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Mech Dev
2003 Apr 01;1204:415-28. doi: 10.1016/s0925-4773(03)00018-2.
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The germ cell nuclear factor is required for retinoic acid signaling during Xenopus development.
Barreto G
,
Borgmeyer U
,
Dreyer C
.
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The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor that functions as a transcriptional repressor and is transiently expressed in mammalian carcinoma cells during retinoic acid (RA) induced neuronal differentiation. During Xenopus laevis development, the spatiotemporal expression pattern of embryonic GCNF (xEmGCNF) suggests a role in anteroposterior specification of the neuroectoderm. Here, we show that RA treatment of Xenopus embryos enhances xEmGCNF expression. Moreover, we present evidence for the relevance of this finding in the context of primary neurogenesis and hindbrain development. During early development of the central nervous system, RA signals promote posterior transformation of the neuroectoderm and increase the number of cells undergoing primary neurogenesis. Our loss-of-function analyses using a xEmGCNF-specific morpholino antisense oligonucleotide indicate that xEmGCNF is required for the effect of RA on primary neurogenesis. This may be caused by transcriptional regulation of the gene encoding the RA-degrading enzyme CYP26, since this gene is derepressed after depletion of xEmGCNF and an antimorph of xEmGCNF directly activates transcription of CYP26, also in absence of protein synthesis. The effect of xEmGCNF knockdown on hindbrain patterning is similar to conditions of reduced RA signaling, which may be caused by a reduction of RAR gamma expression specifically in the presumptive hindbrain.
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12676320
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Fig. 1. Retinoic acid enhances the expression of xEmGCNF. Embryos were continuously treated with 1026 M RA from stage 8 until the stages indicated. (A)
RA posteriorises the expression pattern of xEmGCNF. Dorsal views (DOR) of embryos subjected to whole mount in situ hybridization with xEmGCNF asRNA
at developmental stages 12, 13 and 17 are shown. Ventral views (VEN) of stage 17 embryos are also shown. Anterior is at the top of each figure. Non-treated
control embryos (CON, left panels), RA-treated specimens (RA, right panels). (B) RA shifts the peak of xEmGCNF transcription to an earlier developmental
stage. Normalized GCNF-specific signals quantified by densitometry after RT-PCR of non-treated (CON) and RA-treated embryos (RA) at neurula stages 14,
17 and 20 were plotted. (C) The amount of xEmGCNF protein is enhanced by RA and reduced by Citral, an inhibitor of RA signaling. Protein extracts of nontreated
(CON, lanes 1, 4, 7), RA-treated (RA, lanes 2, 5, 8) and citral-treated embryos (CI; lanes 3, 6, 9) at the indicated developmental stages were analyzed by
Western blot for xEmGCNF (arrow) and nucleolin (arrowhead) as a loading control.
Fig. 2. The Morpholino antisense oligonucleotide specifically inhibits the
translation of the xEmGCNF mRNA. Embryos were injected at the two-cell
stage with 42 ng of a standard morpholino oligonucleotide as control (STA:
lane 2) or a xEmGCNF-specific antisense Morpholino oligonucleotide
(MOR: lanes 3) into each blastomere. Protein extracts of injected embryos at
stage 17 were analyzed by Western blotting, using an antiserum against
xEmGCNF [DEF] (David et al., 1998). The positions endogenous
xEmGCNF (arrow) is indicated at the left. The lowest band (arrowhead)
detected by the antiserum is non-related to GCNF and can be used as a
loading control.
Fig. 3. The effect of RA on primary neurogenesis requires xEmGCNF. (A)
Schematic representation of the experimental system. Embryos were
injected at the two-cell stage into each blastomere. Animal caps were
explanted at stage 8.5 and cultivated until stage 17 equivalent. Total RNA
was extracted and semi-quantitative RT-PCR was performed. (B) Knockdown
of xEmGCNF abolishes the increasing effect of RA signaling on the
expression of primary neurogenesis markers. Injection of 40 pg noggin
mRNA directed the ectoderm explants to an anterior neural fate (Nogg,
lanes 2). Further posterior specification of the neuroectoderm by an
enhanced RA signaling (RA, lanes 3) was achieved by coinjection of
mRNAs encoding the retinoid receptors xRARa2 (250 pg) and xRXRb
(250 pg) and treatment of the neuralized explants with 1027 M RA. In
addition, embryos were co-injected with 42 ng of the STA (lanes 1) or
42 ng of the MOR (lanes 4). Expression of markers of neuronal end
differentiation (NST; XDelta-1; Xngnr1 and NeuroD), pan-neural markers
(N-CAM and Nrp1) was monitored by RT-PCR using gene-specific
primers. Loading control ODC RT, negative control ODC 2 RT, whole
embryo WE. (C) Whole mount in situ hybridization of stage 18 embryos
to detect NST (C,D) and XDelta-1 mRNA (E and F) after co-injection of
42 ng of asMOR and 150 pg of b-galactosidase mRNA into one cell at the
two-cell stage. b-Galactosidase staining in red identifies the injected
hemispheres to the right. Anterior is at the top of each figure. D and F show
details of C and E, respectively.
