. To test whether FoxD3 represses melanogenesis in early migrating neural crest cells, we knocked down expression in cultured neural crest with antisense oligonucleotides and in vivo by treatment with morpholino antisense oligonucleotides. Both experimental approaches resulted in an expansion of the melanoblast lineage, probably at the expense of neuronal and glial lineages. Conversely, persistent expression of FoxD3 in late-migrating neural crest cells using RCAS viruses resulted in the failure of melanoblasts to develop.
We suggest that FoxD3 plays two important roles in neural crest development. First, it is involved in the segregation of the neural crest lineage from the neuroepithelium. Second, it represses melanogenesis, thereby allowing other neural crest derivatives to differentiate during the early stages of neural crest patterning.
This 1999 edition of The Neural Crest contains comprehensive information about the neural crest, a structure unique to the vertebrate embryo, which has only a transient existence in early embryonic life. The ontogeny of the neural crest embodies the most important issues in developmental biology, as the neural crest is considered to have played a crucial role in evolution of the vertebrate phylum. Data that analyse neural crest ontogeny in murine and zebrafish embryos have been included in this revision. This revised edition also takes advantage of recent advances in our understanding of markers of neural crest cell subpopulations, and a full chapter is now devoted to cell lineage analysis. The major research breakthrough since the first edition has been the introduction of molecular biology to neural crest research, enabling an elucidation of many molecular mechanisms of neural crest development. This book is essential reading for students and researchers in developmental biology, cell biology, and neuroscience.
Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.
This chapter discusses the manipulations of neural crest cells or their migratory pathways. The formation of the embryo involves intricate cell movements, cell proliferation, and differentiation. The neural crest has long served as a model for the study of these processes, because neural crest cells undergo extensive migrations and give rise to many diverse derivatives. Neural crest cells arise from the dorsal portion of the neural tube. Several unique properties of these cells make the neural crest an ideal system for studying cell migration and differentiation. First, these cells migrate extensively along characteristic pathways. Second, they give rise to diverse and numerous derivatives, ranging from pigment cells and cranial cartilage to adrenal chromaffin cells and the ganglia of the peripheral nervous system. In addition, their characteristic position of premigratory neural crest cells within the dorsal portion of the neural tube makes them accessible to surgical and molecular manipulations during the initial stages in their development.
Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.
A technique by which genes can be introduced into the cells and tissues of developing embryos has great potential for studying the roles of genes during vertebrate embryogenesis. The ‘microelectroporation’ technique, in which DNA is introduced into cells within a restricted area of developing chick embryos with high reproducibility, was developed by the authors. In this review, the advantages and applications of this microelectroporation technique for developmental studies and functional analysis of genes in chick embryos is discussed.
A key feature of vertebrate development is the formation of the neural crest. In the trunk, neural crest cells delaminate from the neural tube shortly after the fusion of the neural folds and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. As neural crest cells leave the neural tube during the initial stages of their migration, they express the extracellular matrix glycoprotein tenascin-C, which is also found in the stroma of many tumors. We have studied the possible role for tenascin-C during neural crest morphogenesis in vivo by microinjecting tenascin-C morpholino antisense oligonucleotides into the lumen of the avian neural tube in ovo and electroporating the morpholino antisense oligonucleotides into the precursors of the neural crest. After 24 hr, tenascin-C immunostaining is reduced around the dorsal neural tube in the experimental microinjected embryos (12 of 13) but not in embryos microinjected with control morpholino antisense oligonucleotides (n = 3) or subjected to electroporation only (n = 2). In each of the 12 tenascin-C knockdown embryos neural crest cells are seen ectopically in the lumen of the neural tube and in the neuroepithelium; cells that do leave the neural tube after the microinjection fail to disperse laterally from the surface of the neural tube into the somites. The observation that neural crest cells must express tenascin-C to migrate normally is consistent with a role for this glycoprotein in contributing to the invasive behavior of neural crest cells. © 2001 Wiley-Liss, Inc.
