Dekker EJ et al. (1994), Overexpression of a cellular retinoic acid bind...

XB-ART-21441

Development 1994 Apr 01;1204:973-85.

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Overexpression of a cellular retinoic acid binding protein (xCRABP) causes anteroposterior defects in developing Xenopus embryos.

Dekker EJ , Vaessen MJ , van den Berg C , Timmermans A , Godsave S , Holling T , Nieuwkoop P , Geurts van Kessel A , Durston A .

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We have isolated the first Xenopus laevis cDNA coding for a cellular retinoic acid binding protein (xCRABP). xCRABP contains a single open reading frame, coding for an approximately 15 x 10(3) M(r) protein. Northern blot analysis shows that this cDNA hybridizes to a mRNA that is expressed both maternally and zygotically and which already reaches maximal expression during gastrulation (much earlier than previously described CRABP genes from other species). In situ hybridisation showed that at the onset of gastrulation, xCRABP mRNA is localised at the dorsal side of the embryo, in the ectoderm and in invaginating mesoderm. xCRABP expression then rapidly resolves into two domains; a neural domain, which becomes localised in the anterior hindbrain, and a posterior domain in neuroectoderm and mesoderm. These two domains were already evident by the mid-gastrula stage. We investigated the function of xCRABP by injecting fertilized eggs with an excess of sense xCRABP mRNA and examined the effects on development. We observed embryos with clear antero-posterior defects, many of which resembled the effects of treating Xenopus gastrulae with all-trans retinoic acid. Notably, the heart was deleted, anterior brain structures and the tail were reduced, and segmentation of the hindbrain was inhibited. The effects of injecting xCRABP transcripts are compatible with the idea that xCRABP overexpression modulates the action of an endogenous retinoid, thereby regulating the expression of retinoid target genes, such as Hox genes. In support of this, we showed that the expression of two Xenopus Hoxb genes, Hoxb-9 and Hoxb-4, is strongly enhanced by xCRABP over-expression. These results suggest that xCRABP expression may help to specify the anteroposterior axis during the early development of Xenopus laevis.

