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Expression of exogenous genes trasnferred into the avian limb in ovo
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Neuroscience Letters 210 (1996) 99-102
Expression of exogenous genes transferred into the avian limb in ovo Franck Perez*, Elettra Ronchi CNRS URA 1414, Ecole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, France Received 5 February 1996; revised version received 29 April 1996; accepted 29 April 1996
Abstract
We report on a simple method of direct gene transfer which allows the ectopic expression of proteins and the study of mesodermspecific genes in the chick embryo. We microinjected into the avian embryonic limb several plasmids containing reporter genes under the control of various promoter sequences, including a minimal chicken muscle acetylcholine receptor tz-subunit promoter [Klarsfeld, A., Daubas, A., Bourachot, B. and Changeux, J.P., Mol. Cell. Biol., 7 (1987) 951-955]. Gene expression is detectable for 3 days, is reproducible, is restricted to the site of injection, and correlates with the amount of DNA injected. Our observations indicate that it is possible to transfer and express genes in ectodermal and mesodermal ceils of the chick limb by direct DNA injection and that the method can be used to analyze promoter sequences in vivo during specific windows of development.
Keywords: Gene transfer; Embryonic limb; Chick
In the past few years, direct injections of naked DNA have been used successfully for the transfer of genes into several systems. Since the original report by Wolff et al. [15] demonstrating the expression of reporter genes following the direct injection of plasmid DNA into rodent muscle, this technique has been used to transfer genes in skeletal and cardiac muscle of rodents [1,5], skeletal muscle of other mammals [10] and skeletal muscle of fish and Xenopus [6]. In adult rodents, striated muscle is the only tissue found to be capable of taking up and expressing reporter genes injected in the form of plasmid DNA, and this method can be used to evaluate physiological regulation of gene expression in muscle [6]'i Furthermore, since the injected DNA appears to persist as an extrachromosomal circular plasmid, and does not integrate, injections usually do not produce adverse side effects [10]. Here, we show that direct gene transfer can also be applied to the avian embryo and can be a useful tool for the study of the regulation of gene expression in the avian limb. We undertook this study to examine the regulation of specific mesodermal genes in the avian limb during embryonic development, in particular at the time of innervation. The direct gene transfer technique was appealing to * Corresponding author.
us for various reasons. First, this method had proven effective for the specific transfer of genes into skeletal muscle in vivo, thus we were hoping to target specifically embryonic mesoderm. Second, we wished to study well defined periods or 'windows' in development. Third, this method had the potential of being a safe and inexpensive alternative to viral-based gene transfer. For the initial characterization of the system we used three promoter-reporter gene constructs: (i) pCMVluc, which contains the firefly luciferase gene under the control of the CMV promoter (gift of B. Whalen, Institut Pasteur); (ii) pSDAPSV40, which contains the human placental alkaline phosphatase (PAP) under the control of SV40 promoter (gift of J. Brokes, UK); (iii) pMMuLVnlslacZ, which encodes a nuclear localization signal fl-galactosidase (lacZ) fusion protein under the control of a murine leukemia virus (MLV) promoter (gift of B. Dujon, Institut Pasteur; data not shown). Plasmids were prepared by standard maxiprep procedures using Qiagen DNA purification columns (DIAGEN, Hilden, Germany) and ethanol precipitation. Plasmids were dissolved at a concentration of 5/zg//zl or 10#g//zl in a solution containing 25% sucrose in phosphatebuffered saline (sucrose/PBS; pH 7.3) [5] and injected into the thigh and anterior limb in ovo through standard glass capillary femtotips (Eppendorf, Hamburg, Germany) that had been delicately wedged in PBS under a
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2681-X
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F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
microscope. In preliminary experiments the concentrate was mixed with Di-I/methanol at 1:20 dilution to label the injection site (see Fig. 1E). As controls, we routinely used the contra-lateral limb that had not been injected or that had been injected with the sucrose/PBS concentrate alone. Eggs were sealed with tape and returned to the incubator for 24--48 h or just before hatching, after which embryos were fixed, stained in toto for lacZ and/or PAP as described by Cepko et al. [4], and in some cases 55/zm cryostat sections were collected after preliminary overnight embedding in gelatin (7.5% gelatin in 15% saccarose-PBS). In a first series of experiments we injected the pSDAPSV40 vector in the anterior and posterior limbs of chick embryos at various stages (E4-E9). Embryos were sacrificed 12 and 48 h after injection and were stained for PAP activity. PAP staining was detected at the site of injection in the whole mount preparations (Fig. 1A), which indicates that the injected gene had been successfully transferred and expressed. Furthermore, we observed that PAP staining was in general confined to the site of injection and was detected, based on histological criteria, in dermal and mesodermal cells (Fig. 1C). In our system, the most superficial cells stained with PAP were easily attributable to the epidermis because of the characteristic appearance at the light microscope of its stacked layers of larger and flatter cells. PAP-positive cells in
