Boy S et al. (2004), XSEB4R, a novel RNA-binding protein involved in...

XB-ART-4074

Development 2004 Feb 01;1314:851-62. doi: 10.1242/dev.00983.

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XSEB4R, a novel RNA-binding protein involved in retinal cell differentiation downstream of bHLH proneural genes.

Boy S , Souopgui J , Amato MA , Wegnez M , Pieler T , Perron M .

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RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression but so far they have not been studied extensively in the context of developmental processes. We report on the molecular cloning and spatio-temporal expression of a novel RNA-binding protein, XSEB4R, which is strongly expressed in the nervous system. This study is focused on the analysis of Xseb4R in the context of primary neurogenesis and retinogenesis. To study Xseb4R function during eye development, we set up a new protocol allowing in vivo lipofection of antisense morpholino oligonucleotides into the retina. The resulting XSEB4R knockdown causes an impairment of neuronal differentiation, with an increase in the number of glial cells. By contrast, our gain-of-function analysis demonstrates that Xseb4R strongly promotes neural differentiation. We also showed a similar function during primary neurogenesis. Consistent with this proneural effect, we found that in the open neural plate Xseb4R expression is upregulated by the proneural gene XNgnr1, as well as by the differentiation gene XNeuroD, but is inhibited by the Notch/Delta pathway. Altogether, our results suggest for the first time a proneural effect for a RNA-binding protein involved in the genetic network of retinogenesis.

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Species referenced: Xenopus
Genes referenced: neurod1 neurog2 notch1 rbm24 rbm38 tubb2b
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Phenotypes: Xla Wt + neurod1 (fig.7.c, d) [+]

