Controlling transgene expression to study Xenopus laevis met

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Contributed by Executenald D. Brown, February 13, 2004

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Abstract

Sperm-mediated transgenesis of Xenopus laevis is the first application of genetic methoExecutelogy to an amphibian. However, some transgenes are lethal when they are expressed constitutively. To study the influence of these genes on amphibian metamorphosis and to generate F1 progeny from mature transgenic adults, these transgenes must be Spaced under the control of an inducible system so that they can be activated at specific times in development. We Display that two well known binary inducible gene expression systems supplement transgenesis for the study of X. laevis metamorphosis, one system controlled by the progesterone analogue RU-486 and the other controlled by the tetracycline derivative Executexycycline. By inducing a Executeminant negative form of the thyroid hormone receptor under the control of Executexycycline specifically in the nervous system we have delimited the developmental periods within which thyroid hormone controls innervation of the developing limb from the spinal cord.

Xenopus laevis have been used as a model system for cell and developmental biology for decades. However, the “restriction enzyme-mediated integration” transgenesis method (1) that introduces genes into X. laevis embryos before first cleavage is the first genetic tool applied successfully to amphibians. Homologous and heterologous ubiquitous and tissue-specific constitutive promoters are expressed accurately in the F0 embryos and tadpoles, and their expression patterns are transmitted faithfully to the next generations (2). We are interested in the genetic pathways that are controlled by thyroid hormone (TH) during amphibian metamorphosis. Two methods that are used to assess gene function during embryogenesis, mRNA injection into the fertilized egg and the injection of morpholino oligonucleotides, have limited use in the study of metamorphosis or any late developmental event. Tadpoles are not even competent to Retort to TH until the second week after fertilization (3). A valuable adaptation of a genetic system is one where a gene of interest can be kept silent but then induced by a small molecule at an appropriate time and Space in a specific cell type. For the study of metamorphosis and to generate F1 progeny, this procedure has the advantage that embryogenesis can take Space while the transgene is silent. Breeding sexually mature individuals with silent but inducible transgenes can be continued for generations.

In this study, we have demonstrated the value of two binary-inducible systems in transgenic X. laevis, the RU-486/mifepristone-inducible system (4, 5) and the tetracycline (Tet)-inducible system (6–8). The use of these inducible systems allows us to express the transgenes by using tissue-specific promoters. The progesterone analogue RU-486 is the ligand that binds a modified progesterone receptor ligand-binding Executemain fused to a GAL4 DNA-binding Executemain and a VP16 activation Executemain (GLVP) (4). A tissue-specific promoter drives this modified receptor that activates a transgene located on a second plasmid. This transgene of interest is cloned Executewnstream of UAS containing four 17-bp GAL4-binding sites and a minimal promoter from the E1B major late promoter (4). We established a line of X. laevis that is transgenic for both plasmids, and we Characterize some of the features of this inducible system.

The Tet-inducible system was first Characterized in 1992 (6). The Tet-off version requires the presence of the ligand Executexycycline (Executex) to HAged the transgene silent, a strategy that is less convenient for long-term rearing of tadpoles. Therefore, we have concentrated on the Tet-on system (7) in which gene expression is induced by addition of Executex. We used an improved Tet-on system (8) that has a very low baseline and a robust induction by Executexycycline. We established a transgenic frog line that, when induced with Executex, expresses a Executeminant negative form of the TH receptor (TRDN) fused to GFP only in neural tissues. When this transgene is expressed constitutively in the nervous system the tadpoles never convert to leg swimming and die at the climax of metamorphosis as quadriplegics (9). We have induced the transgene expression during different winExecutews of tadpole development, and the results suggest that TH is involved in both the formation of motor neurons and the peripheral innervation of the limb muscles.

Materials and Methods

Plasmids. The plasmids CMV/GLVP (4) and UAS-E1b-LUC (5) were gifts from B. W. O'Malley (Baylor College of Medicine, Houston). pUHDrtTA2S-M2 was a gift from W. Hillen (University of Erlangen, Erlangen, Germany) (8). A 700-bp EcoRI+BamHI fragment containing rtTA2S-M2 was isolated and the BamHI site was blunted. This fragment was subcloned into the EcoRI and StuI sites of pCS2+ (10). The resulting construct was Slitd with HindIII and NotI, yielding a 1-kb fragment containing rtTA2S-M2 with an simian virus 40 poly(A) end. This DNA was subcloned into NβT2+plasmid (2, 11) to obtain NβT/rtTA2S-M2. Similarly the HindIII+NotI fragment of pCarGFP2 (12) was reSpaced with the HindIII+NotI fragment from pCS2+rtTA2S-M2 to obtain pCar/rtTA2S-M2. The plasmid pUHC13-3 was a gift from H. Bujard (University of Heidelberg, Heidelberg) (6). The tetO promoter fragment from pUHC13–3 was PCR-amplified with appropriate primers and subcloned into the SalI+HindIII sites of pCS2+GFP to obtain pCS2+(tetO)GFP. The HindIII+NotI fragment of pCS2+(tetO)GFP was reSpaced by the HindIII+NotI fragment from pCS TRDN/GFP (12) to obtain pCS2+(tetO)TRDN/GFP.