deeper tissue layers include mesodermal cells, since at later embryonic stages, staining is found in myofibers that can be easily recognized at the light microscope by the lined up, fused and multinucleate appearance of its cells (Fig. 1B). Previous studies in other systems had emphasized that direct D N A gene transfer is primarily limited to muscle. However, gene transfer to human and pig epidermal keratinocytes by this method has been recently documented [7,9]. Occasionally, labelled cells were found at some distance from the injection site, especially in preparations of E4 embryos and after 48 h survival; an indication, presumably, of local cell movements and migration (Fig. 1E). In a second series of experiments we tested the level and duration of gene expression. For this purpose we selected the pCMVluc vector since luciferase activity can be easily quantified. In these experiments, limbs or muscle (in E9 embryos) were removed 24-48 h after gene transfer and placed in 200/zl of ice-cold lysis buffer, BLUC (Tris-H3PO4 25 mM (pH 7.8), MgCI 2 10 mM, 1% Triton X, 15% glycerol, 1 mM EDTA, 1 mM DTT) and homogenized with a hand-held homogenizer with a plastic pestle (Kontes) for 1 min. The mixture was allowed to sit on ice for 30 min, vortexed briefly and centrifuged at 7000 × g for 5 min at 4°C. Luciferase activity was measured on 100/d of supernatant, to which was added 150/zl
Fig. 1. Pattern of PAP and nlsflgal expression in limbs of E5-E7 (stages 28-30 according to Hamburger and Hamilton) chick embryos. (A) Whole mount staining for PAP of the posterior limb at E5, 24 h after injection of 5/ag of pSDAPSV40;staining is confined to the site of injection and can be detected in epidermal and muscle cells. (B) Whole mount staining for PAP of an anterior limb at E7, 48 h after injection of 10/ag of pSDAPSV40; gene expression from this promoter is extensive and, in this embryo, is primarily detected in muscle fibers. (C) Low power Nomarsky optic view of 55/am sagittal cryostat section of the limb of an E5 embryo 12 h after injection of 5/ag of pSDAPSV40; staining is confined to isolated mesodermal and dermal cells at the site of injection. (D) Low power Nomarsky optic view of 55/am sagittal cryostat section of the limb of an E7 embryo 24 h after injection of 5/ag of AchR-nisLacZ;flgal staining appears distributed along single muscle fibers. (E) Whole mount staining for PAP of the posterior limb at E7/8, 2 days after injection of 10/ag of pSDAPSV40 mixed with Dye-I to label the site of injection; note stained cells at some distance from the site of injection.
101
F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
of reconstituted luciferase assay substrate (ATP 1.25 mM, D-luciferin 85/~g/ml dissolved in BLUC buffer). The relative luciferase units were measured, starting 3-5 s after mixing for a period of 10 s in a L U M A C luminometer (LB9501, Berthold). Results are expressed as group means +_.SEMs of luciferase units/muscle normalized for protein levels (measured with MicroBCA Protein Assay Kit, Pierce). Luciferase reporter gene activity correlated with the amount of DNA injected (Fig. 2A) and decreased within 48 h (Fig. 2B). Thus, with this method gene activity in the chick embryo, contrary to reports on gene transfer in adult tissues, is short-lived. Since the injected vector is expected to remain episomal, this is most likely a consequence of the high levels of embryonic mitotic activity, which would reduce the stability of the episomal vector. Gene activity was also dependent on the age of the injected embryo, since luciferase activity was twice as strong at E5 than at E9 (Fig. 2C). Between days 8 and 14, embryos undergo a period of rapid myogenesis and growth [2]. Thus, our results may be also here a product of the enhanced mitotic activity at this stage. In addition, we found that survival was optimal between E4 and E7, and diminished at E9. Taken together, these results demonstrate that with this procedure it is possible to target gene expression in developing mesodermal derivatives and that this expression is consistent and reproducible. Since expression is shortlived, this method is most useful for studies of the effects of pulse-like ectopic expression of proteins during short periods of embryonic development. We further asked whether we could use our method to study the regulation of the minimal chicken muscle
acetylcholine receptor a-subunit (aAchR) promoter of 850 bp that had been previously shown to confer muscle specificity in cell cultures [11] and that contains sequence elements responsive to denervation in the mouse [12], but that had never been studied in chick embryos. We were hoping to use this promoter as a marker for our studies on gene regulation at the time of muscle innervation in the chick embryo. For this purpose, we injected plasmids containing either a nlslacZ or a luciferase reporter gene under the control of the minimal chicken ctAchR promoter (gifts of J.L. Besserau, Institut Pasteur) in embryos ranging from E4 to E9. Our results indicate that expression of nlsflgal is restricted to developing muscle and that gene expression is muscle-specific (Fig. 1D). Furthermore, levels of reporter gene (luciferase) activation followed the known pattern of expression of the endogenous a-subunit m R N A [11,12], since the activity declined with age (Fig. 2D). Although this can also be due to an age-related decrease in the efficiency of gene transfer or gene expression, our results suggest that the injected a A c h R promoter fragment is functional after injection since, as expected, it is activated exclusively in muscle cells, driving the expression of reporter genes in these cells. Since the a A c h R promoter is known to be regulated by neuromuscular activity we tested whether the levels of reporter gene activity of the injected aAchR-vector would change if we blocked neuromuscular activity. We repeated the injections with the a A c h R promoter fragment and induced a temporary paralysis in half of the embryos at E6 and E8 with 100/zl of a 0.5 mg/ml solution of curare (alcuroniumchlorid diethanolaminum; Alloferine, Roche), which was added on top of the embryos after the
