Kee N et al. (2008), Neogenin and RGMa control neural tube closure a...

XB-ART-38583

J Neurosci 2008 Nov 26;2848:12643-53. doi: 10.1523/JNEUROSCI.4265-08.2008.

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Neogenin and RGMa control neural tube closure and neuroepithelial morphology by regulating cell polarity.

Kee N , Wilson N , De Vries M , Bradford D , Key B , Cooper HM .

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In humans, neural tube closure defects occur in 1:1000 pregnancies. The design of new strategies for the prevention of such common defects would benefit from an improved understanding of the molecular events underlying neurulation. Neural fold elevation is a key morphological process that acts during neurulation to drive neural tube closure. However, to date, the molecular pathways underpinning neural fold elevation have not been elucidated. Here, we use morpholino knock-down technology to demonstrate that Repulsive Guidance Molecule (RGMa)-Neogenin interactions are essential for effective neural fold elevation during Xenopus neurulation and that loss of these molecules results in disrupted neural tube closure. We demonstrate that Neogenin and RGMa are required for establishing the morphology of deep layer cells in the neural plate throughout neurulation. We also show that loss of Neogenin severely disrupts the microtubule network within the deep layer cells suggesting that Neogenin-dependent microtubule organization within the deep cells is essential for radial intercalation with the overlying superficial cell layer, thereby driving neural fold elevation. In addition, we show that sustained Neogenin activity is also necessary for the establishment of the apicobasally polarized pseudostratified neuroepithelium of the neural tube. Therefore, our study identifies a novel signaling pathway essential for radial intercalation and epithelialization during neural fold elevation and neural tube morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: cacna1b cadm1 en1 neo1 nusap1 plxna1 rgma st13 tjp1
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	Figure 1. Xneog is expressed in the deep layer of the lateral and intermediate neural plate during neurulation. Xneog is expressed (A) in the deep layer of the lateral regions of the st13 posterior neural plate (pnp) (Ai, Aiii, arrows) and the anterior neural plate(anp)(Aii),(B) inthest16 posterior NFs(pnf)(Bi, Biii, arrows) and anterior NFs(anf)(Bii) with expression expanding intothemedial neural plate and floor plate(fp)(Biii, arrows), and(C) inthe apposed dorsal NFs(Ci, ii, iii, arrows).(i), Dorsal view; (ii), frontal view; (iii), transverse view at the level of the white dashed line in above panels. Dashed red lines indicate midline. A, Anterior; bp, blastopore; de, dermamyotome; D, dorsal; dnt, dorsal neural tube; me, mesoderm; mhb, midbrain-hindbrain junction; nc, notocord; P, posterior; V, ventral; vnt, ventral neural tube.
	Figure 2. Loss of Xneog results in neural tube closure defects. A, Injection of 16 ng of XneogPan MO but not ContMO results in delayed neural tube closure (dorsal view). Hours indicate time since embryos were at st11. Dashed white lines indicate the elevating NFs. Dashed black lines indicate midline. Aiii, Arrows: NF apposition. Avii, Arrowheads: NFs have failed to reach apposition.B,Quantification ofthe number of embryos exhibiting a neurulation delaywhen injectedwith ContMO, XneogPanMO, XplexATG MO or XnpnATG MO.C, Percentage of XneogPan-injected embryos at each stage of neurulation when 90% of ContMOinjected embryos have reachedst18. D, Percentage of embryos exhibiting a neurulation delay when injected with 5UTR and ATG MOsspecific forXneog1aandXneog1b.E, Coinjection of XneogPan with full-length Xneog1b cRNA increase the mean neurulation stage comparedwith injection of XneogPan alone. The number of embryos in each condition is given atthetop of each graph.Data are represented as mean SEM. **p 0.001; p 0.05.
