Zernicka-Goetz M et al. (1996), An indelible lineage marker for Xenopus using a...

XB-ART-17328

Development 1996 Dec 01;12212:3719-24. doi: 10.1242/dev.122.12.3719.

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An indelible lineage marker for Xenopus using a mutated green fluorescent protein.

Zernicka-Goetz M , Pines J , Ryan K , Siemering KR , Haseloff J , Evans MJ , Gurdon JB .

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We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed.

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Species referenced: Xenopus
Genes referenced: drosha eomes

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	Fig. 1. Whole embryos or larvae labelled by injecting 1 ng GFP.RN3 mRNA into the animal hemisphere of each of the first two blastomeres. (A) Stage 8.5; (B) stage 20; (C) stage 25; (D) stage 37; (E) stage 47; (F) epithelial cells of stage-47 tadpole (all are GFPpositive, but some are brighter than others). (G) Lens of stage-49 tadpole; (H) same view as G, but of unlabelled control tadpole. The black patches in A-D (and also in Fig. 2A-C) are due to pigment. The yellow regions in E are due either to gut autofluorescence or to the photographic consequence of very high intensity green fluorescence associated with the lens and residual sucker (see legend to Fig. 3D).
	Fig. 2. GFP.RN3- or BFP-labelling of single blastomere lineages. (A,B) 1 ng of GFP.RN3 mRNA injected into one of first two blastomeres; (A) stage 8, animal pole; (B) stage 18, right half of neural plate. (C-F) Injection of a single tier A blastomere at the 32- to 64-cell stage with GFP.RN3 mRNA; the descendants of this cell become progressively dispersed; (C) stage 8, animal pole; (D) stage 18 (labelled cells in left-hand neural fold); (E) stage 24; (F) stage 35 (labelled cells in anterior (head) region in E and F). (G,H) Injection of two adjacent tier A blastomeres on one side of a 16- to 32-cell embryo, one with with BFP, and the other with GFP.RN3; (G) stage 18; (H) stage 26, higher magnification. (I,J) Injection of a single blastomere in tier B at the 32- to 64-cell stage with GFP.RN3 mRNA. Note labelling of individual muscle cells in each segment (I) and in interspersed or alternating segments (J). I and J both show the tail region of stage-40 tadpoles (head out of view to the left in I, right in J). The white lines outline the shape of the tail and two single somites containing at least 100 muscle cells.
	Fig. 3. Transplantation of a single stage-8, GFP.RN3-labelled cell into the blastocoel of an unlabelled stage-8 host. (A) stage 10; (B) stage 12; (C) stage 38; (D) stage 45. (A-C) Animal cap cell; (D) equatorial cell. In D the intestine appears yellow due to autofluorescence that develops in the tadpole gut. This autofluorescent yellow is unrelated to green fluorescence and is very clearly distinguishable when viewed under the microscope. The yellow spots in the middle of the single green cells in A-C are an artefact of photographic film, and are seen under the microscope as maximum intensity green (but not yellow) fluorescence.
	Fig. 4. Use of GFP.RN3 to determine the fate of cells overexpressing Eomes. 2-cell embryos were injected with a mixture of 1 ng GFP.RN3 mRNA and 2 ng Eomes mRNA into one blastomere at the 2-cell stage. This was done in order to achieve animal caps of which only part was induced to form muscle, the rest serving as a no-muscle control. Embryos were cultured to stage 8, when animal caps were removed and placed face to face in pairs, and cultured to control stage 26. (A) An animal cap double conjugate showing GFP.RN3 fluorescence in tissue projections caused by Eomes overexpression. (B) Transverse section of conjugate in A stained with 12/101 muscle specific antibody; (C) same sample under green fluorescence. Note that the strongly fluorescent extensions (A and C) are heavily stained with the muscle antibody (B), but that the central part of the conjugate (not extended) contains few GFP.RN3-containing cells and no 12/101 muscle antibody staining. The dark periphery of the central body is attributable to pigment and not to antibody staining.