30o00
50000
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CMVIuc 5ktg
CMVluc10~tg
CMVluc Ctd 24 hrs
CMVIuc Ctrl 48 hrs
CMVIuc Ctrl E5
CMVluc Ctrl E9
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E6
~,
+
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E8
Fig. 2. Luciferase gene expression in chicken limb extracts after direct plasmid DNA injection. (A) Luciferase activity recovered 24 h after injection of 5/~g (n = 7) or 10/*g (n = 5) of a CMVIuc plasmid in the limb of E5 embryos (stage 28 according to Hamburger and Hamilton). Gene expression increases with the amount of DNA injected. (B) Luciferase gene expression 24 h (n = 7) and 48 h (n = 4) after injection of 5/ag CMVluc in the limb of E5 embryos. Gene expression decreases with time. (C) Gene expression recovered from muscle extracts of E9 embryos (n = 7; stage 34/35 according to Hamburger and Hamilton) as compared to total limb extracts of E5 embryos (n = 7) 24 h after injection of 5/~g CMVluc. (D) AchR-luc transgene expression, measured as iuciferase units in limb extracts of E6 (n = 17) and E8 (n = 9) embryos (stage 28/29 and stage 34 according to Hamburger and Hamilton), 24 h after the injection of 5/~g of AchR-luc plasmid DNA. Activation, which tends to decrease with age, responds to treatment with curare (Cur.; n = 5 for both groups). This indicates that the promoter is recognized by muscle-specifictranscription factors and contains sequences sensitive, most likely, to electrical activity in the early chick embryo.
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F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
injection. The results indicate that the treatment with curare produces an increase in the aAchR promoter-driven expression (Fig. 2D). At the age we selected to study, neuromuscular connections have already started forming in the posterior limbs of the chick embryo [13], and spontaneous activity can be recorded as early as 3-5 days of development [8]. That the blockage of spontaneous activity alone can cause a significant increase in the mRNA levels of the aAchR gene has been documented in primary cultures of chicken myotubes in the absence of any nerve connections [11]. Recently, a similar up-regulation of expression was observed for the aAchR promoter fragment of 850 bp transduced in myotubes treated with tetanus toxin in tissue culture (Dr. Besserau, Institut Pasteur, pers. comm.). Thus, it is likely that the effect we observe may be activity-dependent. Since earlier literature based on binding to a-bungarotoxin seems to suggest that a curare-dependent increase in Ach receptor numbers is first measurable at El5 [2,3], our promoter fragment may be regulated differently from the endogenous gene. On the other hand, in contrast with these earlier studies, by using the DNA injection method we were able to directly address the effects of paralysis at E6 on gene expression. Further research is clearly necessary to elucidate the effects of curare on endogenous AchR gene expression at this early embryonic stage and the factors which contribute to the increased expression of our episomal vector; however, this was not within the scope of our current experiments. In conclusion, we show in this study that direct DNA injection is a powerful method for the ectopic expression of proteins in the developing chick limbs. It represents a good alternative to the use of recombinant viruses and to the transfection of cells or the injection of proteins. This method is safe, inexpensive and has the advantage that the same vectors can be used for cell transfections and in vivo experiments. All of the promoters tested so far (CMV, SV40, MLV, and RSV) worked in our system. We also show that this method can be used to follow the regulation of muscle-specific gene promoters in the mesoderm of the avian limb in ovo during specific stages of development. We report for the first time the regulated expression of the 850 bp chicken aAchR promoter fragment in the chick embryo. Our results demonstrate that the 850 bp promoter fragment contained in the plasmid injected in the embryo is recognized by muscle-specific transcription factors and is regulated, most likely, by neuromuscular activity. This chicken promoter had primarily been studied in the mouse because of the limitations of in vivo gene transfer in chick embryos. Therefore, although our method restricts gene transfer to the site of injection and does not provide long-lasting gene expression when used in early embryos, it represents a good alternative to the labor-intensive generation of transgenic animals and to the use of recombinant adenoviral or retroviral vectors. We are now using this method to over-express or locally inhibit the expression of developmental genes in the limb during specific stages of development.
