Ryffel GU et al. (2003), Tagging muscle cell lineages in development and...

XB-ART-5461

Nucleic Acids Res 2003 Apr 15;318:e44. doi: 10.1093/nar/gng044.

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Tagging muscle cell lineages in development and tail regeneration using Cre recombinase in transgenic Xenopus.

Ryffel GU , Werdien D , Turan G , Gerhards A , Goosses S , Senkel S .

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The use of Cre and FLP recombinases to analyze embryogenesis and organogenesis in Xenopus has not been applied so far. We report on the generation of transgenic Xenopus animals containing a Cre-activated reporter gene cassette expressing blue fluorescent protein that can be switched over to yellow fluorescent protein expression upon Cre-mediated recombination. By injecting Cre mRNA into the two-cell stage embryo we show that Cre-mediated activation of the yellow fluorescent protein gene occurs. In addition, we observe upon injection an extinction of blue fluorescence in animals expressing the transgene and the induction of blue fluorescence in larvae containing a silent reporter gene. By crossing the reporter strains with animals expressing a muscle-specific Cre transgene we obtained an efficient and specific recombination of the reporter gene that leads to yellow fluorescence in myotomes and myofibrils of the developing larvae. Removal of the tail tips of these larvae allows the continuous recording of muscle cell differentiation in the regenerating tail. We detect a dramatic increase in transgene expression at the site of tissue removal in the tail stump. In the regenerated tail, yellow fluorescence is restricted to the myotomes thus excluding transdifferentiation of muscle cells.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a tbx2
Lines/Strains:

Xla.Tg(LCMV:eCFP,eYFP)2Ryff Xla.Tg(actc1:Cre)Ryff Xla.Tg(CMV:GFP,dsRED)Amaya Xla.Tg(CMV:eCFP,eYFP)1Ryff