	Figure 3. Xrgmaexpression in the deep layer of the medial neural plate.Xrgmais expressed (A) in the deep layer of the st13 medial posterior neural plate (Ai, Aiii, arrows) and the medial anterior neural plate (Aii), (B) between the midline and the posterior NFs (Bi, Biii, arrows), as well as between the midline and the anterior NFs (Bii) of the st16 neurula, and (C) in the st19 ventral neural tube (Ci,Cii,Ciii). (i), Dorsal view; (ii), frontal view; (iii), transverse view at the level of the dashed white line in above panels; (iv), schematic of complementaryXneogandXrgmaexpression domains. lnp, Lateral neural plate; mdl, midline; mnp, medial neural plate.
	Figure 4. Loss of Xrgma results in neural tube closure defects. A, Injection of 16 ng of XrgmaATG MO or XrgmaUTR MO produces a neurulation delay phenotype. B, Coinjection of 8 ng of XneogPan and XrgmaATG MOs enhances the delay in neurulation. Data are represented as meanSEM.C, XneogPan MO and XrgmaATG MO generate equivalent decreases inthe rate of progression through neurulation. Data are represented as mean SD.
	Figure 5. Loss of Xneog perturbs NF elevation and cell polarity. Transverse sections of st17 embryos immunostained with the anti--tubulin antibody, E7. A, Injection of ContMO into one cell oftwo-cellstage embryos did not affect anterior NF elevation on the ContMO (rightside, arrow) or the wild-type (wt)side (leftside) of the embryo. B, Injection of XneogPan MO disrupts anterior NF elevation on the morphant side of the embryo (right side, arrow). Hinge-points are indicated by arrowheads. C–F, Higher magnification images of the deep layer (dl) in B on the wild-type side (C,E) and XneogPan MO side (D,F) where deep cells have losttheir apicobasal polarity(arrowheads).G–H, Highermagnification images ofthe deep(arrowheads) andsuperficial layers(sl, asterisks) in B atthe hinge-point onthe wild-typeside(G) andthe XneogPan MOside(H). I–K, Posterior NF elevation is inhibited on the XneogPan MOside (I, arrow; K ) but not the wild-typeside (I, leftside;J). Scale bars: A, B, I, 39m;C, D, G, H, 2m;E,F, 1m; J, K, 16m. Ke
	Figure 6. Loss of Neogenin disrupts localization of ZO-1 to the apical domain of neuroepithelial cells inthe zebrafish neuralrod. A, ZO-1 protein(green) isrestrictedto a domain adjacent to the apical surface of neuroepithelial cells at the midline (ml) of the neural rod of embryos injected with zfcont MO. B, C, Injection of the zfneogATG MO resulted in loss of an obvious midline. Neuroepithelial cells wererounded and no longer aligned in an apicobasal orientation. In addition, ZO-1 immunoreactivity was distributed around the entire cell surface. Insets are high-magnification images of cells exhibiting a polarized distribution of ZO-1 in control embryos (A) or a nonpolarized distribution of ZO-1 in morphant cells (B,C). The lipophilic dye, DiI (red) was used to visualize the cell shape of the neuroepithelium. Scale bars: A–C, 20m.
	Figure 7. Xneog and Xrgma are required for the establishment of the neuroepithelium of the neural tube. Transversesections ofthest35 anterior neuraltube labeled with DAPI (Ai, Bi,Ci) andthe Xen1 antibody (Aii, iii, Bii, iii,Cii, iii). Injection of XneogPan MO (B) or XrgmaATG MO (C) but not ContMO (A) disrupts the neuroepithelium of the neural tube. Aiii, Biii andCiii are higher magnification of images of boxed areas in Aii, BiiandCii, respectively. Scale bars: Ai–Cii, 29m; Aiii–Ciii, 8m.
	Figure 8. Coinjection ofXneog1bcRNA with XneogPan MO rescues the formation of the neuroepithelium in the neural tube. Transverse sections of the st35 anterior neural tube labeled with DAPI (Ai, Bi,Ci) and the Xen1 antibody (Aii, iii, Bii, iii,Cii, iii). Injection of XneogPanMO(B) but not ContMO(A) perturbsthe neuroepithelium ofthe neuraltube.C, Coinjection ofXneog1bcRNA with XneogPanMOrescues neuraltubemorphology. Aiii,BiiiandCiiiare highermagnification of images of boxed areas in Aii,Bii andCii, respectively. Scale bars: Ai–Cii, 39m; Aiii–Ciii, 17m.

References [+] :

Connor, Expression and role of Roundabout-1 in embryonic Xenopus forebrain. 2002, Pubmed, Xenbase