This work was carried out in the laboratory of Dr. Alain Prochiantz, to whom we are very grateful for the support and assistance provided at all stages of the project and for critical reading of the manuscript. We thank Dr. R. Whalen, Dr. J.-F. Nicolas, Dr. J.L. Besserau (Institut Pasteur, Paris, France) and Dr. J. Brockes (Ludwig Institute for Cancer Research, London, UK) for the gift of the plasmids used in this study. We also thank Dr. G. Cossu (Rome, Italy), Dr. H. Davis (Ottawa, Canada) and Dr. Marion Wassef (Paris, France) for encouragement and valuable discussion. This work was supported by Association Franqaise de Lutte contre les Myopathies and Fondation pour la Recherche M6dicale. [1] Acsadi, G., Jiao, S.S., Jani, A., Duke, D., Williams, P., Chong, W. and Wolff, J.A., Direct gene transfer and expression into rat heart in vivo, New Biol., 3 (1991) 71-81. 12] Burden, S., Development of the neuromuscular junction in the chicken embryo: the number, distribution, and stability of acetylcholine receptors, Dev. Biol., 57 (1977) 317-329. [3] Betz H., Bourgeois J.P. and Changeux J.P., Evolution of cholinergic proteins in developing slow and fast skeletal muscles in chick embryo, J. Physiol., 302 (1980) 197-218. [4] Cepko, C.L., Ryder, E.F., Austin, C.P., Walsh, C. and Fekete, D.M., Lineage analysis using retrovirus vectors. In P. Wasserman and M. De Pamphilis (Eds.), Methods in Enzymoiogy: A Guide to Techniques in Mouse Development, Academic Press, Orlando, FL, 1993, pp. 933-960. [5l Davis, H.L., Whalen, R.G. and Demeneix, B.A., Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression, Hum. Gene Ther., 4 (1993) 151-159. [6] De Luze, A., Sachs, L. and Demeneix, B.A., Thyroid hormonedependent transcriptional regulation of exogenous genes transferred into Xenopus tadpole muscle in vivo, Proc. Natl. Acad. Sci. USA, 90 (1993) 7322-7326. 17] Fynana, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C. and Robinson, H.L., Protective immunization by parenteral, mucosal and gene gun inoculations, Proc. Natl. Acad. Sci. USA, 90 (1993) 11478-11482. [8] Hamburger, V., Embryonic mobility in vertebrates. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, Rockefeller University Press, New York, 1970, pp. 141-151. [9] Hengge, U.R., Chan, E.F., Foster, R.A., Walker, P.S. and Vogel, J.C., Cytokine gene expression in epidermis with biological effects following injection of naked DNA, Nature Genet., 10 (1995) 161-166. [10] Jiao, S., Williams, P., Berg, R.K., Hodgeman, B.A., Liu, L., Repetto, G. and Wolff, J.A., Direct gene transfer into non-human primate myofibers in vivo, Hum. Gene Ther., 3 (1995) 21-33. [lll Klarsfeld, A. and Changeux J.P., Activity regulates the levels of acetylcholine receptor a-subunit mRNA in cultured chicken myotubes, Proc. Natl. Acad. Sci. USA, 82 (1985) 4558-4562. [12] Klarsfeld, A., Daubas, A., Bourachot, B. and Changeux J.P., A 5' flanking region of the chicken receptor a-subunit gene confers specificity and developmental control of expression in transfected cells, Mol. Cell. Biol., 7 (1987) 951-955. [13] Landmesser, L. and Morris, D.G., The development of functional innervation in the hind limb of the chick embryo, J. Physiol., 249 (1975) 301-326. [14] Merlie, J.P. and Komhauser, K.M., Neural regulation of gene expression by an acetylcholine receptor promoter in muscle of transgenic mice, Neuron, 2 (1988) 1295-1300. [15] Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Feigner, P.L., Direct gene transfer into mouse muscle in vivo, Science, 247 (1990) 1456-1468.
