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Shaggy is a downstream component of the wingless and Notch signaling pathways which operate during Drosophila development. To address the role of glycogen synthase kinase 3 beta (GSK3 beta), a mammalian homologue of Shaggy, in vertebrate embryogenesis, it was overexpressed in Xenopus embryos. Microinjection of rat GSK3 beta mRNA into animal ventral blastomeres of 8-cell-stage embryos triggered development of ectopic cement glands with an adjacent anterior neural tissue as evidenced by in situ hybridization with Xotx2, a fore/midbrain marker, and NCAM, a pan-neural marker. In contrast, animal dorsal injection of the same dose of GSK3 beta mRNA caused eye deficiencies, whereas vegetal injections had no pronounced effects on normal development. Using several mutated forms of rat GSK3 beta, we demonstrate that the observed phenotypes are dose-dependent and tightly correlate with GSK3 beta enzymatic activity. Lineage tracing experiments showed that the effects of GSK3 beta are cell autonomous and that ectopic cement glands and eye deficiencies arose directly from cells containing GSK3 beta mRNA. Molecular marker analysis of ectodermal explants overexpressing GSK3 beta has revealed activation of Xotx2 and of cement gland marker XAG-1, but expression of NCAM and XIF-3 was not detected. Phenotypic effects of mRNA encoding a Xenopus homologue of GSK3 beta were identical to those of rat GSK3 beta mRNA. We hypothesize that GSK3 beta mediates the initial steps of neural tissue specification and modulates anteroposterior ectodermal patterning via activation of Otx2 transcription. Our observations implicate GSK3 beta in signaling pathways operating during neural tissue development and during specification of anteriorectodermal cell fates.
Fig. 1. Developmental effects of microinjected GSK3b mRNA. In vitro synthesized GSK3b mRNA was
injected into a single blastomere of the 8-cell-stage embryos. The injected embryos were photographed at
stage 36. (A) Animal-ventralblastomere injected. The arrow indicates an ectopic cement gland. (B) Animaldorsal
blastomere injected. Defective eyes are apparent (compare with A or C). (C) Animal ventralblastomere was injected with mRNA encoding DAB deletion mutant of GSK3b.
Fig. 2. Cement gland formation at neurula stage caused by GSK3b
overexpression. (A) GSK3b mRNA was injected into a single ventral
blastomere at the 4-cell stage and the embryo was left to develop
until stage 21. The arrow indicates a large band of ectopic cement
gland in the ventrolateral region. (B) Control sibling embryo.
Fig. 3. Kinase activity and expression of different myc-GSK3b
constructs in injected embryos. Both blastomeres of 2-cell-stage
embryos were injected with 2 ng of mRNAs encoding wild-type
GSK3b (GSK), Y216F point mutant (F216), DE and DH deletion
mutants. The injected embryos were lysed at stage 9 for
immunoprecipitation and for western blotting with anti-myc 9E10
antibodies. (A) Immunoprecipitation and kinase activity analysis
using myelin basic protein (MBP) as a substrate. (B) Western
blotting with anti-myc antibodies. Uninjected embryos (lane C) were
used as a negative control in both experiments.
Fig. 4. Histological analysis of GSK3b mRNA-injected embryos. A single
blastomere of the 8-cell-stage embryos was injected with GSK3b mRNA or with
DAB mRNA. The injected embryos which developed until stage 39 were fixed,
sectioned and stained with hematoxylin/eosin. (A) Animal-dorsal blastomere
injected with GSK3b mRNA. One eye deficiency is apparent. Malformed neural
retina-like cells in the brain are indicated by the arrow. This section is cut at
somewhat different plane than the corresponding section in B, such that
notochord is not visible. (B) Animal-dorsal blastomere injected with DAB
mRNA. The injected embryos are morphologically similar to uninjected control
embryos. (C,D) Animal ventralblastomere injected with GSK3b mRNA.
Ectopic cement glands are visible. Arrow in D indicates malformed neural tube.
The uninjected side of this embryo is indistinguishable from that in normal
tadpoles. Abbreviations: bv, brain vesicle; cg, cement gland; nt, neural tube; nc,
notochord; mu, muscle. Bar in D (also applies to A, B and C) is 150 mm.
Fig. 5. Injection of GSK3b causes ectopic expression of anterior ectodermal and neural markers. Albino embryos injected with mRNAs from
rat (A) or Xenopus GSK3b (B) were analyzed by whole-mount in situ hybridization using XAG1 (A), Xotx2 (B). The bottom rightembryo in A
is a normal embryo for comparison with the other embryos that have ectopic cement gland. The arrowhead in B indicates ectopic Xotx2
expression, which is absent in the top control sibling embryo. Localization of muscle actin (C), Xotx2 (D) and NCAM (E) transcripts in wildtype
embryos injected with Xenopus GSK3b mRNA is shown in sections prepared after whole-mount in situ hybridization. After whole-mount
analysis, embryos at stage 24 (C and E) and at stage 33 (D) were sectioned. Ectopic neural tissue (indicated by arrows) expresses Xotx2 (D)
and NCAM (E). Abbreviations are as in Fig. 4, except n, notochord; s, somite. Scale bar is 150 mm.
Fig. 6. Cell autonomous effects of GSK3b overexpression on ectodermal cell fate. Lineage
tracing was carried out by injecting a single blastomere of the 8- to 16-cell-stage embryos
with 1 ng of b-gal mRNA alone (top embryo in A and in B) or with 0.8 ng of b-gal mRNA
and 0.5 ng of GSK3b mRNA (three bottom embryos in each group). The injected embryos
were cultured until sibling embryos developed to stage 36, fixed and stained for b-gal
activity. (A) Animal-ventral blastomere injected. Staining is concentrated in ectopic cement
glands. (B) Animal dorsal blastomere injected with b-gal. Head area is b-gal positive.
Some embryos also show staining in ectopic cement glands.
Fig. 7. Overexpression of GSK3b mRNA activates transcription of anterior ectodermal markers
in animal caps. Animal pole region of both blastomeres of the 2-cell embryos was injected with 2
ng of XGSK3b RNA (lane 1), with 0.4 ng of rat GSK3b RNA (lane 2), or with 0.05 ng of Noggin
RNA (lane 3). Animal caps were excised from blastulae at stage 8 and cultured until the
equivalent of stage 28 (A) or stage 11 (B), then, total explant RNA was extracted for northern
analysis. Lane 4, control animal caps from uninjected embryos; lane 5, control sibling embryos.
Cytoplasmic actin RNA cross-reacting with a muscle-specific actin probe (two upper bands) in A
and fibronectin RNA in B indicate equal loading. RNA from 10 animal caps or from 2 embryos
was loaded in each lane.
