Moore KB et al. (2018), C8orf46 homolog encodes a novel protein Vexin t...

XB-ART-54654

Dev Biol 2018 May 01;4371:27-40. doi: 10.1016/j.ydbio.2018.03.003.

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C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

Moore KB , Logan MA , Aldiri I , Roberts JM , Steele M , Vetter ML .

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Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program.

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Species referenced: Xenopus laevis
Genes referenced: atoh7 barhl2 calb1 cdkn1b cdknx ebf2 ebf3 elavl3 hes4 hes5.1 hes5.2 myc neurod1 neurog2 notch1 nrarp pou4f2 rbpms2 rlbp1 sox2 tub tubb2b vxn
GO keywords: cell proliferation [+]
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	Fig. 1. Alignment and phylogenetic tree of c8orf46 orthologs. (A) The predicted protein sequence encoded by the Xenopus laevis gene c8orf46.L (vxn) was aligned to predicted protein sequences encoded by c8orf46 orthologs from other vertebrate species (see methods for accession numbers) using the webPRANK program in Jalview2 (Waterhouse et al., 2009). Residues conserved in at least 70% of the aligned sequences are indicated with dark blue shading while light blue shading indicates residues that are 50–69% conserved. (B) The webPRANK alignment was used to generate a phylogenetic tree using BLOSUM62.
	Fig. 2. Vexin is expressed in the developing Xenopus nervous system. (A) RT-PCR analysis of vxn expression using Xenopus whole embryo cDNA. EF1α was used as a loading control. Control PCR was performed using no cDNA template (no templ). (B–E) Whole-mount in situ hybridization analysis of vxn expression at stage 15 (B), stage 19 (C), and at stage 27 (D, dorsal view; E, anterior view). (F-I) Shown are stage 25 and stage 33 d with axial levels for the sections in F-I (Nieuwkoop and Faber, 1994). In situ hybridization analysis of vxn expression on transverse sections of the neural tube at stage 25 (F), and 33 (G), and of the head including the optic vesicle at stage 25 (H) and the developing retina at stage 33 (I). (J-M) Analysis of vxn expression in the stage 41 retina. (J) Nuclear Hoechst staining shows the three layers of post-mitotic neurons (RGC, INL, ONL) within the central retina, as well as the peripheral ciliary marginal zone (CMZ) that contains retinal progenitors. (K) Fluorescent in situ hybridization shows vxn expression (red) in the CMZ. (L) Anti-BrdU immunostaining (green) labels proliferating retinal progenitors in the CMZ. (J) A merged image of (J-L) shows that vxn expression overlaps with BrdU-positive progenitors (arrows), and is also detected in early differentiating post-mitotic cells more centrally. Abbreviations: L, lens; RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.
	Fig. 3. Vxn protein localizes to the nucleus and the cell membrane. (A, B) Anti-myc immunostaining for myc-tagged Vxn (Myc-Vxn, green) in isolated animal cap explants (A) or anti-Vxn (green) staining of endogenous protein in the neural plate (B) shows localization in the nucleus (labeled by ToPro, red), and at the cell membrane. C) Myc-Vxn (red) within the nucleus is localized to subnuclear domains that stain weakly with Hoechst (blue). (D) At the membrane, Myc-Vxn (red) colocalizes with mCherry-CAAX (green). (E) Mutation of critical lysine residues to alanine within the candidate nuclear localization sequence (NLSmut). (F) Vxn-NLSmut-Myc detected by immunostaining with α-myc localizes to both the nucleus and the cell membrane.