Fig. 4. Expression of CYP26 is regulated by xEmGCNF. (A) Using neuralized animal caps, the expression level of RALDH2 and CYP26 was monitored by
means of RT-PCR in the presence or absence of xEmGCNF after injection of 42 ng of the STA (STAndard control morpholion) (lanes 1) or 42 ng of the MOR (lanes 2), respectively. The
experimental strategy was as outlined in Fig. 3A. Loading control ODC RT, negative control ODC 2 RT, whole embryo WE. (B) Detection of CYP26
expression by whole mount in situ hybridization of late gastrula embryos co-injected at the two-cell stage into one blastomere with 42 ng of the MOR and
150 pg b-galactosidase mRNA as lineage tracer (upper panels). The injected side is to the right as identified by the red b-gal staining. The alkaline phosphatase
color reaction was abbreviated to visualize the differences between injected and non-injected hemisphere. The insets show the normal expression pattern of
CYP26 at midgastrula. Middle and lower panels show whole mount in situ hybridization to detect CYP26 transcripts in midneurula stage 17 embryos after
injection of 42 ng of the STA (middle panels) or 42 ng of the MOR (lower panels) into each blastomere at the two-cell stage. Brackets in the middle panels
indicate the posterior and dorsal region. Dorsal (DOR), posterior (POST), and anterior (ANT) aspects are shown in each column from left to right. (C) Early
ectoderm explants of embryos injected at the two-cell stage into each blastomere with 42 ng STA (lane 1), 42 ng MOR (lane 2) or 200 pg VP16GCNF
mRNA (VP16, lanes 3) were treated with 10 mg/ml cycloheximide (CHX, lane 4) from stage 10 to 10.5 equivalent (2 h at 22 8C). Total RNA was extracted
and expression levels of CYP26, Xlim1 and ODC were monitored by means of RT-PCR. (D) Expression of hGCNF precedes that of hCYP26 during RAtriggered
neuronal differentiation of embryonic carcinoma cells. NT2 cells were cultured for 7 days in the absence (lane 1) or in the presence of RA for the
times indicated at the top (lanes 2). After total RNA extraction, expression of hHboxb6, hOct4, hCYP26 and hGCNF was monitored by RT-PCR. Loading
control EF1a RT, negative control EF1a 2 RT.
Fig. 5. Function of xEmGCNF is required for normal hindbrain development. (A) Whole mount in situ hybridization of neurula embryos at stages 17 and 20 to
detect the expression of the anterior marker Otx2, the posterior marker Gbx2, the marker for rhombomeres 3 and 5 Krox20 and the MHB marker En2. Albino
embryos (1 MOR, right panels) were unilaterally injected at the two-cell stage with 42 ng of the MOR and 150 pg b-galactosidase mRNA as lineage tracer.
The injected side is to the right as identified by the red b-gal staining. The inset shows Krox20 expression pattern after unilateral depletion of xEmGCNF
without b-gal staining. Non-injected control embryos (CON, left panels) show the normal expression patterns of each marker. (B, C) Depletion of xEmGCNF
causes a hindbrain-specific inhibition of RARg transcription. (B) Dorsal, posterior, and anterior aspects of a stage 18 neurulaembryo injected with 42 ng of
MOR into one blastomere at the two-cell stage. The injected side (to the right) was identified by lineage tracing. (C) Lateral and dorsal aspects (lowest panels)
of whole mount in situ to detect RARg expression in stage 35 embryos that were injected at the two-cell stage into each blastomere with 42 ng of STA (left
panels) or MOR (right panels).
nr6a1 (nuclear receptor subfamily 6, group A, member 1) gene expression in Xenopus laevis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anteriorleft.