One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems. This article describes a highly efficient method of delivering morpholino oligos into adherent and nonadherent cultured cells. In this system, a nonionic morpholino oligo is paired to a complementary DNA “carrier.” The DNA is then bound electrostatically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol. This article describes optimization of delivery in HeLa cells and provides the basis for delivery in any cultured endocytic cell type. genesis 30:94–102, 2001. © 2001 Wiley-Liss, Inc.
After the vertebrate lens is induced from head ectoderm, lens-specific genes are expressed. Transcriptional regulation of
the lens-specific αA-crystallin gene is controlled by an enhancer element, αCE2. A gene encoding an αCE2-binding protein,
L-maf(lens-specific maf), was isolated.L-maf expression is initiated in the lens placode and is restricted to lens cells. The gene product L-Maf regulates the expression
of multiple genes expressed in the lens, and ectopic expression of this transcription factor converts chick embryonic ectodermal
cells and cultured cells into lens fibers. Thus, vertebrate lens induction and differentiation can be triggered by the activation
of L-Maf.
Slug, a vertebrate gene encoding a zinc finger protein of the Snail family, is expressed in the neural crest and in mesodermal
cells emigrating from the primitive streak. Early chick embryos were incubated with antisense oligonucleotides to chick Slug.
These oligonucleotides specifically inhibit the normal change in cell behavior that occurs at the two sites in the emerging
body plan in which the gene is expressed. This change, which is the transition from epithelial to mesenchymal character, occurs
at the formation of mesoderm during gastrulation and on emigration of the neutral crest from the neural tube.
Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors.
Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular
heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone had no effect on embryonic development, whereas together
they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development.
The metameric pattern of the vertebrate axial skeleton, defined by structures such as the vertebral bodies and ribs, is a result of segmentation events that occur during embryogenesis. The key event in axial segmentation is somite formation. This study examines the role of Pax-1, a member of the paired-box containing Pax gene family, in chick somite development. To investigate whether misexpression of Pax-1 during somite development is functionally related to abnormal axial patterning, antisense methodologies were used to perturb Pax-1 expression. An antisense, phosphorothioate-modified oligodeoxynucleotide (ODN) was designed based on the mouse Pax-1 paired-box sequence, and was either injected into or directly applied topically to early, somitic stage chick embryos. Abnormalities in somite structure and pattern were subsequently observed and scored, including loss of somites (18% of injected embryos, 35% of embryos treated by topical application), fusion of somites (25% of injected, 6% with topical application), and shortened body axis (0% of injected, 11% with topical application). Control embryos receiving sense ODN or non-sense ODN (a scrambled sequence with base composition identical to the antisense ODN) showed substantially fewer somite anomalies, indicating that the effects were sequence-specific. These developmental abnormalities were analyzed using standard histological methods. Whole mount in situ hybridization was carried out to analyze the three-dimensional pattern of Pax-1 expression in whole embryos. In control, untreated embryos, the expression was localized to the entire epithelial somite, and as the somite matured, the expression was limited to its ventromedial region. With Pax-1 antisense ODN treatment, embryos with fused somites retained expression over the entire fused somite, and embryos that had complete loss of somites had greatly reduced expression of Pax-1 transcript. The results presented here provide strong evidence that Pax-1 is functionally important during somitogenesis and morphogenesis of the vertebral column. The spatial pattern of gene expression appears to delineate different populations of cells in the developing embryo (i.e., somite from somite, sclerotome from dermomyotome), and is consistent with the hypothesis that Pax-1 is involved in forming or maintaining boundaries at specific times and locations during development.
By using three nonviral transfection methods, i.e., microparticle bombardment, lipofection and electroporation, the transfection efficiency and the expression intensity of a lacZ reporter gene were compared in developing chicken embryos in ovo. Of the three transfection methods employed, electroporation conferred the strongest expression of the bacterial lacZ gene with similar transfection efficiency. The results suggest that as far as transient gene expression is concerned, electroporation would provide a useful and efficient nonviral means of foreign gene transfection to somatic cells of living chicken embryos in ovo.
Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by technical problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. Herein is an overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs.