	Fig. 4. Vxn is required for primary neuron differentiation. (A–E) Injection 20 ng of vxn translation-blocking morpholino (MO-A) at the 4-cell stage (4CS) causes reduced N-tubulin (N-tub) positive primary neurons at stage 14 (A), with increased inhibition at increasing doses of MO-A (D), while injection of 20 ng of inverted morpholino (Inv MO-A) does not (B). Primary neurogenesis is rescued by co-injection of vxn MO-A with 1 ng of mouse vxn mRNA (C, D). (E-G) Primary neurogenesis is inhibited by injection of 20 ng of MO-B at the 4CS (E), with efficient inhibition with injection of 10–40 ng of vxn MO-B at the 2 or 4 CS (G). Loss of N-tub can be rescued by co-injection of 1 ng of Xenopus 5’myc-vxn (F) or mouse (G) vxn mRNA. (H-O) Injection of vxn MO-B inhibits expression of other markers of differentiating primary neurons, including elavl3 (H) and ebf2 (I), but has no effects on expression of neurogenic bHLH genes such as neurog2 (J) or neurod1 (K). (L, M) Injection of vxn MO-A or MO-B causes expansion of progenitors in the neural plate as revealed by expansion of sox2 (L) or the neural plate domain bounded by hes4 (M). (N, O) Injection of vxn MO-B causes no change in the expression of Notch pathway genes such as hes5.1 (N) and nrarp (O). For all experiments, mRNA encoding either GFP or β-galactosidase was co-injected to label the injected side (right). X-gal staining is light blue.
	Fig. 5. Vxn overexpression enhances primary neuron differentiation. (A-C) Injection of 1 ng vxn mRNA at the 2-cell stage (2CS) causes expansion of primary neurons (brackets) labeled by N-tub (A), ebf3 (B) and elavl3 (C). (D–E) Injection of 1 ng vxn mRNA causes reduction of progenitors in the neural plate as revealed by narrowing of the sox2 domain (D) or the neural plate domain bounded by hes4 (E). (F, G) Differentiation of N-tub labeled primary neurons is blocked by injecting mRNA encoding Atoh7-EnR (F), and this cannot be overcome by co-injection of vxn mRNA. (H–J) Injection of low doses of neurod1 mRNA (100 pg) causes expansion of N-tub labeled primary neurons (H, bracket), which is enhanced by co-injection of 1 ng vxn mRNA (bracket in I, J). (K-N) Injection of 50 pg mRNA encoding the intracellular domain of Notch (NICD) blocks differentiation of N-tub labeled primary neurons (K, M), and this is not rescued by co-injection of 1 ng vxn mRNA (L, M). Injection of 1 ng vxn mRNA does not change expression of the Notch target gene hes5.1 (N). For all experiments, mRNA encoding either GFP or β-galactosidase was co-injected to label the injected side (right). X-gal staining is light blue.
	Fig. 6. Vxn is required for retinal neuron differentiation. (A–C) mRNA for GFP (300 pg) was injected alone or together with 3 ng of vxn mismatch MO-A or vxn MO-A into 1 dorsal blastomere at the 32-cell stage. Embryos were cryosectioned at stage 41, and GFP-labeled retinal cell types were counted. vxn MO-A caused a significant decrease in all retinal neuron types, and a significant increase in Muller glia (MG) and/or neuroepithelial cells (NEP) (A). A section of stage 41 retina showing many GFP-labeled retinal cells with the morphology of Muller glia and/or NEPs (B). Immunostaining for CRALBP to distinguish MG from NEPs showed that both populations increased significantly (C). (D–I) Injection of 20 ng of vxn MO-B at 8CS did not alter atoh7 expression in stage 34–35 embryos (D, E), but prevented expression of hermes (F, G) and barhl2 (H, I) on the injected (inj) side (brackets; E, G,I) when compared to the uninjected (uninj) side (D, F, H). RGC, retinal ganglion cells; HC, horizontal cells; AC, amacrine cells; BP, bipolar cells; PR, photoreceptor cells; MG, Muller glial cells; NEP, neuroepithelial cells. *p < 0.001 and #p < 0.01 by Student's t-test.
