Method for electroporation for the early chick embryo

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.     

Misexpression of genes in brain vesicles by in ovo electroporation

Transfection to living chick embryos in ovo by electroporation has been recently developed. In this mini-review, misexpression in brain vesicles is introduced. To transfect, expression plasmid is inserted in the brain vesicle, and the square pulse of 25 V, 50 ms was charged five times. The translation product of the transfected gene is detected 2 h after electroporation, and reaches the peak at 24 h after electroporation. Transfection is so effective that this method is contributing greatly to the study of the molecular mechanisms of morphogenesis.     

Targeted Gene Misexpression in Chick Limb Buds Using Avian Replication-Competent Retroviruses

The methods and applications for using avian replication-competent retroviruses to target gene misexpression in the developing limb bud of the chicken are described. These viruses provide the means to exploit the strengths of the chick as a model system in experimental embryology in conjunction with a genetic approach for ectopically expressing a gene of interest. The applications and strengths of the system are detailed. All the steps required to produce a virus carrying a transgene of interest and the methodologies behind designing and carrying out misexpression strategies are outlined, and some useful techniques for analyzing infected embryos are described.     

Chick homeobox gene cbx and its role in retinal development

Homeobox genes play important roles in animal development. We isolated a chick homeobox gene, cbx, and studied its function during embryonic development. The deduced Cbx protein contained 376 amino acid residues. Its homeodomain was related (with 65-71% sequence identity) to that of human Crx, human Cart-1, and chick Alx-4. On searching the human genome sequence, a human homologue was found, which had 78% overall sequence identity and a 100% identical homeodomain. In the developing chick retina, cbx was expressed in a small fraction of post-mitotic cells residing at anatomical locations typical of bipolar cells. These cells were Goalpha(+) and protein kinase C(-), suggesting that they were probably cone bipolar cells. cbx mRNA was also detected outside the retina, particularly in the tectum and Rathke's pouch. Replication-competent retrovirus was used to drive misexpression of cbx and of an Engrailed repression construct. Engrailed-mediated repression of Cbx was embryonic lethal, while misexpression of cbx itself was tolerated. In the retina, misexpression of cbx resulted in fewer PKC(+) bipolar cells. Our data suggest that cbx is essential for embryonic survival and may participate in the development of bipolar, probably cone bipolar, cells in the retina.     

Somatic transgenesis in the avian model system

The chick embryo is a versatile model system, in which classical embryology can be combined with modern molecular approaches. In the last two decades, several efficient methods have been developed to introduce exogenous genes into the chick embryo. These techniques allow alteration of gene expression levels in a spatially and temporally restricted manner, thereby circumventing embryonic lethality and/or eliminating secondary effects in other tissues. Here, we present the current status of avian somatic transgenic techniques, focusing on electroporation and retrovirus-mediated gene transfer. Electroporation allows quick and efficient gain-of-function studies based on transient misexpression of genes. Retroviral vectors, which are capable of integrating exogenous genes into the host chromosome, permit analysis of long-term effects of gene misexpression. The variety of methods available for somatic transgenesis, along with the recent completion of the chicken genome, are transforming the chick embryo into one of the most attractive model systems to examine function of genes that are important for embryonic development.     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk

Neural crest cells migrate along two discrete pathways within the trunk of developing embryos. In the chick, early migrating crest cells are confined to a ventral pathway medial to the dermamyotome while later cells migrate on a dorsal pathway lateral to the dermamyotome. Here we show that Slits are expressed in the dermamyotome, that early migrating crest cells express the Slit receptors Robo 1 and Robo 2, that Slit2 repels migrating crest cells in an in vitro assay, and that the misexpression of a dominant-negative Robo1 receptor induces a significant fraction of early crest cells to migrate ectopically in the dorso-lateral pathway. These findings suggest that Slits, most likely those expressed in the dermamyotome, help to confine the migration of early crest cells to the ventral pathway.     

Activation of Ras-ERK pathway by Fgf8 and its downregulation by Sprouty2 for the isthmus organizing activity

In the previous studies, we showed that strong Fgf8 signaling activates the Ras-ERK pathway to induce cerebellum. Here, we show importance of negative regulation following activation of this pathway for proper regionalization of mesencephalon and metencephalon in chick embryos. ‘Prolonged’ activation of ERK by misexpression of Fgf8b and dominant-negative Sprouty2 (dnSprouty2) did not change the fate of the mesencephalic alar plate. Downregulation of ERK activity using an MEK inhibitor, U0126, or by tetracycline-dependent Tet-off system after co-expression of Fgf8b and dnSprouty2 forced the mesencephalic alar plate to differentiate into cerebellum. We then paid attention to Mkp3. After misexpression of dnMkp3 and Fgf8b, slight downregulation of ERK activity occurred, which may be due to Sprouty2, and the mesencephalon transformed to the isthmus-like structure. The results indicate that ERK must be once upregulated and then be downregulated for cerebellar differentiation and that differential ERK activity level established by negative regulators receiving Fgf8 signal may determine regional specificity of mesencephalon and metencephalon.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

Microbubble-enhanced sonoporation: efficient gene transduction technique for chick embryos

The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed.     

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.     

Immunohistochemical study of the appearance of somatotropic hormone in the adenohypophysis of chick embryos

The differentiation of somatotropocytes was studied in the Leghorn chick embryos during 11 to 18 days of incubation and in chickens during the first week of life using the immunohistochemical method of nonlabelled antibodies with PAP complex and antiserum against human somatotropic hormone (STH). Unlike in humans, the fixation of pituitaries in the Carnoy mixture is optimal for STH to be immunohistochemically estimated in the chickens and chick embryos. STH was found in adenohypophysis from 12-13 days of development. Besides predominant localization of somatotropocytes in the adenohypophysis caudal lobe, individual STH-positive cells are also present in the cephalic lobe of chickens and chick embryos. The results obtained suggest a relatively late appearance of STH during histotypical development of adenohypophysis in chick embryos.     

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.     

Immunofluorescence study of the formation of evolutionary stable lens proteins in chick embryos]

In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.     

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.