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Figure 1. Distribution of the Epidermal Antigen Defined by 2F7J.Z’ on Sections of Early X. laevis Embryos
Ten p cryostat sections from TCA-fixed embryos embedded in acrylamide were stained by indirect immunofluorescence with 2F7C7 and FITCconjugated
RAM IgG. (a) stage 7, early blastula; (b) stage 12%, late gastrula; (c) stage 14, early neuruta; (d) stage 19, late neurula. Abbreviations
are as follows: a, archenteron; ap, animal pole; b, blastocoel; e, endoderm; ep, epidermal ectoderm; g, graft; m, mesoderm; np, neural plate; nt,
notochord; pnp, presumptive neural plate; s, somites. Bar = 190 f~
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Figure 2. Expression of the Epidermal Marker on Animal, but Not Vegetal Explants
Explants were removed from whole embryos by dissection and were incubated in MBS until control embryos reached stage 19. Cryostat sections
of 10 microns were cut from TCA-fixed explants embedded in acrylamide and were stained by indirect fluorescence with 2F7C7 and FlTCconjugated
RAM IgG. Phase contrast (a and c) and fluorescence (band d) paired images of the same regions are shown. (a and b) Stage 9 animal pole explant;
(c and d) stage 9 vegetal pole explant. Bar = 75 w
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Figure 3. Expression of the Epidermal Marker on Explants from the Inner and Outer Layers of Ectoderm
Inner and outer layers of ectoderm from the animal pole region were separated and cultured in MBB until control embryos reached stage 19. Explants
were fixed, embedded, and stained as in Figure 2. All parts show antibody fluorescence. (a) Explant from the outer layer of ectoderm from embryo
at stage 10; (b) explants from the inner layer of ectoderm from embryos at stage 10. Arrows indicate some of the internal, isolated positive cells.
Bar = 75 fi
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Figure 4. Epidermal Differentiation in Disaggregated Embryos
Animal caps were dissected from demembraned embryos and were incubated in Ca2+- and Mg2+-free NAM until control embryos reached stage
19. The disaggregated cells were then fixed in 2% TCA and were stained by indirect fluorescence with 2F7C7 and FITC-conjugated RAM IgG. (a
and b) Stage 6. late cleavage; (c and d) stage 7, early blastula; (e and f) late blastula. Phase contrast (a, c, and e) and fluorescence (b, d, and f)
paired images of the same field are shown. (g) Whole embryo put in Ca2+. and Mg*+-free medium at the l-cell stage and reaggregated by addition
of Cazf when control embryos reached stage 8. Bar = 75 r~ (a-f), 50 ~1 (g).
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Figure 5. s5S-Methionine Incorporation into Disaggregated and
CytochalasinTreated Embryos
Embryos from stages 6 to 12Vz were demembraned and incubated either
in Gas+- and Mgz+-free Barths’ saline or in cytochalasin S at 10
pgglml in complete Barths’ saline until control embryos reached stage
12%. At this point 25 &i %-methionine was added to each well, and
the embryos were incubated for 1 hr more. The %S incorporation into
TCA-precipitable radioactivity was then determined. Radioactivity,
mean counts of triplicate samples, is expressed as counts per minute
per microgram of protein. The bar represents the stages from which
neither treatment prevents subsequent 2F7.C7 binding. The dotted
bar represents stages when very small, but detectable, numbers of
cells express 2F7.C7.
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Figure 6. Epidermal Differentiation of Ectodermal Caps in the Presence of Cytochalasin B
Ectodermal caps were dissected from demembraned embryos from stages 6% to IO and were incubated in cytochalasin B at 10 &ml until control
embryos reached stage 19. Cells were then fixed, stained, and analyzed as described in Figure 1. All parts show indirect fluorescent staining of
2F2.G’. (a) Stage 6, late cleavage; (b) stage 7, early blastula; (c)stage 6, midblastula; (d) stage 9, late blastula; (e) stage 10, early gastrula; (f) control
animal pole cap in the absence of cytochalasin 8. (a) and (b) are exposed to show the background fluorescence of cells negative for 2F7.G’. This
diffuse cytoplasmic staining is in fact yellow. (c, d, e, and 1) The characteristic pattern of intense surface fluorescence of 2F7C7 on cytochalasin
B-treated cells (c, d, and e) or on an intact animal cap explant (f) taken from an untreated stage 6% embryo. Bar = 75 ,u
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Figure 7. Epidermal Differentiation of Ectodermal Caps in the Presence of Actinomycin D
Ectodermal caps were dissected from demembraned embryos from stages 9 to 12 and were incubated in actinomycin D at 100 pg/ml until control
embryos reached stage 22. Cells were then fixed, stained, and analyzed as described in Figure 1. All parts show indirect fluorescent staining of
2F7.G’. (a) Stage 10, early gastrula; (b) stage Wh, midgastrula; (c)stage 12, late gastrula; (d) stage 12M, late gastrula. Caps dissected from before
stage 10 gave a result identical with stage 10 caps. Bar = 75 r~.
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Figure 8. Summary Diagram of Periods of Insensitivity to Cell Disaggregation,
Rescue of Disaggregation by Caz+ Addition, and Independence
from Cell Division and Actinomycin D
The stages of development are from Nieuwkoop and Faber (1957), and
the time scale is from their normal table at 23%. The solid lines show
periods over which the treatments indicated allow the appearance of
the epidermal marker at about the normal time. The Ca2+ rescue experiment
shows the time at which Ca2+ can be added to embryos disaggregated
from the l-cell stage to allow appearance of the epidermal
marker. The dotted bar represents stages when very small but detectable
numbers of cells express 2F7C7.
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