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The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.
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10559480
???displayArticle.link???Mech Dev
Fig. 2. The phenotypes displayed by stage 28 and 40 embryos after expression
and stimulation of 5-HT1aR. (A) Control embryo injected with 4.6 ng
of t5-HT1aR mRNA into the ventral one blastomere at the 4-cell stage and
stimulated with 1 mMof 5-HT for 2 h at stage 8 showed normal morphology
at stage 28. Embryos injected with 5-HT1aR mRNA into the dorsal one
blastomere and stimulated with 5-HT also developed normally. (B) Embryo
injected with 4.6 ng of 5-HT1aR mRNA into the ventralblastomere at the 4-
cell stage and stimulated with 1 mM of 5-HT at stage 8 developed incomplete
secondary axis. Same phenotypes were obtained when ventrally
injected embryos were stimulated at stage 10 or 11 with lower frequencies.
Primary (18) and secondary (28) dorsal axes are indicated. (C,D) Stage 40
embryos treated in the same way as (A) and (B), respectively. Note that an
absence of head structure in ectopically induced second axis (D). Transverse
sections of a control (E) and an embryo injected ventrally with 5-
HT1aR mRNA (F) ®xed at stage 28. Note the presence of neural tube,
muscle structures and gut tube in the secondary axis of 5-HT1aR mRNAinjected
embryo.
Fig. 3. Expression and stimulation of 5-HT1aR partially rescues dorsal axis in ventralized embryos by UV-irradiation. UV-irradiated embryos were injected
into one blastomere with 4.6 ng of 5-HT1aR or t5-HT1aR mRNA. At stage 8, injected embryos were treated with 1 mM of 5-HT for 2 h and allowed to develop
until uninjected sibling embryos reached at stage 33. (A) Representative UV-irradiated control (top embryo) and partially dorsal-rescued by 5-HT1aR
stimulation (bottom embryo) embryos are shown. (B) The dorsal anterior index (DAI) of the embryos was scored, and the average DAI for each sample is
shown, with the sample size indicated above each bar. (C) Expressions of marker genes were analyzed by RT-PCR with 3 embryos sampled at stage 11.5. U,
UV irradiation and no other treatment; U/tR/5, UV irradiation, t5-HT1aR mRNA injection and 5-HT treatment; U/R/5, UV irradiation, 5-HT1aR mRNA
injection and 5-HT treatment.