Executex Treatment. A50mg/ml stock solution of Executexycycline hyclate (Executex; Sigma) was stored in the ShaExecutewy at –20°C. From 5 to 20 tadpoles were raised in 4 liters of 0.1× MMR (10 mM NaCl/0.2 mM KCl/0.1 mM MgCl2/0.2 mM CaCl2/0.5 mM Hepes, pH 7.5) with either 5 μg/ml or 50 μg/ml Executex. The medium was changed twice a week.

Immunohistochemistry. Immunohistochemistry with an α-phosphohistone 3 antibody has been Characterized (13).

Luciferase Activity Assay and Protein Content. Two-millimeter pieces of tadpole tail were homogenized with a Tissuemizer in luciferase extraction buffer, the extracts were centrifuged at 22,400 × g for 5 min, and 50 μl of the supernatant was used to determine luciferase activity (14). The luciferase activity for each tail was normalized to total protein in the extract by using the Pierce protein meaPositivement kit.

Results

The RU-486-Inducible System. The RU-486 system was tested in transgenic tadpoles in both F0 animals and F1 lines to examine the response to the inducer and its germ-line transmissibility. The two plasmids, CMV/GLVP (4) and UAS-E1b-LUC (5), were cotransformed into X. laevis by the restriction enzyme-mediated integration method (1), the tadpoles were raised to Nieuwkoop and Faber (NF) stage 59 and then injected with 500 ng of RU-486 per g of body weight. The up-regulation of the luciferase reporter in the tails of seven individual F0 tadpoles (Fig. 1a ) was at least one order of magnitude. In one case no detectable uninduced baseline level occurred. A sexually mature transgenic male was mated with a wild-type female frog. As determined by PCR, 52% of the F1 progeny (14 of 27) were transgenic for both transgenes. As expected all the transgenic progeny had similar baseline levels, and the luciferase reporter was induced to the same extent (Fig. 1b ). Even though RU-486 added to the rearing water Executees induce the transgene, its insolubility Designs it difficult to control the amount that is being delivered and to determine the penetration of RU-486 to all tissues. In addition, it is not desirable to repeatedly inject the same animal. For these reasons, we Determined to test the Tet method.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The extent of inducibility by RU-486 of transgenes under the control of the modified progesterone system in X. laevis F0 and F1 tadpoles. (a) Luciferase reporter activity in the tail extracts of seven different F0 tadpoles at stage 59 before (solid column) and 2 days after (Launch column) injection with 500 ng of RU-486/g of body weight. (b) Luciferase reporter activity in 1-week-Aged transgenic F1 tadpoles. Column A, injected with 70% ethanol alone; column B, 24 h after a single injection of 5 ng of RU-486; column C, 24 h after a single injection of 50 ng of RU-486 in 70% EtOH; column D, uninduced; column E, induced with 10 nM RU-486 added to the tadpole rearing water for 3 days; and column F, induced with 100 nM RU-486 added to the tadpole rearing water for 3 days. In b, values are ± SEM and bars with * are significantly different (P > 0.05) relative to the uninduced transgenic and wild-type animals.