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	Fig. 2. XSeb4R is strongly expressed in the developing central nervous system. (A) Embryo (stage 10.5) presented in a posterior view, with dorsal up, shows a ring-like expression around the blastopore. (B) Transverse section (as indicated in A) showing Xseb4R in the mesoderm (me). (C) Embryo (stage 14) presented dorsally, with anterior up, revealing three bilateral stripes, medial (m), intermediate (i) and lateral (l), of Xseb4R expression in the open neural plate, as well as expression in the trigeminal placode (tp) and in the presumptive ventral midbrain/forebrain (vmfb) area. (D) Transverse section (as indicated in C) showing Xseb4R signals in both the sensorial layer (sl) of the neuro-ectoderm and in the mesoderm (me) as well as the notochord (no). (E,F) Embryos (stage 17 and 20, respectively) presented dorsally, with anterior up, showing additional Xseb4R expression in the olfactory placode (op), eye (e), forebrain (fb), midbrain (mb), hindbrain (hb) and spinal cord (sc). (G,H) Embryos (stage 24 and 32, respectively) placed laterally with anterior left showing Xseb4R expression in the tail bud (tb) and tail tip (tt). (I) Transverse section (as indicated in H) showing Xseb4R signal in the subventricular zone (svz) of the neural tube. (J) Lateral view showing a weak expression of Xseb4R expression in the developing pronephros (pn). (K) Two bilateral stripes of Xseb4R expression in cells associated with blood islands (bi), and a strong signal in the liver (li). (L) Expression during the characteristic Y-shape formation of blood islands. (M) Xseb4R-expressing cells in the area associated with duodenum (du) formation. (N,O) Embryos presented in the ventral view showing pigmented eye (pe) and Xseb4R expression in the presumptive rectum/anus (r/a).
	Fig. 3. In the retina, Xseb4R is expressed in retinoblasts and undifferentiated postmitotic neurons. (A-D) In situ hybridisation showing the spatio-temporal expression pattern of Xseb4R on retinal sections at various developmental stages. (A) At stage 28, Xseb4R is expressed in the whole presumptive neural retina that contains dividing retinoblasts (arrow). (B) At stage 32, Xseb4R is expressed in the whole neural retina containing mainly dividing precursor cells (arrow). (C) At stage 34, Xseb4R is most strongly expressed in the lens (arrowhead) and in the peripheral region of the neural retina containing dividing precursors (arrow). (D) At stage 37, Xseb4R expression is restricted to the CMZ (arrow) and the lens (arrowhead). (E-G) Staining for BrdU uptake (green; F) and Xseb4R expression (blue; E) at stage 39. Double staining (G) shows that BrdU-positive cells in the peripheral CMZ are Xseb4R negative (green arrowhead). In the central CMZ, BrdU-positive cells also express Xseb4R (black arrowhead). Some cells are BrdU negative and stained with Xseb4R (arrow). Scale bar in A: 50 μm. (H-J) Retinoblasts of stage 34 embryos were transfected at stage 18 with Xseb4R-Flag DNA. On sections, immunostaining was performed with an anti-FLAG antibody revealing the presence of XSEB4R-FLAG (red; H). (I) Higher magnification of this retinoblast. Hoescht staining was performed to visualise the nucleus. (J) Double staining showing that XSEB4R-FLAG is mainly cytoplasmic. The nucleus (arrow) and cytoplasm (arrowhead) are indicated. Scale bar in H: 30 μm.
	Fig. 6. Xseb4R is involved in the regulation of primary neurogenesis in Xenopus embryos (A-C) Dorsal views of embryos injected with control lacZ mRNA (A), 200 pg of Xseb4R mRNA plus lacZ mRNA (B), or 50 pg of Xseb4R mRNA plus lacZ mRNA (C), and stained for N-tubulin expression (dark blue). β-galactosidase activity is shown in light blue. Whereas the high dose (200 pg) of Xseb4R represses N-tubulin expression on the injected side (arrow in B), the low dose (50 pg) leads to the expansion of the N-tubulin domain (arrow in C). (DG) Dorsal views of embryos after injection of the fusion construct Xseb4R-GR. Induction with dexamethasone was performed between stage 9.5 and 10 (D), or between stage 10.5 and 11 (E). F and G show non-induced control embryos. Whereas induction of Xseb4RGR between stage 9.5 and 10 represses N-tubulin expression in the injected side (arrow in D), induction of Xseb4R-GR between stage 10.5 and 11 leads to expansion of the N-tubulin domain (arrow in E). (H,I) Dorsal views of embryos after injection of control Mo (H) or Xseb4R Mo1 (I). Xseb4R Mo1 leads to a repression of N-tubulin expression in the injected side (arrowheads in I). (J) Effects of various doses of control Mo and Xseb4R Mo1 on N-tubulin expression. Injections of 10 or 20 ng of Xseb4R Mo1 lead to a decrease of N-tubulin expression compared with control embryos.
	Fig. 1. XSEB4R is an RRM-type protein. Alignment of XSEB4R (Accession Number AY289193) with its homologues in mouse (mSEB4, Accession Number NP062420), human (hSEB4B, Accession Number CAA53064) and C. elegans (C. e-X, Accession Number T33034), and with the Xenopus muscle specific XSEB4 protein (Accession Number AF223427). Dashes represent identical amino acids; dots represent gaps. The RNA recognition motif (RRM) is shaded in grey and the characteristic RNP consensus motifs (RNP1 and RNP2) are boxed. Two additional conserved domains (CD1 and CD2) are indicated. Sequence comparison is indicated as percentage (%) of amino acid similarity.
	Fig. 4. Xseb4R overexpression promotes early differentiation of retinal cells. (A,B) Typical sections of retinas co-transfected with GFP plus a control plasmid pCS2 (A), or GFP plus Xseb4R (B). The white bracket in B indicates the inner nuclear layer, where very few Xseb4R transfected cells are present compared with the control. (C) Percentage of retinal cell types observed in retinas co-lipofected with GFP plus pCS2, GFP plus Xseb4R, or GFP plus Xseb4R-flag. Statistical analysis was performed using the Student's t-test. ***, P<0.0001. Scale bar in A: 50 μm.
	Fig. 5. Morpholino oligonucleotides lipofection in the retina. (A) Retinal lipofection of GFP Mo interferes with GFP translation. Embryos were lipofected at stage 18 with GFP and a control Mo plus CD2, or with GFP and GFP Mo plus CD2. In stage 41 embryos, the intensity of GFP fluorescence in retinal cells was analysed. We used a filter to decrease the fluorescence light of the microscope so that low GFP fluorescent cells are below the detectable threshold. We then counted the number of lipofected retinas (CD2 positive) that also contain GFP-positive cells. While 79% (n=43 retinas) of CD2-positive retinas also contained GFP-positive cells when lipofected with a control Mo, only 19% (n=42 retinas) contained GFP-positive cells when lipofected with GFP Mo. (B) Xseb4R Mo block the translation of Xseb4R mRNA. Synthetic Xseb4R mRNA (containing the complementary sequence of Mo1) was injected into two-cell stage embryos together with Xseb4R Mo1 or control Mo. Stage 10 embryo lysates were then analysed by western blotting with a polyclonal anti-XSEB4R antibody and a monoclonal anti-αtubulin antibody (control). In the presence of control Mo, the anti-XSEB4R antibody recognises the 22 kD XSEB4 protein. The presence of Xseb4R Mo1 specifically abolishes Xseb4R translation. (C) Xseb4R loss-of-function delays differentiation of retinal cells. The proportion of retinal cell types observed in retinas co-lipofected with GFP plus a control Mo, GFP plus Xseb4R Mo1, or GFP plus Xseb4R Mo2 was determined. Both Mo give the same results. The statistical analysis was performed using the Student's t-test. , P<0.05; , P<0.001; **, P<0.0001.
	Fig. 7. Xseb4R is regulated by proneural and neurogenic genes. (A,C,E) Dorsal views of embryos after injection of the indicated genes and stained for Xseb4R expression (dark blue). β-galactosidase activity is shown in light blue. (B,D,F) One representative embryo for each injection series is shown at high magnification. (A,B) XNgnr1 strongly activates Xseb4R transcription in neural and non-neural ectoderm. (C,D) XNeuroD equally induces ectopic Xseb4R expression. (E,F) Activation of Notch signalling by injection of ICD-Notch reduces the expression of Xseb4R.
	rbm38 (RNA binding motif protein 38) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
	rbm38 ( RNA binding motif protein 38) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, lateral view, dorsal up, anterior left.
	rbm38 (RNA binding motif protein 38) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, dorsal up, anterior left.