	Fig. 7. Targeted retinal overexpression of vxn promotes early retinal cell differentiation. (A, B) GFP (300 pg) was lipofected alone or together with Xenopus vxn (250 pg) or Xenopus atoh7 into the optic vesicle of stage 18 embryos. Embryos were cryosectioned at stage 41, and GFP-labeled retinal cell types were counted. Atoh7 expression caused a significant increase in RGCs. Vxn expression caused a significant increase in RGCs and photoreceptors, that were confirmed to be calbindin-positive cones, which are born early (B). Later born bipolar cells and Mueller glia were significantly reduced. p < 0.001 relative to GFP, * p < 0.001 relative to vxn + GFP. (C) Injection of vxn mRNA into 1 cell of a 32- cell embryo produced a 2.5-fold increase in the number of GFP-labeled RGCs as compared to injection of GFP mRNA control alone, while vxn + atoh7 caused almost 90% of labeled cells to differentiate as RGCs. p < 0.001 relative to GFP alone, * p < 0.001 relative to vxn + GFP. (D) Schematic of 3 retinal layers: the outer nuclear layer (ONL), the inner nuclear layer (ONL), and the retinal ganglion cell (RGC) layer. Injection of GFP mRNA alone reproducibly labeled neuronal subtypes within all 3 layers. Vxn + GFP mRNA injection resulted in an increased number of GFP-positive cells in the RGC layer (arrowheads). Vxn + atoh7 + GFP mRNA caused almost all labeled cells to differentiate as RGCs. (E) Injection of mRNA for mouse vxn had a similar effect, causing a significant increase in early born cell types (GCs, HCs and PRs) at the expense of later born cell types (BPs, AMs). * p < 0.001, # p < 0.01, @p < 0.02. (F) Injection of mRNA for either Xenopus or mouse vxn together with GFP mRNA into blastomere V1.2.1 at the 32-cell stage resulted in reduced labeled retinal clone size in stage 41 retina, consistent with reduced retinal progenitor proliferation. *p < 0.01, as compared to GFP alone. RGC, retinal ganglion cells; HC, horizontal cells; AC, amacrine cells; BP, bipolar cells; PR, photoreceptor cells; MG, Muller glial cells.
	Fig. 8. Vxn functions within the nucleus to promote neural differentiation. (A) Addition of NLS sequence at the N-terminus of Myc-Vxn to create NLS-myc-vxn. (B) Anti-myc immunostaining for myc-tagged NLS-Vxn (green) shows localization exclusively in the nucleus (labeled by ToPro, red). (C) Overexpression of NLS-myc-vxn mRNA promotes expansion of N-tubulin labeled primary neurons in the neural plate on the injected side (right). (D) mRNA for GFP (300 pg) was injected alone or together with mRNA for NLS-myc-vxn (250 pg) into 1 dorsal blastomere at the 32-cell stage. Embryos were cryosectioned at stage 41, and GFP-labeled retinal cell types were counted. NLS-myc-vxn expression caused a significant increase in RGCs. p < 0.001. (E) Addition of NES sequence at the C-terminus of 5’ myc-vxn to create 5’myc-vxn-NES. (F) Anti-myc immunostaining for myc-tagged Vxn-NES (green) shows localization exclusively at the membrane and excluded from the nucleus (labeled by ToPro, red). (G) Overexpression of 5’myc-vxn-NES mRNA inhibits differentiation of N-tubulin labeled primary neurons in the neural plate on the injected side (right). (H) mRNA for GFP (300 pg) was injected alone or together with mRNA for 5’myc-vxn-NES (250 pg) into 1 dorsal blastomere at the 32-cell stage. Embryos were cryosectioned at stage 41, and GFP-labeled retinal cell types were counted. Vxn-NES expression caused a significant increase in Mueller glia/NEPs. p < 0.001.
	Fig. 9. Vxn and p27Xic1 work together to regulate cell cycle exit and neurogenesis. (A-E) Injection of 1 ng GFP mRNA at 2CS (A) does not change pH3 labeling (B), while co-injection of 1 ng vxn mRNA causes significant reduction of pH3 labeling on the injected side (bracket in D; E). * = p < 0.01. (F, G) Co-injection of p27Xic1 morpholino (p27MO) with vxn mRNA rescues the pH3 levels (F, *p < 0.001), and prevents expanded N-tubulin (G) on the injected side. (H) Injection of p27Xic1 mRNA at 2CS promotes expanded N-tubulin, which is reversed by co-injection of 20 ng vxn MO-B. (I) Western blot (WB) to detect Myc-Neurog2 when expressed alone or together with vxn or p27Xic1. β-actin was detected as a loading control. For all experiments, mRNA encoding GFP was co-injected to label the injected side (right).

References [+] :

Agathocleous, A directional Wnt/beta-catenin-Sox2-proneural pathway regulates the transition from proliferation to differentiation in the Xenopus retina. 2009, Pubmed, Xenbase