L ;:t
ELSEVIER
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1
Neuroscience Letters 210 (1996) 99-102
Expression of exogenous genes transferred into the avian limb in ovo Franck Perez*, Elettra Ronchi CNRS URA 1414, Ecole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, France Received 5 February 1996; revised version received 29 April 1996; accepted 29 April 1996
Abstract
We report on a simple method of direct gene transfer which allows the ectopic expression of proteins and the study of mesodermspecific genes in the chick embryo. We microinjected into the avian embryonic limb several plasmids containing reporter genes under the control of various promoter sequences, including a minimal chicken muscle acetylcholine receptor tz-subunit promoter [Klarsfeld, A., Daubas, A., Bourachot, B. and Changeux, J.P., Mol. Cell. Biol., 7 (1987) 951-955]. Gene expression is detectable for 3 days, is reproducible, is restricted to the site of injection, and correlates with the amount of DNA injected. Our observations indicate that it is possible to transfer and express genes in ectodermal and mesodermal ceils of the chick limb by direct DNA injection and that the method can be used to analyze promoter sequences in vivo during specific windows of development.
Keywords: Gene transfer; Embryonic limb; Chick
In the past few years, direct injections of naked DNA have been used successfully for the transfer of genes into several systems. Since the original report by Wolff et al. [15] demonstrating the expression of reporter genes following the direct injection of plasmid DNA into rodent muscle, this technique has been used to transfer genes in skeletal and cardiac muscle of rodents [1,5], skeletal muscle of other mammals [10] and skeletal muscle of fish and Xenopus [6]. In adult rodents, striated muscle is the only tissue found to be capable of taking up and expressing reporter genes injected in the form of plasmid DNA, and this method can be used to evaluate physiological regulation of gene expression in muscle [6]'i Furthermore, since the injected DNA appears to persist as an extrachromosomal circular plasmid, and does not integrate, injections usually do not produce adverse side effects [10]. Here, we show that direct gene transfer can also be applied to the avian embryo and can be a useful tool for the study of the regulation of gene expression in the avian limb. We undertook this study to examine the regulation of specific mesodermal genes in the avian limb during embryonic development, in particular at the time of innervation. The direct gene transfer technique was appealing to * Corresponding author.
us for various reasons. First, this method had proven effective for the specific transfer of genes into skeletal muscle in vivo, thus we were hoping to target specifically embryonic mesoderm. Second, we wished to study well defined periods or 'windows' in development. Third, this method had the potential of being a safe and inexpensive alternative to viral-based gene transfer. For the initial characterization of the system we used three promoter-reporter gene constructs: (i) pCMVluc, which contains the firefly luciferase gene under the control of the CMV promoter (gift of B. Whalen, Institut Pasteur); (ii) pSDAPSV40, which contains the human placental alkaline phosphatase (PAP) under the control of SV40 promoter (gift of J. Brokes, UK); (iii) pMMuLVnlslacZ, which encodes a nuclear localization signal fl-galactosidase (lacZ) fusion protein under the control of a murine leukemia virus (MLV) promoter (gift of B. Dujon, Institut Pasteur; data not shown). Plasmids were prepared by standard maxiprep procedures using Qiagen DNA purification columns (DIAGEN, Hilden, Germany) and ethanol precipitation. Plasmids were dissolved at a concentration of 5/zg//zl or 10#g//zl in a solution containing 25% sucrose in phosphatebuffered saline (sucrose/PBS; pH 7.3) [5] and injected into the thigh and anterior limb in ovo through standard glass capillary femtotips (Eppendorf, Hamburg, Germany) that had been delicately wedged in PBS under a
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2681-X
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F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
microscope. In preliminary experiments the concentrate was mixed with Di-I/methanol at 1:20 dilution to label the injection site (see Fig. 1E). As controls, we routinely used the contra-lateral limb that had not been injected or that had been injected with the sucrose/PBS concentrate alone. Eggs were sealed with tape and returned to the incubator for 24--48 h or just before hatching, after which embryos were fixed, stained in toto for lacZ and/or PAP as described by Cepko et al. [4], and in some cases 55/zm cryostat sections were collected after preliminary overnight embedding in gelatin (7.5% gelatin in 15% saccarose-PBS). In a first series of experiments we injected the pSDAPSV40 vector in the anterior and posterior limbs of chick embryos at various stages (E4-E9). Embryos were sacrificed 12 and 48 h after injection and were stained for PAP activity. PAP staining was detected at the site of injection in the whole mount preparations (Fig. 1A), which indicates that the injected gene had been successfully transferred and expressed. Furthermore, we observed that PAP staining was in general confined to the site of injection and was detected, based on histological criteria, in dermal and mesodermal cells (Fig. 1C). In our system, the most superficial cells stained with PAP were easily attributable to the epidermis because of the characteristic appearance at the light microscope of its stacked layers of larger and flatter cells. PAP-positive cells in
