Gurdon JB et al. (1985), Activation of muscle-specific actin genes in Xe...

XB-ART-29162

Cell 1985 Jul 01;413:913-22.

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Activation of muscle-specific actin genes in Xenopus development by an induction between animal and vegetal cells of a blastula.

Gurdon JB , Fairman S , Mohun TJ , Brennan S .

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Muscle gene expression is induced a few hours after vegetal cells of a Xenopus blastula are placed in contact with animal cells that normally develop into epidermis and nerve cells. We have used a muscle-specific actin gene probe to determine the timing of gene activation in animal-vegetal conjugates. Muscle actin RNA is first transcribed in a minority of animal cells at a stage equivalent to late gastrula. The time of muscle gene activation is determined by the developmental stage of the responding (animal) cells, and not by the time when cells are first placed in contact. The minimal cell contact time required for induction is between 1 1/2 and 2 1/2 hr, and the minimal time for gene activation after induction is 5-7 hr.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a sp6
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	Figure 1. Design of Induction Experiments The diagram shows the position of cuts used to separate animal, vegetal, and equatorial regions of a blastula, as well as the preparation of animal-vegetal conjugates and exconjugates.
	Figure 2. Morphology of Embryo Fragments before and after Induction Whole views of animal (A, B) and vegetal (C, D) regions cultured in isolation or as conjugates (E, F), compared to complete embryo controls (G, H). Views on the left were taken 30 min after the time of isolation or conjugation (stage 8), and those on the right 15 hr later, when controls (H) had reached stage 18. Results of this kind have been obtained in all experiments (over 20 series with eggs of different females).
	Figure 3. Transcription of Muscle-Specific Actin Genes in Conjugates RNA was extracted from conjugates (AnNeg), or from isolated animal (An), vegetal (Veg), or equatorial (Eq) regions, of stage 8 blastulae, cultured until control (Whole) embryos had reached stage 20. (A) $1 nuclease protection analysis using an M13 DNA probe specific for Xenopus laevis cardiac actin RNA, as described by Mohun et al. (1984) and Gurdon et al. (1984). DNA markers (Mkr) were obtained from a Hinf I digest of +X174. (B) RNAase protection analysis using an SP6 probe specific for Xenopus laevis cardiac actin RNA (see text and Experimental Procedures). Lanes 4 and 5 each show the analysis of a single whole embryo (control) from two experiments. RNA markers (lane 6) were run-off transcripts of the pSP65 vector linearized at various sites downstream of the SP6 promoter. Results of the kind illustrated have been obtained in over 20 experimental series with eggs of different females.
	Figure 4. Synthesis of Muscle-Specific o~-Actin in Conjugates Conjugates (A, B) and animal tissue alone (C) were prepared from stage 8 blastulae and incubated in 3SS-methionine until controls had reached stage 18. At this time, conjugates were roughly separated into animal (A) and vegetal (B) parts, and all samples were analyzed by twodimensional gel electrophoresis, e: muscle-specific actins. -(, fl: cytoskeletal actins. Arrows indicate reference proteins used to confirm the location of ,~-actin. The small amount of o~-actin in B is attributable to residual animal cells left on the vegetal part when separated after 15 hr of contact. Each analysis is of ten or more equivalent conjugates or animal pieces pooled for extraction.
	Figure 5. Cellular Distribution of a Muscle- Specific Protein Sections on the left (A, C, E, G) show the use of Hoechst nuclear stain; sections on the right (B, D, F, H) were viewed with rhodamine attached indirectly to the 12/101 antibody. (B) The monoclonal antibody 12/101 binds only to muscle cells of Xenopus embryos reared from fertilized eggs; transverse section, stage 26. (D) The same antibody binds to one or two regions in the animal part of conjugates cultured until controls reached stage 26; transverse section. (F) Shows the clustering of cells into myotomes characteristic of Xenopus embryo muscle; longitudinal section. (H) An animal region cultured alone failed to bind the antibody. All photographs were taken under appropriate fluorescence conditions. Positive antibody binding in conjugates, and the absence of it in animal only controls, has been seen in all six samples of each kind which have been serially sectioned throughout. Each sample yielded over 50 sections, all of which were examined after antibody treatment.
	Figure 6. Stage Specificity of Induction; Examples of SP6 Analyses Pieces of animal or vegetal tissue, isolated as shown in Figure 1 from the blastula or early gastrula stages indicated, were used to prepare conjugates and cultured until controls reached stage 18. Extracted RNA was analyzed with our SP6 probe, which gives a protected fragment of 285 nucleotides when hybridized to cardiac actin mRNA (~,-) and of 135 nucleotides with cytoskeletal actin mRNA (0), as explained in Experimental Procedures. The presence of cytoskeletal actin RNA is an important internal control for the absence of cardiac actin RNA in the right-hand lane of each group.
	Figure 7. Stage Specificity of Induction; Summary of Results Seventy-seven SP6 analyses of the design shown in Figure 6 are summarized. A cardiac actin RNA band was classified as clearly positive (+), within a factor of 2 of the controls; clearly negative (-), at 1east 10 times less than a positive signal; or weakly positive (+_), 5-10 times less than a positive signal. Each plus or minus indicates the result of a single conjugate analysis. N+F stage refers to Nieuwkoop and Faber (1956).
	Figure 8. The Time of Gene Activation after Conjugation at Different Stages Pieces of animal tissue were taken from blastulae or very early gastrulae (stage 7 or 10), combined with stage 8 vegetal tissue, and incubated until frozen at the final stage indicated. Analyses made use of an SP6 probe (see Experimental Procedures). Conjugates started at stage 7 transcribe cardiac actin genes at stages 12-13, 2 hr before those started at stage 10 (gene transcripts first seen at stage 14). Cardiac, Cytoskel., see legend to Figure 6.
	Figure 9. The Time of Gene Activation Is Related to the Developmental Stage of the Responding Cells Animal tissue at stage 7, 9, or 10 was combined with stage 8 vegetal tissue and incubated for the times shown. The upper group of lines indicates results with fertilized eggs. +, -+, and - represent the amount of cardiac actin RNA as explained in the legend to Figure 7, each sign being the average of two analyses from two independent experiments that were in close agreement.
	Figure 10. Separation of Animal and Vegetal Parts of Conjugates Vegetal cells were taken at stage 8 from embryos injected at the one-cell stage with 3H-thymidine and were combined with unlabeled stage 8 animal tissue. All figures are autoradiographs of sections. (A) A conjugate incubated until control stage 18. Nuclei of vegetal origin (large yolk platelets) are intensely labeled, and nuclei of animal origin almost unlabeled (enlarged view in B). (C, D) The animal part of a conjugate fixed as soon as isolated 5 hr after conjugation; a single contaminating vegetal cell is easily seen (enlarged view in D). (E, F) Two animal exconjugates separated after 3 hr and cultured until control stage 18; note the absence of any contaminating labeled vegetal cells.
	Figure 11. The Minimum Cell Contact Time Required for Gene Activation Stage 8 animal and vegetal tissues were placed in contact and separated after the times stated, using 1251-labeledv egetal cells to monitor complete vegetal cell removal (see text). RNA was analyzed by our SP6 probe. Note the cytoskeletal actin RNA in the 1 hr analyses. Two other similar experimental series have been analyzed. Altogether eight samples with a contact period of 40 min to 11/2 hr were negative; seven samples with a 21/2-3 hr contact period, and numerous samples in contact for 5-10 hr, were positive.