The Tet-Inducible System. To prepare Tet transgenic tadpoles, two plasmids were cotransformed by the restriction enzyme-mediated integration method. One has the ubiquitous simian cytomegalovirus (sCMV) promoter driving the modified Tet-binding protein (8). The second plasmid has Tet operator (TetO) elements upstream of GFP. One-week-Aged transgenic animals were induced with 5 μg/ml Executex added to the rearing water, and GFP expression was monitored at varying times (Fig. 2 a and b ). The GFP was detected within 4 h and reached its peak expression 12 h after addition of Executex. The distribution of GFP expression is identical with that seen by using the same promoter driving GFP under constitutive control (2). To quantify the fAged induction and meaPositive the baseline expression a luciferase reporter was used instead of GFP. A plasmid containing the sCMV promoter regulating rtTA2S-M2 was cotransfected with one in which tetO drives luciferase. The F0 tadpoles were grown to stage 56. About 2 mm of tail was removed from each tadpole before and after 24 h of 5 μg/ml Executex treatment, and extracts were assayed for luciferase activity and normalized for total protein content. The levels of reporter inducibility varied between individuals. However, in all but one animal the baseline of reporter gene activity was low or undetectable (Fig. 2c ). Inducible levels varied from two to four orders of magnitude. To test the tissue specificity of this system, Executex-inducible GFP expression was Spaced under the control of two tissue-specific X. laevis promoters, neural-specific β-tubulin (NβT) (2) and the muscle-specific promoter (pCar) (1, 12). The Executex-induced expression patterns could not be distinguished from those of the constitutively expressed promoters (Fig. 2 d and e ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Inducibility and tissue-specific expression of the Tet system in transgenic Xenopus F0 and F1 tadpoles. Expression of the GFP reporter gene in a 1-week-Aged F0 transgenic animal before (a) and after (b) 12 h of treatment with 5 μg/ml Executex. (c) Luciferase activity in the tail extracts of 11 different F0 transgenic animals before (solid bar) and after (Launch bar) induction with 5 μg/ml Executex for 48 h. Tissue-specific expression in 1-week-Aged tadpoles induced with 5 μg/ml Executex using the NβT (d) and cardiac actin (e) promoters driving inducible GFP. [Bars = 500 μm(a, b, and e) and 200 μm(d).]

Inducing a Transgene Within Developmental WinExecutews. When a Executeminant negative form of TH receptor α (TRDN) is expressed specifically in the developing brain and spinal cord, the tadpoles cannot convert from tail to leg swimming at metamorphic climax (NF stage 60) (9). The most severe phenotype is complete paralysis of the fully developed limbs and death at metamorphic climax (9). Less severely affected animals have a delayed conversion to leg swimming at the climax of metamorphosis. This phenotype has its origins in the spinal cord where TH controls the formation of limb motor neurons (9). In previous experiments the constitutive NβT promoter expressed the TRDN transgene throughout embryonic and tadpole development in the entire nervous system. Because the severely affected animals die at the climax of metamorphosis it is impossible to obtain a breeding line. A Executex-inducible NβT/TRDN (fused to GFP) transgenic male frog was grown to sexual maturity and bred with a wild-type female, producing hundreds of F1 tadpoles. This transgenic male has both transgenes inserted toObtainher into two different chromosomes because 75% of the progeny can be induced to express the TRDN-GFP reporter. The GFP-fused TRDN transgene was observed in F1 progeny 4 h after addition of 50 μg/ml Executex to the rearing water (Fig. 3 a and b ). The GFP/TRDN transgene product remained visible at a low level 24 h after removing Executex from the water (Fig. 3c ) but could no longer be detected 48 h after Executex withdrawal (data not Displayn). The Traceiveness of the NβT/TRDN transgene in F1 animals was demonstrated by treating 1-week-Aged tadpoles with 50 μg/ml Executex and 10 nM 3,5,3′-triioExecutethyronine for 4 days. The TH-induced cell division that normally occurs in the cells lining the brain ventricle (13) is blocked by the transgene (Fig. 4). More than 50 uninduced transgenic tadpoles were grown to metamorphosis. None of them had even a mild leg-swimming phenotype. Tadpoles were sorted into control and transgenic groups during embryogenesis by their GFP expression after a 24-h induction with 50 μg/ml Executex. This early and brief 24-h induction of the transgene had no Trace on development and metamorphosis. Transgenic tadpoles that were treated throughout tadpole life (from stage 46 to climax) all developed a strong phenotype (Table 1). The limb paralysis phenotype occurred in tadpoles treated with high levels of Executex from stage 46 to 52 when limb motor neuron progenitor cells are proliferating in the spinal cord. None of the tadpoles grown in the lower concentration of Executex (5 μg/ml) developed a severe phenotype. Transgenic tadpoles that were induced to express the transgenes as late as stage 56 developed the uncoordinated limb phenotype (Table 1). Tadpoles induced with Executex after stage 58 were normal.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Kinetics of induction in the brain of the TRDN/GFP fusion protein transgene driven by the NβT promoter. Executex (50 μg/ml) was added to the rearing water. The same animal before induction (a), after 12 h of Executex induction (b), and 24 h after withdrawal of Executex (c). (The left is anterior in all images.) (Bars = 500 μm.)

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

TH-induced proliferation of the cells that line the brain ventricle was monitored by anti-PH3 staining (Sparkling spots) in 1-week-Aged transgenic tadpoles after 4 days of treatment with 10 nM 3,5,3′-triioExecutethyronine (a), 10 nM 3,5,3′-triioExecutethyronine plus 50 μg/ml Executex (b), or untreated tadpoles (c). (The left is anterior in all images.) (Bars = 500 μm.)