deeper tissue layers include mesodermal cells, since at later embryonic stages, staining is found in myofibers that can be easily recognized at the light microscope by the lined up, fused and multinucleate appearance of its cells (Fig. 1B). Previous studies in other systems had emphasized that direct D N A gene transfer is primarily limited to muscle. However, gene transfer to human and pig epidermal keratinocytes by this method has been recently documented [7,9]. Occasionally, labelled cells were found at some distance from the injection site, especially in preparations of E4 embryos and after 48 h survival; an indication, presumably, of local cell movements and migration (Fig. 1E). In a second series of experiments we tested the level and duration of gene expression. For this purpose we selected the pCMVluc vector since luciferase activity can be easily quantified. In these experiments, limbs or muscle (in E9 embryos) were removed 24-48 h after gene transfer and placed in 200/zl of ice-cold lysis buffer, BLUC (Tris-H3PO4 25 mM (pH 7.8), MgCI 2 10 mM, 1% Triton X, 15% glycerol, 1 mM EDTA, 1 mM DTT) and homogenized with a hand-held homogenizer with a plastic pestle (Kontes) for 1 min. The mixture was allowed to sit on ice for 30 min, vortexed briefly and centrifuged at 7000 × g for 5 min at 4°C. Luciferase activity was measured on 100/d of supernatant, to which was added 150/zl
Fig. 1. Pattern of PAP and nlsflgal expression in limbs of E5-E7 (stages 28-30 according to Hamburger and Hamilton) chick embryos. (A) Whole mount staining for PAP of the posterior limb at E5, 24 h after injection of 5/ag of pSDAPSV40;staining is confined to the site of injection and can be detected in epidermal and muscle cells. (B) Whole mount staining for PAP of an anterior limb at E7, 48 h after injection of 10/ag of pSDAPSV40; gene expression from this promoter is extensive and, in this embryo, is primarily detected in muscle fibers. (C) Low power Nomarsky optic view of 55/am sagittal cryostat section of the limb of an E5 embryo 12 h after injection of 5/ag of pSDAPSV40; staining is confined to isolated mesodermal and dermal cells at the site of injection. (D) Low power Nomarsky optic view of 55/am sagittal cryostat section of the limb of an E7 embryo 24 h after injection of 5/ag of AchR-nisLacZ;flgal staining appears distributed along single muscle fibers. (E) Whole mount staining for PAP of the posterior limb at E7/8, 2 days after injection of 10/ag of pSDAPSV40 mixed with Dye-I to label the site of injection; note stained cells at some distance from the site of injection.
101
F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
of reconstituted luciferase assay substrate (ATP 1.25 mM, D-luciferin 85/~g/ml dissolved in BLUC buffer). The relative luciferase units were measured, starting 3-5 s after mixing for a period of 10 s in a L U M A C luminometer (LB9501, Berthold). Results are expressed as group means +_.SEMs of luciferase units/muscle normalized for protein levels (measured with MicroBCA Protein Assay Kit, Pierce). Luciferase reporter gene activity correlated with the amount of DNA injected (Fig. 2A) and decreased within 48 h (Fig. 2B). Thus, with this method gene activity in the chick embryo, contrary to reports on gene transfer in adult tissues, is short-lived. Since the injected vector is expected to remain episomal, this is most likely a consequence of the high levels of embryonic mitotic activity, which would reduce the stability of the episomal vector. Gene activity was also dependent on the age of the injected embryo, since luciferase activity was twice as strong at E5 than at E9 (Fig. 2C). Between days 8 and 14, embryos undergo a period of rapid myogenesis and growth [2]. Thus, our results may be also here a product of the enhanced mitotic activity at this stage. In addition, we found that survival was optimal between E4 and E7, and diminished at E9. Taken together, these results demonstrate that with this procedure it is possible to target gene expression in developing mesodermal derivatives and that this expression is consistent and reproducible. Since expression is shortlived, this method is most useful for studies of the effects of pulse-like ectopic expression of proteins during short periods of embryonic development. We further asked whether we could use our method to study the regulation of the minimal chicken muscle
acetylcholine receptor a-subunit (aAchR) promoter of 850 bp that had been previously shown to confer muscle specificity in cell cultures [11] and that contains sequence elements responsive to denervation in the mouse [12], but that had never been studied in chick embryos. We were hoping to use this promoter as a marker for our studies on gene regulation at the time of muscle innervation in the chick embryo. For this purpose, we injected plasmids containing either a nlslacZ or a luciferase reporter gene under the control of the minimal chicken ctAchR promoter (gifts of J.L. Besserau, Institut Pasteur) in embryos ranging from E4 to E9. Our results indicate that expression of nlsflgal is restricted to developing muscle and that gene expression is muscle-specific (Fig. 1D). Furthermore, levels of reporter gene (luciferase) activation followed the known pattern of expression of the endogenous a-subunit m R N A [11,12], since the activity declined