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	Figure 1. Muscle-speciï¿½c activation of the EYFP gene. (A) Schematic drawing of the reporter gene LCMV:ECFP(loxP)(FRT)EYFP containing the CMV promoter driving the expression of the ECFP and EYFP genes. The ECFP gene with its polyadenylation signal is ̄anked by loxP and FRT sites allowing Cre and FLP recombinase-mediated deletion of the ECFP gene and thus leading to the expression of the EYFP gene. (B and C) Transgenic Xenopus generated by simultaneously introducing the reporter gene LCMV:ECFP(loxP)(FRT)EYFP and the Cre expression vector CAR-Cre that drives Cre activity by the muscle-speciï¿½c actin promoter (11). (B) Lateral view of a living larvae using ï¿½lters to detect blue (ECFP, left) or yellow (EYFP, right) ̄uorescence. (C) Detailed ventral view of another larvae using the same ï¿½lters. M, myoï¿½brils of the jaws; My, myotomes of the tail; G, gall bladder with auto ̄uorescence. The scale bars are 1 mm. The two double transgenic animals shown were derived from an experiment using 2900 injected eggs. We obtained 118 larvae of stage 35 (20) and 51 larvae were transgenic as assayed by ECFP ̄uorescence. Fifteen transgenic animals reached the swimming larval stage and two showed EYFP expres- sion in the muscle. The larvae in (B) has abnormal morphology. The use of egg extract in this experiment explains the low frequency of healthy tadpoles.
	Figure 1. Muscle-specific activation of the EYFP gene. (A) Schematic drawing of the reporter gene LCMV:ECFP(loxP)(FRT)EYFP containing the CMV promoter driving the expression of the ECFP and EYFP genes. The ECFP gene with its polyadenylation signal is Ì„anked by loxP and FRT sites allowing Cre and FLP recombinase-mediated deletion of the ECFP gene and thus leading to the expression of the EYFP gene.
	Figure 2. Founder animals at larval stage and F1 embryos. (A) Blue ̄uores- cence of two Xenopus larvae derived from a transgenic reaction with LCMV:ECFP(loxP) (FRT)EYFP. (B) Early tail bud stage embryos derived from the founder female C5 under normal light or blue ̄uorescence show that ~30% of the embryos are blue. The intensity of the blue ̄uorescence in F1 animals differs. The scale bars are 1 mm.
	Figure 3. Cre action in F1 larvae of C5 containing EYFP as an activated marker gene. Eggs of female C5 were fertilized with wild-type sperm and injected with a mixture of Cre (one part) and DsRed2 (three parts) mRNA at the two-cell stage. To get a high level of recombination we injected a high amount of Cre mRNA known to interfere with development in the tail bud stage. Injecting 364 two-cell stage embryos we obtained 42 swimming tadpoles (stage 45) (20) expressing the marker DsRed2. Pictures of living larvae are given. (A) Red, blue and yellow ̄uorescence of the lateral view of the middle part of an injected larvae. Twelve tadpoles showed such yellow myotomes. (B) Blue and yellow ̄uorescence of the lateral view of the middle part of an uninjected larvae. (C) Dorsal view of a swimming larvae in normal light as well as in red and blue ̄uorescence. Eight tadpoles showed this phenotype of extinction of blue ̄uorescence in the injected part. (D) Lateral view of a tail bud embryo in normal light as well as in red and blue ̄uorescence. This `blue-induction' phenotype was seen in 21 animals from a sample of 30 larvae derived from injected non- ̄uorescent two-cell stage embryos. All these animals died before reaching the swimming tadpole stage. The scale bars are 2 mm.
	Figure 4. Muscle-speciï¿½c recombination in crossings of Xenopus strains. (Aï¿½C) Eggs from reporter females containing the LCMV:ECFP- (loxP)(FRT)EYFP transgene were fertilized with sperm of transgenic Cre founder males expressing Cre under the control of the muscle actin pro- moter. Lateral (A and B) and ventral (C) views of larvae are given. The age of the larvae and the name of the founder animals are indicated for each panel. (D) Tail tip of a 28 day larvae in the region of gradual muscle differ- entiation. (E) Lateral view of a larvae obtained by the crossing of the repor- ter founder female C5 containing the LCMV:ECFP(loxP)(FRT)EYFP transgene with a control male lacking Cre recombinase. The left and right panel show the blue and yellow ̄uorescence, respectively. The scale bars are 1 mm.
	Figure 5. Muscle cell labeling in the regenerating Xenopus tail. Larvae derived from crossings between C5 and A6 that contain yellow muscle were grown up to stage 52 (day 28) and the tail tip was removed (see Fig. 4D). (Aï¿½C) Pictures in normal light, blue and yellow ̄uorescence are given at various times after tail removal. The border between the tail stump and the regenerate is marked by an arrow. (D) Close-up of a regenerated tail tip in blue (top) and yellow ̄uorescence (bottom) at day 16. The scale bars are 1 mm.
	Y2 construct cartoon. Y2: Contains the Cre reporter CMV:ECFP(loxP)EYFP. The larvae show blue fluorescence that turns into yellow fluorescence upon Cre recombinase action (Ryffel et al., 2003).
	Muscleâ€specific activation of the EYFP gene. (A) Schematic drawing of the reporter gene LCMV:ECFP(loxP)(FRT)EYFP containing the CMV promoter driving the expression of the ECFP and EYFP genes. The ECFP gene with its polyadenylation signal is flanked by loxP and FRT sites allowing Cre and FLP recombinaseâ€mediated deletion of the ECFP gene and thus leading to the expression of the EYFP gene. (B and C) Transgenic Xenopus generated by simultaneously introducing the reporter gene LCMV:ECFP(loxP)(FRT)EYFP and the Cre expression vector CARâ€Cre that drives Cre activity by the muscleâ€specific actin promoter (11). (B) Lateral view of a living larvae using filters to detect blue (ECFP, left) or yellow (EYFP, right) fluorescence. (C) Detailed ventral view of another larvae using the same filters. M, myofibrils of the jaws; My, myotomes of the tail; G, gall bladder with autofluorescence. The scale bars are 1 mm.

References [+] :

Amaya, A method for generating transgenic frog embryos. 1999, Pubmed, Xenbase