Roffers-Agarwal J , Xanthos JB , Kragtorp KA , Miller JR .

HD07480 NICHD NIH HHS , T32 HD007480-09 NICHD NIH HHS , T32 HD007480 NICHD NIH HHS

	Fig. 1. Spatial distribution of Xena protein within the neural plate during Xenopus development. (A) Xena is localized to sites of cell–cell adhesion at stage 14 in a dorsal view of the neural plate (arrowheads). (B) Xena and vinculin colocalize at sites of cell–cell adhesion in the neural plate (arrowheads). (C–F) Xena protein was detected in cross-sections using a polyclonal antibody at (C) stage 14, (D) stage 16, (E) stage 18, (F) and stage 20. Xena is enriched apically in cells of the neural plate undergoing apical wedging (arrowheads). Scale bar in panels A and B = 25 μm. Scale bar in panels C–F = 100 μm.
	Fig. 2. Xena knockdown disrupts neural tube formation. (A) Schematic showing target site of the Xena sMO. Binding of Xena sMO to the splice donor site of exon 2 is predicted to cause mis-splicing of exon 1 to exon 3 and a frameshift, resulting in an early stop codon and production of a non-functional, truncated protein. (B) RT-PCR analysis shows that injection of 50 ng of Xena sMO causes mis-splicing of Xena transcripts. The predicted mis-spliced product was confirmed by sequencing. (C) Xena depletion results in failure of neural tube closure. Xena sMO (25 ng) injected embryos at stage 15 (mid-neurulation) and stage 18 (late neurulation) displaying neural tube closure defects. Dashed lines indicate the borders of the neural folds. (D) Defects caused by Xena sMO injection can be rescued by Xena-GFP mRNA injection (500 pg). Injection of Xena sMO together with 250 pg of GFP mRNA produced a majority of embryos with moderate to severe neural tube closure defects at stage 19. Injection of Xena sMO together with Xena-GFP mRNA reduced the severity of the phenotype and produced a majority of embryos with mild neural tube closure defects. Embryos were scored as described in Materials and methods.
	Fig. 3. Xena knockdown disrupts neural plate morphogenesis. (A) Xena sMO injected embryos have wider domains of Sox2 expression compared to control embryos. (B) Quantification of the length–width ratio (LWR) of Sox2 staining demonstrates that the LWR of Xena sMO injected embryos is significantly smaller than that of GFP injected controls (GFP n = 21, Xena sMO n = 25, student's t-test p = 6.02678 × 10− 09). (C) Pax3 expression domains are significantly wider in Xena sMO injected embryos compared to GFP-injected controls. (D) Quantification of distance between neural folds as measured by Pax3 expression (GFP n = 17, Xena sMO n = 23, student's t-test p = 4.22892 × 10− 08).
	Fig. 4. Xena is required for changes in cell shape and apical constriction during neurulation. (A) Stage 15 GFP injected control embryo stained for GFP and β-tubulin to outline cells. In control embryos cells are elongated and apically constricted. (B) In Xena sMO injected embryos, cells appear rounded and fail to undergo apical constriction. Scale bar = 25 μm.
	Fig. 5. Xena is required for apical accumulation of actin during apical constriction. (A) Distribution of actin during neural tube closure in control embryos. Actin accumulates apically (arrowheads) in cells undergoing apical constriction and persists apically until the neural tube is completely fused. (B) Stage 18 GFP injected control embryo. Actin is concentrated apically in cells undergoing apical constriction (arrowhead). (C) Stage 18 Xena sMO/GFP injected embryo. No apical accumulation of actin is observed in Xena sMO/GFP positive cells. Xena knockdown cells remain cuboidal and fail to undergo apical constriction (bracket). Notice the higher density of apical actin in GFP negative cells (arrowheads). Scale bar in panel A = 100 μm. Scale bar in panels B and C = 20 μm.
	Fig. 6. Xena is required for cell–cell adhesion. (A) Reaggregation assays show that dissociated cells from control explants form numerous tightly adherent aggregates whereas (B) cells from Xena sMO injected embryos fail to reaggregate and mostly stay as single cells. (C) Neural plate of stage 15 GFP-injected control embryo. α-Catenin is localized apically at the cortex of cells in the neuroepithelial cells of neural plate (arrowheads). (D) Neural plate of stage 15 Xena sMO/GFP injected embryo. α-Catenin is diminished at cell–cell junctions and is distributed cytoplasmically in cells containing Xena sMO/GFP (arrowheads). (E) Neural plate of stage 15 Xena sMO/Xena-GFP injected embryo. α-Catenin is localized cortically in cells containing Xena sMO/Xena-GFP (arrowheads). Notice that α-catenin is enriched at sites of cell–cell adhesion when compared to levels in surrounding uninjected cells. Scale bar in panels C–E = 50 μm.

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