June 1, 2000;
Dissecting GHRH- and pituitary adenylate cyclase activating polypeptide-mediated signalling in Xenopus.
The highly conserved neuropeptide pituitary
adenylate cyclase activating polypeptide (PACAP
) has been implicated in a broad variety of physiological processes. The PACAP
precursor protein gives rise to three different peptides, the cryptic peptide, GHRH
, and PACAP
, respectively, and here we dissect their functional properties using Xenopus as model system. PACAP
but not the cryptic peptide directly neuralize animal caps. In contrast to GHRH
, the neuralizing effect mediated by PACAP
is independent of the PKA pathway. Moreover, PACAP
but not GHRH
behaves like a BMP-4 antagonist. Blastocoel
injection of PACAP
-38 but not of the closely related peptides PACAP
-27 and VIP
leads to strong anteriorization of the injected embryos suggesting the possible involvement of a novel PACAP
[+] show captions
Expression of PACAP mRNA in Xenopus embryos. (A) Expression of PACAP and its type-I-receptor (P-I-R) was studied at different stages (indicated by numbers) of Xenopus development by RT-PCR. (B) In early gastrula embryos, PACAP showed a widespread expression pattern with elevated expression in the dorsal marginal zone (DMZ) and the vegetal cap (VC). (C) Treatment of Xenopus embryos with lithium (Li) lead to strong upregulation of PACAP, while UV-irradiation (UV) did not influence the expression of PACAP. Abbreviations: AC, animal caps; VC, vegetal caps; VMZ, ventral marginal zone; LMZ, lateral marginal zone; DMZ, dorsal marginal zone; E, untreated control embryo; −RT, minus reverse transcription control sample.
PACAP dorso-anteriorizes embryos. Embryos at stage 8 were injected in the blastocoel with (A,C) carrier solution or (B,D) 1.3 pmol PACAP-38. Injection of PACAP-38 leads to anteriorization and cement gland enlargement (compare frontal view in D and C) Embryos depicted in (A,B) are three days old, while st.21 embryos are shown in (C,D). (E) cDNA constructs used for the in vitro transcription of mRNA microinjected into Xenopus embryos. Abbreviations: UTR, 5′-untranslated region; SP, signal peptide; CRY, cryptic peptide. (F) Animal cap assay: 4-cell embryos were microinjected into the animal region of all blastomeres with 0.5 ng mRNA per blastomer of preprolactin (PPL), CRY, GHRH, and PACAP. Animal cap fragments were cut from late blastulae, cultivated until stage 31 and analyzed by RT-PCR for marker gene expression. Co, animal cap fragment from uninjected embryo; E, uninjected control embryo; −RT, minus reverse transcription control sample.
PACAP induces secondary embryonic axes. Four-cell embryos were (A) uninjected (control) or (B) injected in two ventral blastomeres with 0.5 ng PACAP mRNA per blastomere. Ventral injection of PACAP mRNA led to development of an incomplete secondary axis. (C–F) Four-cell embryos were injected in the animal poles of all blastomeres. Final doses of mRNA per blastomere were (C) 0.125 ng BMP-4, (D) 0.125 ng BMP-4 and 0.25 ng PACAP, (E) 25 pg pCSKA-Wnt 8 (DNA), and (F) 0.25 ng PACAP and 25 pg pCSKA-Wnt 8 (DNA). While PACAP was able to rescue the BMP-4 phenotype (D), it could not antagonize the posteriorizing effects of late Wnt signalling (F).
PACAP has dorsalizing and neuralizing activity that is not dependent on the PKA pathway. (A) VMZ assay: 4-cell embryos were radially microinjected into all blastomeres. The final doses of mRNA per blastomere were 0.5 ng preprolactin (PPL), CRY, GHRH, and PACAP or 0.25 ng of transdominant-negative BMP-4 receptor (tBR) mRNA. In addition, stage 8 embryos were injected with 1.3 pmol PACAP-38 peptide in the blastocoel. Dorsal (DMZ) or ventral marginal zone (VMZ) fragments were cut from early gastrulae and analyzed at stage 11.5 by r-PCR for marker gene expression. − RT (minus reverse transcription control sample). Note, that PACAP, but not GHRH or CRY dorsalizes ventral marginal zones. (B) Animal cap assay: 4-cell embryos were microinjected into the animal region of all blastomeres. The final dose of mRNA per blastomere were 0.5 ng PACAP, dominant-negative PKA (dnPKA), and GHRH mRNA alone or in combination as indicated. Animal cap fragments were cut from late blastulae, cultured until stage 31 and analyzed by RT-PCR for the expression of marker genes as indicated. Co, animal cap from uninjected embryo; E, untreated control embryo; −RT, minus reverse transcription control sample.