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Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functions in the determination of the dorsal-ventral body axis. We have cloned two xnf7 cDNAs, xnf7-O and xnf7-B, that have a different temporal pattern of expression. The cDNAs differ by 39 amino acid residues scattered throughout the molecule. Most of the changes were conservative in nature. Using gene specific probes we found that xnf7-O transcripts were abundant in oocytes and decreased until the neurula stage, after which they increased in abundance. Xnf7-B transcripts were in low abundance in oocytes and were expressed at high levels at the neurula stage and in adult brain. Both xnf7-O and xnf7-B transcripts at the neurula stage were localized in the dorsal region of the embryo, including the neural folds and somites. Xnf7 was not expressed in ventralized embryos that lacked dorsal structures, thereby substantiating its dorsal localization in the embryo. The promoter region of the xnf7-O gene does not possess a TATA box but does contain E2F, USF, Sp1-like and AP1 binding sites within the first 421 bp from the transcription initiation site. A 62 bp fragment of the xnf7-O promoter containing the Sp1-like and E2F sites can direct proper spatial expression of a transgene in embryos.
Fig. 3. Developmental expression of the xnfl-0 and xnfl-B mRNA.
10u g of total RNA isolated from oocytes and different stage X. laevis
embryos was separated by electrophoresis and blotted onto Nytran
membrane. The northern blot was hybridized with random-prime labeled
xnf7 cDNA probes (specific activity 1 X lo9 cpm/,ug) 00. oocytes;
different embryonic stages are indicated by the numbers at the
top of the figure (Nieuwkoop and Faber, 1967). The top panel was from
a blot hybridized with a probe produced from the 5' untranslated leader
sequence of the oocyte clone. The second panel is the same blot rehybridized
with a brain specific probe from the 3' untranslated trailer of
the brain clone. The equivalence and integrity of the RNA was monitored
by ethidium bromide staining of the gel prior to blotting (not
shown) and by rehybridization of the blot with a Xenopus histone H4
and EF1 alpha probes.
Fig. 4. Spatial pattern of localization of xnff by in situ hybridization and Northern blot analysis. A. The spatial distribution of xnt7 mRNA in stage 17
and stage 26 embryos was analyzed by whole-mount in situ hybridization according to Harland (1991). The stage 17 embryo is observed from above
showing the neural folds which have just closed. The stage 26 embryo is shown from the side (upper panel) and from the dorsal aspect (lower panel).
The arrow points to the anterior of the embryos. Control embryos hybridized with the sense strand xnfl probe did not show any specific hybridization
(third panel under stage 26). The color reaction was carried out for the same time for experimental and control embryos. B. Sections of embryos at
stage 17 and 26 hybridized with the xnff probe. N-notochrod; NT neural tube; S- somites; NP neural plate. C. Localization in dissected embryos.
Ten ug of total RNA isolated from dorsal (D) and ventral (V) regions of stage 25 or stage 45 embryos was electrophoresed, blotted onto Nytran membrane
and hybridized with whole xnf7 cDNA probe. Ethidium bromide staining (bottom) shows equal amounts of RNA loaded into each lane of the
gel. Diagram to the right represents the approximate plane of dissection into dorsal (D) and ventral (V) regions.
Fig. 8. Whole mount in situ hybridization analysis of the spatial expression of the xnf7 constructs. Fertilized eggs were injected with the xnf7delta-421 construct and analyzed by whole mount in situ hybridization uising a sense (S) and antisense (AS) probe that will recognize transcripts from the CAT reporter gene. A. The top panel is a control stage 24 embryo hybridized with the S probe; the middle panel is a stage 24 embryo hybridized with the CAT AS probe; the bottom panel is a stage 33 embryo hybridized with the AS probe. B. This is a view from the dorsal region of a stage 24 embryo that was injected into one blastomere at the two cell stage showing the distribution of the transcripts in only one-half of the embryo. A, anterior; D, dorsal; P, posterior; V, ventral
xnf7 (Nuclear factor 7) gene expression in a Xenopus laevis embryo, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left