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Species referenced: Xenopus laevis
Genes referenced: crabp2 hoxa9 hoxb4 hoxb9 hoxc9 lsamp sp6 tbx2 tubb2b
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	Fig. 1. The corresponding amino acid sequences for xCRABP and the xCRABP cDNA probes and artificial xCRABP mRNAs used in this study. (A) Alignment of one letter code protein sequence of the Xenopus laevis cellular retinoic acid binding protein (xCRABP) with those of known CRABPs from other species. Xenopus laevis CRABP is compared with bovine CRABPI (bCRABPI) (Shubeita et al. 1987), and with mouse CRABPI (mCRABPI) (Vaessen et al., 1990a), mouse CRABPII (mCRABPII) (Kitamoto et al., 1989) and a partial chicken CRABPI amino acid sequence (chCRABP-I) (Vaessen et al., 1990b). Boxed sequences indicate non-conservative amino acid substitutions. (B) Restriction map of the xCRABP cDNA clone. The black box indicates the open reading frame which is the region homologous to other known CRABPs (shown in Fig. 1A). The lower panel shows the probes used in our experiments. For northern blot analysis and in situ hybridisation a 1050 bp EcoRI/SalI open reading frame containing fragment (1) and a 950 bp SalI/EcoRI non-coding fragment (2) of xCRABP cDNA were used as probes. A 138 bp template (SalI-AvaII fragment) was used to generate antisense RNA xCRABP probe for RNase protection assays (3). (C) Diagrammatic representation of the sense (1), antisense (2), and nonsense (3) Sp6 or T7 RNA polymerasegenerated mRNAs used for injection into 1-cell-stage Xenopus embryos and used in the rabbit reticulocyte lysate assay. The nonsense mRNA contains an in-frame 270 bp human parathyroid hormone receptor (PTHR) AvaI fragment (Karperien et al., unpublished data) cloned in the AvaI site of xCRABP.
	Fig. 2. Developmental expression of xCRABP mRNA. Northern blot of total RNA from embryos at the stages indicated (Nieuwkoop and Faber, 1967), hybridised to both xCRABP and histone H3 probes. Each lane represents 15 mg of total RNA. The equivalence and integrity of the RNA was monitored by ethidium bromide staining of the gel prior to blotting (not shown). The equivalence was also monitored by hybridising the northern blot with the histone H3 probe (Destrée et al., 1984). The location of the xCRABP transcript of 2.3 kb is indicated. A smaller transcript (1.9 kb) which is detectable at a very low level tends to disappear after washing at high stringency, indicating that this transcript is partially homologous to, but not identical with, xCRABP.
	Fig. 3. xCRABP in situ hybridisation. Whole embryos were hybridised to antisense xCRABP probes. Hybridisation was visualised using an alkaline phosphatase reaction with nitro blue tetrazolium (NBT) as the substrate. (A) Stage 10.5 (early gastrula stage), cross section. Staining was observed in the animal hemisphere and in the marginal zone above the dorsal lip (DL). (B) Stage 11.5 (late gastrula embryo), dorsal view. Staining is evident in the presumptive anterior (hindbrain region) part of the embryo. A semicircle of staining was also found in the dorsal lip (DL) of the blastopore, in the posterior region of the developing embryo. It is notable that, already at this stage, the midline of the neural plate (notoplate) shows lower xCRABP expression. (C) Stage 14/15 (early neurula stage), dorsal-lateral view. Staining was found in the presumptive hindbrain and tail regions. There are also small lateral and anterior patches of staining, which possibly correspond with the future nasal pit and branchial arch regions. The midline of the neural plate is negative for xCRABP expression. (D) Stage 25 (tailbud stage), dorsal view. This photo shows regional differences in the distribution of xCRABP mRNA in the CNS. Anteriorly, the forebrain-related structures show very light staining, the midbrain structures show somewhat stronger staining while the hindbrain shows abundant xCRABP expression. The spinal cord also clearly shows positive staining. (E) Stage 25 (tailbud stage), side view (same embryo as shown in Fig. 3D). xCRABP expression is evident in the tail region (in the mesoderm as well as in the central nervous system), in the neural tube, in the hindbrain region, and in the presumptive branchial arch and nasal pit area. (F) Stage 32 (tailbud stage), side view. The hybridisation signal was found in the branchial arches (BA), as well as the hindbrain, in rhombomere 4 above the otic vesicle (OV), in rhombomeres 6, 7 and 8 (rhombomere 5 shows no xCRABP expression) and in the anterior spinal cord. The anterior spinal cord, next to the hindbrain, demonstrates a relatively abundant signal that fades in the more posterior part of the spinal cord. Furthermore, staining can be observed in the region of the nasal pit (NP) and at the boundary between the mesencephalon and the rhombencephalon. The tip of the tail also still contains xCRABP mRNA. (G) An RA-treated stage 14/15 (early neurula stage) embryo, side view. Enhanced expression of xCRABP was observed in the presumptive hindbrain region of the embryo. No staining was observed in the presumptive tail region. This embryo, and the embryo in Fig. 3H, were stained for a shorter time than the embryos in Fig. 3A-G (see below). The anterior hybridisation signal is actually much stronger than in control embryos at the same stage. (H) A RA-treated stage 25 (tailbud stage) embryo, side view. Enhanced expression of xCRABP occurs both in the presumptive hindbrain region and in the tail (staining as in Fig. 3G). In B-H, the embryos are shown with their anterior sides to the left. Note: The embryo depicted in A was stained for 3 days, while the embryos shown in B-F were stained overnight with NBT (see experimental procedures).