with age (Fig. 2D). Although this can also be due to an age-related decrease in the efficiency of gene transfer or gene expression, our results suggest that the injected a A c h R promoter fragment is functional after injection since, as expected, it is activated exclusively in muscle cells, driving the expression of reporter genes in these cells. Since the a A c h R promoter is known to be regulated by neuromuscular activity we tested whether the levels of reporter gene activity of the injected aAchR-vector would change if we blocked neuromuscular activity. We repeated the injections with the a A c h R promoter fragment and induced a temporary paralysis in half of the embryos at E6 and E8 with 100/zl of a 0.5 mg/ml solution of curare (alcuroniumchlorid diethanolaminum; Alloferine, Roche), which was added on top of the embryos after the
30o00
50000
,o,
40~0t
CMVIuc 5ktg
CMVluc10~tg
CMVluc Ctd 24 hrs
CMVIuc Ctrl 48 hrs
CMVIuc Ctrl E5
CMVluc Ctrl E9
+
E6
~,
+
+
E8
Fig. 2. Luciferase gene expression in chicken limb extracts after direct plasmid DNA injection. (A) Luciferase activity recovered 24 h after injection of 5/~g (n = 7) or 10/*g (n = 5) of a CMVIuc plasmid in the limb of E5 embryos (stage 28 according to Hamburger and Hamilton). Gene expression increases with the amount of DNA injected. (B) Luciferase gene expression 24 h (n = 7) and 48 h (n = 4) after injection of 5/ag CMVluc in the limb of E5 embryos. Gene expression decreases with time. (C) Gene expression recovered from muscle extracts of E9 embryos (n = 7; stage 34/35 according to Hamburger and Hamilton) as compared to total limb extracts of E5 embryos (n = 7) 24 h after injection of 5/~g CMVluc. (D) AchR-luc transgene expression, measured as iuciferase units in limb extracts of E6 (n = 17) and E8 (n = 9) embryos (stage 28/29 and stage 34 according to Hamburger and Hamilton), 24 h after the injection of 5/~g of AchR-luc plasmid DNA. Activation, which tends to decrease with age, responds to treatment with curare (Cur.; n = 5 for both groups). This indicates that the promoter is recognized by muscle-specifictranscription factors and contains sequences sensitive, most likely, to electrical activity in the early chick embryo.
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F. Perez, E. Ronchi / Neuroscience Letters 210 (1996) 99-102
injection. The results indicate that the treatment with curare produces an increase in the aAchR promoter-driven expression (Fig. 2D). At the age we selected to study, neuromuscular connections have already started forming in the posterior limbs of the chick embryo [13], and spontaneous activity can be recorded as early as 3-5 days of development [8]. That the blockage of spontaneous activity alone can cause a significant increase in the mRNA levels of the aAchR gene has been documented in primary cultures of chicken myotubes in the absence of any nerve connections [11]. Recently, a similar up-regulation of expression was observed for the aAchR promoter fragment of 850 bp transduced in myotubes treated with tetanus toxin in tissue culture (Dr. Besserau, Institut Pasteur, pers. comm.). Thus, it is likely that the effect we observe may be activity-dependent. Since earlier literature based on binding to a-bungarotoxin seems to suggest that a curare-dependent increase in Ach receptor numbers is first measurable at El5 [2,3], our promoter fragment may be regulated differently from the endogenous gene. On the other hand, in contrast with these earlier studies, by using the DNA injection method we were able to directly address the effects of paralysis at E6 on gene expression. Further research is clearly necessary to elucidate the effects of curare on endogenous AchR gene expression at this early embryonic stage and the factors which contribute to the increased expression of our episomal vector; however, this was not within the scope of our current experiments. In conclusion, we show in this study that direct DNA injection is a powerful method for the ectopic expression of proteins in the developing chick limbs. It represents a good alternative to the use of recombinant viruses and to the transfection of cells or the injection of proteins. This method is safe, inexpensive and has the advantage that the same vectors can be used for cell transfections and in vivo experiments. All of the promoters tested so far (CMV, SV40, MLV, and RSV) worked in our system. We also show that this method can be used to follow the regulation of muscle-specific gene promoters in the mesoderm of the avian limb in ovo during specific stages of development. We report for the first time the regulated expression of the 850 bp chicken aAchR promoter fragment in the chick embryo. Our results demonstrate that the 850 bp promoter fragment contained in the plasmid injected in the embryo is recognized by muscle-specific transcription factors and is regulated, most likely, by neuromuscular activity. This chicken promoter had primarily been studied in the mouse because of the limitations of in vivo gene transfer in chick embryos. Therefore, although our method restricts gene transfer to the site of injection and does not provide long-lasting gene expression when used in early embryos, it represents a good alternative to the labor-intensive generation of transgenic animals and to the use of recombinant adenoviral or retroviral vectors. We are now using this method to over-express or locally inhibit the expression of developmental genes in the limb during specific stages of development.
