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Nuclear orphan receptors are DNA binding proteins that share the domain structure of the nuclear hormone receptor superfamily, although ligands are unknown. We have identified an orphan receptor in Xenopus laevis and named it xGCNF based on its high degree of sequence homology to the previously described murine germ cell nuclear factor (mGCNF). In gel-electrophoresis mobility shift analysis experiments in vitro translated xGCNF and mGCNF proteins both bind specifically as homodimers to the same response element, a direct repeat of the half-site consensus AGGTCA with zero spacing (DRO). Transcripts of xGCNF are found in oocytes and in much smaller amounts in the testes. In developmental Northern blots and RNase protection using RNA from different embryonic stages, zygotic expression of xGCNF peaks at midneurula. From late gastrula to midneurula stages, an anterior to posterior concentration gradient of the RNA was observed in whole mount in situ analysis. This antero-posterior gradient of expression was also observed in exogastrulae, both in the ectoderm and mesoderm. In the midneurula embryo, the mRNA was predominantly found in the neural plate and neural crest. Transcription of xGCNF in animal cap explants occurred independent of mesoderm induction.
Fig. 4. Transcripts of xGCNF analysed by Northern blots. (A) Total
RNA of embryos of the developmental stages as indicated at the top
(lanes l-7). and from young oocytes stage I-III (0), testes (T), kidney
(K), and liver (L), was hybridized to a radioactively labelled cDNA
probe representing nt 291-1495 of the xGCNF cDNA shown in Fig.
1B. Lower panel: the same Northern blot was hybridized with a 32Plabelled
cDNA representing a 1 .I kb KpnbHindIII fragment of Xenopus
j%actin. The low amount of j%actin during blastula and early gastruia
stages was observed previously (Dreyer et al. 1992). The amount of 18s
and 28s rRNA was almost the same at all developmental stages investigated
(not shown). Stage 8, midblastula; stage 10, early gastrula; stage
15 and 17, midneurula; stage 21, late neurula; stage 30, late tailbud. (B)
Total RNA of testes (lane 1), of oocytes at Dumont stage I-III (lane 2).
was hybridized to the same xGCNF probe. This Northern blot was
overexposed to reveal an 8.5 kb transcript found in testes. No transcript
was observed in kidney or liver under this condition (not shown). The
position of the 2.8 and 7.5 kb RNA markers is indicated by bars. Transcripts
of approximately 126.96.36.199, 8.5, and 10 kb length are marked with
arrowheads. Note that in the embryo transcripts of approximately 10
and 8.5 kb prevail, whereas oocytes mainly contain transcripts of 8.5
and 7.5 kb. Transcripts of about 2.4 kb are also found in oocytes and in
Fig. 5. Localisation of xGCNF mRNA in germ cells. (A,B) Whole mount in situ hybridization of oocytes of stages I-V with xGCNF antisense (A) or
sense (B) RNA. Note that pigment is present in oocytes of advanced stages. Stages of oogenesis according to Dumont (1972) are indicated by Roman
numbers. Young and advanced stage IV are shown. (C,D) In situ hybridization on 20pm cryosections of ovary from a young frog (CD) was performed
with xGCNF antisense (C) or sense RNA (D). The maximum signal was obtained in previtellogenic (stage I) oocytes. Note the absence of
signal from somatic cells of the ovary. Bar, 250,~m.
Fig. 6. Localization of xGCNF transcripts in embryos by whole mount in situ hybridization: Whole mount in situ hybridization of albino embryos with
Digoxigenin-labelled xGCNF antisense RNA reveals maximal expression of xGCNF transcripts during neurulation. Stages according to Nieuwkoop
and Faber refer to (A), early gastrula (stage lo), (B), late gastrula (stage 12), (C), early neurula (stage 14), (D), dorsal aspects of midneurula (stage 17)
and (E), dorsolateral aspect of late neurula (stage 21), (F), lateral aspect of early tailbud (stage 23). Anterior is to the left in (B-F). Note the anteroposterior
gradient of xGCNF mRNA expression at stages 12.5-21 (B-E) and labelling of the branchial arches (ba) at stage 23 (F). Bars, 0.5 mm.
Fig. 7. Whole mount in situ hybridization of midneurula stage 16 embryo. Hybridization with digoxygenin-labelled xGCNF antisense RNA shows
highest amount of transcript in the neural plate, neural fold, and neural crest. The specimen was treated as described in Fig. 6, except for a shorter
period of colour development. The dorsal (A), lateral (B), anterior(C) and posterior(D) aspect of a stage 16 midneurula embryo is shown. Note weaker
labelling in the dorsal midline (arrows) as compared to the rest of the neural plate (np) and neural folds (nf), and absence of label from the blastoporal
area (arrowhead in D). a, anterior; p, posterior; d, dorsal; v, ventral. Bar, 0.5 mm. (E,F) In situ hybridization with xGCNF antisense RNA on 100 pm
thin vibratome sections from a wild-type stage 17 midneurula embryo. Only the dorsal parts of transverse sections through the closing neural tube (E)
and more anteriorly, through the open neural plate at the tip of the notochord (F), are shown. Neural fold (nf), neural plate (np). and neural crest (nc)
were predominantly labelled. Lower amounts of mRNA were detected in the notochord (no), and somites (so). Note the comparatively low amount of
mRNA in the prospective floorplate above the notochord in (E). ng, neural groove; ar, archenteron roof; en, endoderm. Sectioning was slightly oblique
with respect to the anteroposterior axis.
Fig. 8. Expression of xGCNF mRNA in exogastrula. Exogastrulation was induced by keeping blastula embryos of stage 8 in 1.2x MBSH until control
siblings had reached late neurula stage 20. All specimens were subjected to whole mount in situ hybridization with digoxygenin-labelled xGCNF antisense
RNA as probe. Note absence of signal from the border between ectoderm (ec) and endomesoderm (en/m), which demarcates the posterior ends
of both the ectoderm and the endomesoderm (arrows). Bar, 0.3 mm.