	Fig. 4. The stability and the translation of the xCRABP mRNA. (A) RNase protection assays were used to examine the stability of injected sense xCRABP mRNA during development. These were used to measure xCRABP mRNA-injected embryos (hatched bars) and controls injected with full-length antisense xCRABP mRNA (open bars) at the stages (Nieuwkoop and Faber, 1967) indicated using a 138 bp xCRABP 3¢ untranslated (SalI-AvaII) fragment as the probe (see Fig. 1B, fragment 3). Xom62/9 was used as an internal standard for quantification. The sense xCRABP-injected embryos maintain a strongly enhanced level of xCRABP mRNA. (B) A Northern blot showing three lanes with total RNA, isolated at stage 20 (late neurula/early tailbud stage) from non-injected (1), sense (2) and antisense xCRABP (3)-injected embryos hybridised to a 950 bp 3¢ untranslated SalI-EcoRI fragment of the xCRABP cDNA clone. This also demonstrates the stability of the 2.0 kb synthetic mRNAs injected into 1-cell-stage embryos. (C) Translation of xCRABP mRNA. A rabbit reticulocyte assay (Promega) and metabolic labeling using [35S]methionine was used to confirm that the sense xCRABP mRNA is translated into an approximately 15´103 Mr protein (lane 1). The antisense xCRABP mRNA is not translated into a protein (lane 2).
	Fig. 5. sense xCRABP mRNA overexpression causes developmental defects. Microinjection of sense xCRABP mRNA (A), TE-buffer (B), antisense xCRABP mRNA (C) and nonsense xCRABP mRNA (D) into 1-cell-stage Xenopus embryos. The injection of sense xCRABP mRNA (A) induced anteroposterior defects in a large percentage (63%) of the injected embryos, while the controls (B-D) failed to develop many defects. The embryos were examined at stage 35 for specific A-P defects, e.g. reduced or fused eyes, reduced cement gland, reduced forebrain structures, tail shortening, reduction of heart and kidneys (see experimental procedures). Values are means ± s.e.m. of 9 independent experiments.
	Fig. 6. Phenotypes of normal and xCRABP-injected embryos. A, anterior; P, posterior. (A) Normal stage 35 (tailbud stage) embryo, side view. (B) xCRABP mRNA-injected stage 35 embryo, side view. (C) Normal stage 47 (tadpole) embryo, side view. (D) xCRABP mRNA-injected stage 47 embryo, side view. (E) Stage 47 embryo, dorsal view. Stained with 2G9, a neural-specific antibody to reveal the central nervous system. FB, forebrain; HB, hindbrain; SC, spinal cord. (F) xCRABP mRNA-injected stage 47 embryo, dorsal view, stained with 2G9. This staining shows that the forebrain (FB) is strongly reduced, the hindbrain (HB) is still detectable, but the spinal cord (SC) ends up in a knot-like structure and does not develop in the posterior (tail) region of the embryo.
	Fig. 7. Graphical reconstructions of a xCRABP mRNA-injected and control stage 47 embryo. Projection on sagittal plane (described by Nieuwkoop, 1947), graphical reconstruction of a stage 47 control (above) and xCRABP mRNA-injected embryo (bottom). Lines in brain part indicate successive brain parts (see below). Black part in the brain indicates inner/outer brain. t., telencephalon; d., diencephalon; m., mesencephalon; r., rhombencephalon; p.b., pineal body; p.e.c., primary eye cup; e., eye; ph., pharynx; le., lens; g.l., gut loops; pe., pericard; h., heart; l., liver; n., notochord; s.c., spinal cord; c.g., cement gland; m.c., mouth cavity: n.p., nasal pit; e.p., ear placode; pa., pancreas; i., intestine; e.s., ectodermal stomodaeum; pr., proctodaeum; t.b., tail bud.
	Fig. 8. xCRABP mRNA overexpression enhances Hoxb gene expression during early development. (A) 1-cell-stage embryos were injected with antisense xCRABP mRNAs (B), sense xCRABP mRNAs (C), and treated with 10-6 M RA continuously from stage 10 onwards (D) and isolated at stage 15 (mid neurula) and 20 (late neurula) together with non-injected embryos at the same stages (A) and from the same batch of embryos. Total RNA was extracted from these embryos and used to perform an RNase protection assay using two Xenopus laevis Hoxb (Hox-2) genes and an internal standard Xom62/9 gene (see experimental procedures). Hoxb-9 (XlHbox6) is the most 5¢ localised Hoxb gene and Hoxb-4 (Xhox-1A) is a 3¢ localised gene in the Hoxb chromosomal complex (Dekker et al., 1992a). Note: We have adopted the Xenopus Hox gene nomenclature according to a recent proposal by M. P. Scott (1993) Cell 71, 551- 553. (B,C) Quantification of Hoxb gene expression. The results shown in A were quantitated by phosphorimaging. Open bars, control embryos; lightly hatched bars, anti-sense xCRABP-injected embryos; heavily hatched bars, sense xCRABP-injected embryos; black bars, RA-treated embryos. Expression of Hoxb-4 (B) and Hoxb-9 (C) was measured by RNase protection at the developmental stages indicated. Xom62/9 was used as internal standard. Data are expressed as percentages relative to the maximum expression of Hoxb-4 and Hoxb-9 during development. The results show that expression of both Hox genes is hyperinduced by xCRABP injection: Hoxb-4 (Xhox-1A) is 3-fold hyperinduced at stage 15, and 2-fold hyperinduced at stage 20, and Hoxb-9 (XlHbox6) is 2-fold hyperinduced at stage 15 and 1.15-fold hyperinduced at stage 20. This effect of xCRABP overexpression resembles the effect of treating gastrula stage embryos with a high concentration (10-6 M) of RA.