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cAMP is a second messenger controlling various cellular processes through cAMP-dependent protein kinase (cAPK, PKA) and cyclic nucleotide-gated ion channels. Recently, the PKA-independent-cAMP-mediated signaling pathway by means of exchange protein directly activated by cAMP (Epac) has been demonstrated. Epac is a guanine nucleotide-exchange factor (GEF) for Rap, a Ras-like small GTPase. To investigate this new target for cAMP in development, we have isolated Xepac, the Xenopus laevis homologue of Epac by cDNA library screening. Xepac (Xepac1) encodes 890 amino acids, which have 57% identity with human Epac1 and 59% with that of rat Epac1 in amino acids. Whole-mount in situ hybridization and reverse transcriptase-polymerase chain reaction analysis show that XEpac is expressed both maternally and zygotically and is restricted within the developing hatching gland. Intriguingly, overexpression of XEpac induces the anterior markers XAG-1 and XOtx2 and can convert ectoderm into cement- and hatching gland-expressing cells. These results suggest that XEpac contains anterior positional information.
Figure 2. Temporal and spatial expression patterns of XEpac. A: Temporal expression patterns. XEpac was expressed both maternally and zygotically. RT(-), lacking reverse transcriptase. B: Spatial expression patterns. XEpac is restricted exclusively within the developing hatching gland, which forms a characteristic inverted Y type and functions as secreting hatching enzyme. It first appeared in the early neurula and persisted as long as the hatching gland existed (a-f, black and open arrowheads); no positive signals were detected with XEpac sense probe (g). a is early neurula stage; b and c are late neurula and its sagittal section, respectively; d, e, and g are tail bud stage; f is tadpole stage. c is a lateral view, g dorsal; others are anterior or dorsoanterior views.
rapgef3 (Rap guanine nucleotide exchange factor (GEF) 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, anterior view, dorsal up.
Fig. 3. XEpac modulates other hatching gland markers and induces anterior markers. A: Reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis was performed with RNA from XEpac
mRNA (2�4 ng) -injected animal caps for expression of other hatching gland markers. Overexpression
of XEpac induced XAG-1 and XA-1 but reduced XHE, a Xenopus hatching enzyme, and Cx30,
Xenopus connexin30. Additionally, the XOtx-2 anterior neural marker was induced by overexpression
of XEpac. Induction of these markers is irrespective of the induction of Xbra, a pan-mesodermal
marker. Ornithine decarboxylase (ODC) is used as loading control. RT( ), no reverse transcriptase
control; WE, whole embryo. B: XEpac induces XAG-1 and XOtx2 in naive ectoderm and
whole embryo. Two to 4 ng of XEpac were injected into the prospective ectoderm in 2-cell Xenopus
embryos. The injected ectoderm and noninjected control ectoderm were dissected at stage 8.5 and
aged until stage 25. Explants and whole embryos were stained for XAG-1 (a� d) and XOtx2 (e� h) by
in situ hybridization. a, c, e, and g are noninjected control; and b, d, f, and h are injected with XEpac.
Overexpression of XEpac mRNA converted ectoderm into XAG-1- and XOtx2-expressiong cells
and led to the expansion of XAG-1 and XOtx2 expression in the whole embryo. The numbers shown
in a, b, e, f indicate the number of explants exhibiting patches of darkly stained cells per total
number of explants analyzed.