Lunatic Fringe (l-Fng) is one of the vertebrate homologues of Drosophila Fringe, which interacts with the Notch signal pathway and regulates activation of the Notch ligands, Delta and Serrate. To elucidate the roles of l-Fng in vertebrate neurogenesis, we transfected chick l-Fng (C-l-Fng) to chick neural tube using the in ovo electroporation technique and examined the subsequent changes in expression of Notch-related genes. We observed downregulation of C-Serrate-1 by ectopic C-l-Fng expression which implied that C-l-Fng acts on the vertebrate Notch pathway to regulate the expression of its ligand.
Experimental embryology has long used manipulation of interacting tissues to examine questions of tissue interaction and differentiation. The potential for specific manipulation of gene expression in such tissues has made the utilization of antisense techniques desirable. However, problems with this methodology have discouraged many investigators from using this approach. Selection of target sequences for antisense oligonucleotides, delivery of oligonucleotides into cells or tissues, and the type of modification of the oligonucleotide to be used all present concerns that must be addressed. This paper describes our approach to selection of target sequence and methods of delivery and describes the synthesis of a methoxyethylamidate-modified antisense oligonucleotide that has proved useful in our studies. This approach has enabled us to explore aspects of tissue interaction in the embryonic heart that would have been difficult to explore in a genetic model.
RNase H-competent phosphorothioates (S-DNAs) have dominated the antisense field in large part because they offer reasonable resistance to nucleases, they afford good efficacy in cell-free test systems, they can be targeted against sites throughout the RNA transcript of a gene, and they are widely available from commercial sources at modest prices. However, these merits are counterbalanced by significant limitations, including: degradation by nucleases, poor in-cell targeting predictability, low sequence specificity, and a variety of non-antisense activities. In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.
Xenopus embryos develop dorsal/ventral and anterior/posterior axes as a result of the activity of a maternal Xwnt pathway, in which beta-catenin is an essential component, acting as a transactivator of transcription of zygotic genes. However, the questions of where and when beta-catenin is required in early embryogenesis have not been addressed directly, because no loss-of-function method has been available. Here we report the use of a novel antisense approach that allows us to target depletion of protein to individual blastomeres. When a "morpholino" oligo complementary to beta-catenin mRNA is injected into early embryos, it depletes beta-catenin protein effectively through the neurula stage. By targeting the oligo to different cleavage blastomeres, we block beta-catenin activity in different areas and at different times. Dorsal vegetal injection at the 2- and 4-cell stages blocks dorsal axis formation and at the 8-cell stage blocks head formation, while A-tier injection at the 32-cell stage causes abnormal cement gland formation. This approach shows the complex involvement of Xwnt pathways in embryonic patterning and offers a rapid method for the functional analysis of both maternal and early zygotic gene products in Xenopus.
Since morpholino oligos were first introduced as a means to inhibit gene function in embryos, in the Spring of 2000, they have been tested in a range of model organisms, including sea urchin, ascidian, zebrafish, frog, chick, and mouse. This review surveys the results of these studies and examines the successes and limitations of the approach for targeting maternal and zygotic gene function. The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.
The ADAMs are a family of proteins containing multiple functional domains. We have cloned the avian orthologue of ADAM 10 and demonstrate that it has metalloprotease activity. Chick ADAM 10 is expressed in the developing dermatome and myotome of the somite, epidermis, gut endoderm, the epithelial tissues of the kidney, liver, and heart, and in neural crest cells. The expression patterns and protein distribution of ADAM 10 suggest it may play a significant role in the morphogenesis of several epithelial tissues. When a dominant-negative metalloprotease-mutant form of ADAM 10 is expressed in the ectoderm or ADAM 10 expression is knocked down with morpholinos, morphogenesis and tissue specification are altered.
The occurrence of localized zones of intensive cell degeneration in the wing bud of the chick embryo between stages 17 and 32 has been studied chiefly by means of Nile blue staining in vivo and in vitro, and has been shown to be correlated spatiotemporally with the topographic distribution of prospective wing parts and with the morphogenetic movements which carve their definitive contours. Particular attention has been directed to the role of cell death in the shaping of the upper arm and forearm and in the elimination of tissues between the digits.
Control of cell behavior during vertebrate development by Slug, a zinc finger gene
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Effective targeted gene 'knockdown' in zebrafish
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