Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. Here we show that CREB is the predominant CRE-binding protein in Xenopus embryos and that it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB that is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation resulted in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, as genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting the abnormal development in both neural and somitic derivatives. In summary, our data show that CREB is essential during several developmental stages of Xenopus embryogenesis.
???displayArticle.pubmedLink???
10525188
???displayArticle.link???Mech Dev
Fig. 1. Presence of CREB-like protein and CREB mRNA in Xenopus embryos. (A) Bandshift experiments using 32P-labeled CRE oligonucleotides and stage
20 embryonic nuclear extracts show the presence of CRE-binding activity (lanes 1 and 2). Anti-CREB antibodies (lanes 3 and 4) and anti-phospho-CREB
antibodies (lane 5) induced strong supershifts, but the antiserum ATF1.1 which is speci®c for ATF1 was unable to produce a supershift (lane 6). Antiserum
ATF1.2 recognizing both CREB and ATF1 produced a strong supershift (lane 8). Adding a 100-fold excess of unlabeled CRE oligonucleotides completely
abolished bandshift signal (lane 9). (B) Northern blot analysis showing a speci®c 2.6 kb band representing Xenopus CREB mRNA.
Fig. 2. Dominant-negative forms of CREB. (A) Schematic representation of the CREB constructs used for injection experiments. Full length constitutive
dominant-negative CREBA133 and leucine zipper mutant CREBDA133, respectively, were fused to the ligand binding domain of the human progesterone
receptor, which contains the C-terminal truncation D892-933 and is, thus, responsive only to the agonist RU486. (B) Transfection experiments showing
that CREBA133PR inhibits CRE-mediated transcription in a RU486-dependent and dose-dependent manner. CAT activity is expressed as percentage of the
maximal activity induced by wild-type CREB. Diagram represents the average of three independent experiments. (C) Western blot analysis to determine
CREBA133PR protein levels in injected embryos. CREBA133PR mRNA was injected into every cell of 4-cell stage embryos. Embryos were grown until the stage
indicated and lysed. An equivalent to two embryos was analyzed, and CREBA133PR protein was detected with anti-CREB antibodies. A strong band at the
expected size of 60 kDa was observed in extracts of injected embryos, but not in uninjected control embryos. Abbreviations: bZIP, basic DNA-binding domain
with leucine zipper; hPR-LBD, human progesterone receptor ligand binding domain; KID, kinase inducible domain containing serine 133 in wild-type CREB.
Fig. 4. Inhibition of CREB function leads to phenotypic changes of developing Xenopus embryos. All blastomeres of 4-cell stage embryos were injected
equatorially with CREBA133PR mRNA (750 pg each), and embryos were treated with 1 mM RU486 at the stages indicated and were cultured until stage 40.
Treatment at stage 8 resulted in strong spina bi®da and shortening of the anteroposterior extension, whereas treatment at stage 12 led to stunted embryos with
microcephaly. Injected embryos without RU486 treatment developed normally. Scale bar: 500 mm.
Fig. 5. Histological analysis of transverse sections at the level of the otic vesicles in the hindbrain. Note the slightly enlarged neural tissue and the decreased amount of somitic structures that is found consistently in injected and RU486-treated embryos (panels B, C and D). Injected, untreated embryos (panel A) did not show any phenotypic changes as compared to wild-type embryos. Scale bar: 100 mm.
Fig. 6. Expression analyses of CREBA133PR injected embryos. Representative embryos are shown that were injected with RNA encoding CREBA133PR, treated with RU486 at stage 12, and used for whole mount in situ hybridization analyses at stage 30 to visualize the expression pattern of MyoD, Otx2 and Shh. Note that in CREBA133PR-injected and RU486-treated embryos the fore- and midbrain expression domains of Otx2 are not well separated from each other and that the MyoD expression domain in the somites is split and does not show the typical V-shaped form of normal somites. The expression of Shh has not changed. Injected embryos without RU486 treatment developed normally and did not show any changes in expression of MyoD, Otx2 and Shh. Scale bar: 500 mm; scale bar for insets: 100 mm.