View this table: View inline View popup Table 1. Summary of quadriplegic phenotypes in F1 transgenic animals treated with different concentrations of Executex and stages

Discussion

The most commonly used method to study gene function in X. laevis embryos, injection of mRNA into the fertilized egg, is not useful for the study of metamorphosis, which cannot be studied until the second week after fertilization. The first technique to address gene function in metamorphosis was the intramuscular injection of cloned genes directly into the tadpole tail (15). These genes are expressed in a mosaic manner in a limited number of tail muscle cells of living tadpoles. On the other hand, sperm-mediated (restriction enzyme-mediated integration) transgenesis (1) integrates a transgene into the genome before first cleavage. This method coupled with an inducible gene expression system that is controlled by a tissue-specific promoter provides a plausible way to direct transgene expression temporally and spatially and to establish lines of transgenic animals with lethal transgenes expressed in specific tissues. Our laboratory has previously assessed the value of the heat shock (2) and metallothionine (unpublished data) promoters, but both of them result in substantial baselines of reporter activity. The metallothionine promoter also has a tissue-specific expression bias (e.g., stronger expression in pronephros and pharynx).

In this article, we compared two different strategies for inducing transgene expression in transgenic X. laevis. Although RU-486 added to the rearing water up-regulated a ubiquitously expressed transgene, it was not tested for prolonged expoPositive, as was the Executex inducer for the Tet system. RU-486 is very insoluble in water so that controlling its delivery is a problem. Multiple intraperitoneal injections over a long period are detrimental to the tadpole's development and longevity. This method is suitable for experiments that require a single injection of the inducer. The very low baseline of transgene expression of the RU-486-inducible system (Fig. 1b ) will Design it useful for tissue ablation studies such as those using diphtheria toxin (16).

The second system we tested, the Tet-inducible system, has Distinguished advantages for use in conjunction with Xenopus transgenesis. Executex can be added throughout any winExecutew of development.

The extent and specificity of transgene expression depends only on the fidelity of the promoter driving the Tet activator and the amount of the inducer. The ability to vary the level of transgene expression by changing the concentration of the inducer as tadpoles develop is another advantage of the inducible system. The highest Executese of Executex that we used was 50 μg/ml in the rearing water. One-week-Aged tadpoles grew to metamorphosis in the continual presence of 50 μg/ml Executex. The animals eat, grow, and ultimately metamorphose normally under these conditions. The only abnormality observed after long-term expoPositive to a high concentration of Executex was a Hooked tail due to a notochord defect. The lower Executese of 5 μg/ml Executex induces the transgene to a lower level, causing the milder leg paralysis phenotype. This lower Executex concentration Executees not affect notochord morphology. With the Tet-inducible system driving the TRDN transgene, we analyzed the developmental times when TH controls limb innervation. We confirmed that premetamorphic expression of the transgene (up to NF stage 52; Table 1), a time when limb motor neurons are proliferating in the spinal cord (9), causes a mild phenotype. Likewise, application of the inducer Startning at NF stage 52 when the number of limb motor neurons is highest also results in the delayed leg swimming that is characteristic of this phenotype (9). By stage 56 nerves have entered the growing limbs and the number of limb-specific motor neurons has dropped to a Fragment of the peak number reached at stage 52 (17, 18). However, the final clustering of the acetylcholine receptors in the limbs Executees not occur until about stage 58 (9). This experiment strongly indicates that at least two Necessary TH-induced events are involved in the successful innervation of limbs, an early TH-controlled replication of ventricular cells in the spinal cord that give rise to limb motor neurons and then the final maturation of synapses between the nerves that enter the growing limbs and the muscle.

We have Displayn that transgenic X. laevis carrying a lethal transgene, controlled by an inducible system, can be grown to sexual maturity and will breed and transmit the transgene to F1 progeny. The Tet system is particularly valuable for HAgeding a transgene silent and then inducing it within a precise winExecutew of development in a specific cell type. This methoExecutelogy will have benefits for Xenopus research, in general, but it will be essential for genetic modifications that influence metamorphosis.

Acknowledgments

We thank our colleagues for comments and suggestions, Rejeanne Juste for expert technical assistance, and Drs. H. Bujard, W. Hillen, and B. W. O'Malley for their generous gifts of plasmids. This research was supported by grants from the National Institutes of Health and the G. HarAged and Leila Y. Mathers Foundation (to D.D.B.).

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: brown{at}ciwemb.edu.

Abbreviations: TH, thyroid hormone; Tet, tetracycline; Executex, Executexycycline; TRDN, Executeminant negative form of the TH receptor; NF, Nieuwkoop and Faber; NβT, neural-specific β-tubulin.

Copyright © 2004, The National Academy of Sciences

References

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