This work was carried out in the laboratory of Dr. Alain Prochiantz, to whom we are very grateful for the support and assistance provided at all stages of the project and for critical reading of the manuscript. We thank Dr. R. Whalen, Dr. J.-F. Nicolas, Dr. J.L. Besserau (Institut Pasteur, Paris, France) and Dr. J. Brockes (Ludwig Institute for Cancer Research, London, UK) for the gift of the plasmids used in this study. We also thank Dr. G. Cossu (Rome, Italy), Dr. H. Davis (Ottawa, Canada) and Dr. Marion Wassef (Paris, France) for encouragement and valuable discussion. This work was supported by Association Franqaise de Lutte contre les Myopathies and Fondation pour la Recherche M6dicale. [1] Acsadi, G., Jiao, S.S., Jani, A., Duke, D., Williams, P., Chong, W. and Wolff, J.A., Direct gene transfer and expression into rat heart in vivo, New Biol., 3 (1991) 71-81. 12] Burden, S., Development of the neuromuscular junction in the chicken embryo: the number, distribution, and stability of acetylcholine receptors, Dev. Biol., 57 (1977) 317-329. [3] Betz H., Bourgeois J.P. and Changeux J.P., Evolution of cholinergic proteins in developing slow and fast skeletal muscles in chick embryo, J. Physiol., 302 (1980) 197-218. [4] Cepko, C.L., Ryder, E.F., Austin, C.P., Walsh, C. and Fekete, D.M., Lineage analysis using retrovirus vectors. In P. Wasserman and M. De Pamphilis (Eds.), Methods in Enzymoiogy: A Guide to Techniques in Mouse Development, Academic Press, Orlando, FL, 1993, pp. 933-960. [5l Davis, H.L., Whalen, R.G. and Demeneix, B.A., Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression, Hum. Gene Ther., 4 (1993) 151-159. [6] De Luze, A., Sachs, L. and Demeneix, B.A., Thyroid hormonedependent transcriptional regulation of exogenous genes transferred into Xenopus tadpole muscle in vivo, Proc. Natl. Acad. Sci. USA, 90 (1993) 7322-7326. 17] Fynana, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C. and Robinson, H.L., Protective immunization by parenteral, mucosal and gene gun inoculations, Proc. Natl. Acad. Sci. USA, 90 (1993) 11478-11482. [8] Hamburger, V., Embryonic mobility in vertebrates. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, Rockefeller University Press, New York, 1970, pp. 141-151. [9] Hengge, U.R., Chan, E.F., Foster, R.A., Walker, P.S. and Vogel, J.C., Cytokine gene expression in epidermis with biological effects following injection of naked DNA, Nature Genet., 10 (1995) 161-166. [10] Jiao, S., Williams, P., Berg, R.K., Hodgeman, B.A., Liu, L., Repetto, G. and Wolff, J.A., Direct gene transfer into non-human primate myofibers in vivo, Hum. Gene Ther., 3 (1995) 21-33. [lll Klarsfeld, A. and Changeux J.P., Activity regulates the levels of acetylcholine receptor a-subunit mRNA in cultured chicken myotubes, Proc. Natl. Acad. Sci. USA, 82 (1985) 4558-4562. [12] Klarsfeld, A., Daubas, A., Bourachot, B. and Changeux J.P., A 5' flanking region of the chicken receptor a-subunit gene confers specificity and developmental control of expression in transfected cells, Mol. Cell. Biol., 7 (1987) 951-955. [13] Landmesser, L. and Morris, D.G., The development of functional innervation in the hind limb of the chick embryo, J. Physiol., 249 (1975) 301-326. [14] Merlie, J.P. and Komhauser, K.M., Neural regulation of gene expression by an acetylcholine receptor promoter in muscle of transgenic mice, Neuron, 2 (1988) 1295-1300. [15] Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Feigner, P.L., Direct gene transfer into mouse muscle in vivo, Science, 247 (1990) 1456-1468.