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Activins are known to be potentially important regulators in Xenopus developmental processes. It has been shown that activins exist maternally in the egg and can induce mesodermal tissues in blastula animal cap explants. However, the blastula ectoderm is known to possess a predisposed local response pattern to activin, and the process of the prepatterning is not understood. We isolated animal hemispheres from late 8-cell-stage embryos and treated them briefly with activin A. Expression of the muscle-specific actin gene was induced after a 30-min activin treatment, even when it was followed by treatment with follistatin, an activin-specific binding protein. This suggests that the animal-half blastomeres become competent to activin A before the 16-cell stage. In the normal embryo, the 8-cell-stage animal dorsal blastomeres populate neural ectoderm and most of the dorsal lip of the gastrula blastopore, the region of Spemann's organizer, and are the major progenitor for dorsal mesodermal tissues. When the dorsal and ventral animal-half blastomeres of the 8-cell stage were isolated and treated with activin independently, significant differences in tissue differentiation were observed. Dorsal blastomeres gave rise to trunk and tail structures containing dorsal mesoderm, whereas the ventral blastomere explants formed spheres containing solely ventral mesoderm. Further, both muscle actin transcription and goosecoid transcription were induced primarily in dorsal blastomeres. Our results suggest that a competence prepattern of response to activin exists as early as the 8-cell stage.
FIG. 1. Examples of blastomeres isolated at the late 8-cell stage. In
all cases a nimal blastomcres of the dorsal side are shown by D and
those of the venLral side are shown by V. (A) An 8-ccll embryo transferred
in PBS at the late 4-cell stage. (B) The isolated animal and
vegetal hemispheres after removal of the vitelline membrane at the
late 8-cell stage. (C) S3llle as A except that the embryo was transferred
to PBS at the 2-cell stage. Note that the blastomeres are not
adhering a t the animal pole. (D) Four animal-half blastomeres isolated
separately.
FIG. 2. lnduct.ion by acti.,.in A of muscle actin gene on the animal
halves isolated at the late 8-cell stage. RNA extracts were prepared
from explants cultured for 2 days and assayed by RNase protection
with ~·P-Iabclcd muscle-specific a -actin ant.isensc RNA. Cytoskeletal
{3-actin RNA is a lso assayed by the same probe and serves as a control
for RNA recovery. (A) Animal-half <:xplants treat.ed with 50 ng/ml
activin A for different times. Lane 1, probe. Lane 2, tailbud stage
whole embryo. Lanes 3-6. Total animal-half explnnts treated with
activio A for 0, SO, 60, and 120 min. RNA from one embryo or five
animal-half explants is loaded in ea.eh la ne. (B) RNA extracts were
prepared from explants treated under different conditions with peptide
factors. The concent ration of activin A was 50 ng/ml and that of
follistatin was 500 ng/ml. Lane 1, control explnnts. Lane 2, explants
cultured in follistatin for 2 days. Lane 3, explants cu ltured in activin A
together wi t h Collistatin for 2 days. Lane 4, explants Lrealed with
activin A for t h•·, washed in SS, and cultured in foll istalin for 2 d~ys.
Lane 5, Explants treated with activin A for 1 hr, was hed, and cultured
in SS for 2 days. I(NA from five animal-half explants is loaded in each
Jane.
FIG. 3. Dilf erential induction or mcsoderm-specilic marker genes in
the ex plants from dorsal and ventral blastomercs. (A) Single ventral
(AV) and dorsal (AD) anima! blastomeres were incubated with or
without 50 ng/ ml activin A for60 min, washed, and cultured in SS for
2 days. RNA was extn•cted and probed by RNasc protl:c~ion with muscle-
specific a--actin antisense 'RNA. RNA frorn 20 bh1stomeres is
loaded in each Ja ne. (B) RNA ex)~acts were prepared from explants
cultured for 10 hr and probed on RNase protection mapping with 82J>-
labeled {lrKlsecoid arid E'Fi-'a a' nt isense RNAs. Lane I. early gast rula
whole embryos. Lanes t.-5, total anima1-half ~xplants treated with 50
ng/ ml activin A for 0, 60, 120, and 180 min. Lanes 6-7, single dorsal
and ventral animal blnslomeres incubated with 50 ng/ml activin A for
180 min. RNA from 10 embryos or 20 explants is loaded in each lane.
FIG. 4. Differential response of dorsal and ventral blastomeres to activin A. Isolated dorsal and ventral blastomeres were incubated with o:r
without 50 ng/ ml activin A for 60 min and cultured for 4 days. Morphology (A-F) and his tological sections (G-L ) of the eXI>Iants are shown. (A,
G) Control dorsal blastomere explants cultured without activin A, which differentiated atypical epidermis and sucker cells. (B, H) Control
ventralblastomere ex plants cultured without activin A, which differentiated atypical epidermis. (C, I) Dorsal blastomere expla.nts treated with
activin A. Notochord, muscle, mesenchyme, and neural tissue are formed. (D, J) Ventralblastomere explants treated with activin A. Ventral
mesoderm such as mesenchyme and coelomic epidermis are formed. (E, K) Dorsal blastomere explants cultured in follistatin after the treatment
with activin A. (F, L) Ventralblastomere cxplanls cultured in follistat in after the treatment with activin A. sue. sucker cells (cement
gland); epi, normal epidermis; neu. neural tissue: not, notochord; mus, muscle; mes, mesenchyme; coe, coelomic epithelium.
