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Characterisation and developmental regulation of the Xenopus laevis CCAAT-enhancer binding protein beta gene.
Kousteni S
,
Kockar FT
,
Sweeney GE
,
Ramji DP
.
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We report here the cloning, characterisation and developmental expression profile of the Xenopus laevis CCAAT-enhancer binding protein beta (xC/EBPbeta) gene. The protein synthesised from the xC/EBPbeta gene interacts specifically with a C/EBP-recognition sequence and acts as a transcriptional activator. Several conserved regions are present in the xC/EBPbeta sequence, including the basic region, leucine zipper, activation domains, three in-frame AUG codons, and a consensus site for mitogen activated protein kinase. The corresponding mRNA is present at high levels in the kidney, liver, lung, muscle and adipose tissue, and at low levels in the ovary, brain and heart. Although the xC/EBPbeta mRNA and protein are present throughout embryogenesis, there is a biphasic increase in their expression levels during development. Whole-mount in situ hybridisation shows a restricted spatial expression profile of the xC/EBPbeta gene during early embryogenesis, with transcripts present around the blastopore lip and in the endodermal cells at the mid-gastrula stage, and, the whole dorsal side at the neurula and early tailbud stage. The expression domain becomes almost ubiquitous during later embryonic development, and includes the brain, spinal cord, somites and regions that give rise to the liver and the heart.
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9831641
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cebpb (CCAAT/enhancer binding protein beta) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage11, vegetal view, dorsal upper left.
cebpb (CCAAT/enhancer binding protein beta) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anteriorright, dorsal up.
cebpb (CCAAT/enhancer binding protein beta) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anteriorright, dorsal up.
Fig. 3. The spatial pattern of xC/EBPb expression during embryogenesis. Whole-mount in situ hybridisation to digoxigenin-labelled antisense (A) and sense probes (F), derived from the N-terminal region of the xC/EBPb gene, was carried out as described in Section 3. (A) mid-gastrulaembryo (stage 11) showing expression around the blastopore lip and in endodermal cells; (B) stage 22 embryo indicating expression in the fore brain and along the whole dorsal side; (C) stage 30 embryo indicating expression in the optic vesicle (ov), branchial arches (b), spinal cord (sc) and somites; (D) close view of a stage 30 embryo showing expression in the somites and the spinal cord; (E) stage 38 embryo, arrow indicates expression in the region that gives rise to the liver and heart; (F) representative control embryos probed with the sense probe, stages 11 (F), 22 (G) and 30 (I).
Fig. 2. Expression pattern of the xC/EBPβ gene. cDNA template was prepared against total RNA from either adult Xenopus kidney (Ki), muscle (Mu), lung (Lu), liver (Li), brain (Br), ovary (Ov), adipose tissue (Ad) and heart (He) (A) or from the following embryonic stages: 2 (cleavage), 7 (blastula), 11 (mid-gastrula), 14 (early neurula), 17 (mid-neurula), 23 (early tailbud), 26 (mid-tailbud), 36 (late tailbud) and 40 (early tadpole) (B). The cDNA was then used for PCR reactions in which primers against either the xC/EBPβ gene or the constitutively expressed β-actin or ODC gene (A and B, respectively) was present. The amplification products were subjected to Southern blot analysis, and their position is indicated by labelled arrows. (C). Western blot analysis was carried out on whole cell extracts from the embryonic stages indicated as described in Section 3. The blotted membranes were probed with rabbit antiserum to rat C/EBPβ ( Poli et al., 1990; Ramji et al., 1993), and the antigen–antibody complexes were detected using the ECL detection system. The positions of the xLAP* and xLAP polypeptides, produced by alternate use of translation initiation codons, are indicated by labelled arrows. The description of changes in band intensity included in the text are based on the replicated outcome of laser densitometric quantification.
