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First published online 29 March 2006
doi: 10.1242/dev.02343

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XNF-ATc3 affects neural convergent extension

¹ Department of Genetics, Stanford University Medical School, Stanford, CA 94062, USA.
² Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.

View larger version (51K): [in a new window]   Fig. 1. Inhibition of XNF-ATc3 affects neural tube closure. (A) Embryos were either injected in the prospective ectoderm at the one-cell stage with 2 ng DN XNF-ATc3 or treated with 4 mM cyclosporin A (CsA) at stage 10 for 1 hour. Both sets were analyzed for Eng, Krox20, Six1, Pax3, Sox2 and HoxB9 expression at late neurula stages (19-21) by in situ hybridization. Neural tube closure defects were seen at concentrations as low as 250 pg DN XNF-ATc3. White arrowheads indicate expansions in the neural marker expression. (B) WT XNF-ATc3 rescues neural CE defects caused by CsA treatment. The graph represents three different experiments (n=731), which were normalized by the percentage of neural CE defects in embryos that were incubated in 400 µM CsA starting at the 64-cell stage. Co-injection of 2 ng WT XNF-ATc3(4) lead to a rescue of neural CE defects.

View larger version (113K): [in a new window]   Fig. 2. Inhibition of NF-AT signaling by DN XNF-ATc3 or CsA does not affect hinge point formation. (A) Phalloidin (red) was used to stain the accumulated apical actin in hingepoints (white arrowhead) of control embryos. White line shows width of the neural tube. (B) Embryos were injected with 2 ng DN XNF-ATc3 and 500 pg EGFP- RNA at the two-cell stage. Although the neural plate appears wider (horizontal line) on the injected side, phalloidin staining (white arrowhead) is not disrupted. Vertical line sketches the midline of the embryo. (C) GFP fluorescence marking the injected side. (D) Transverse section of the embryo in B showing phalloidin staining (arrowheads). (E) GFP fluorescence of D. (F) Phalloidin staining of a control embryo and (G) of an embryo treated with 400 µM CsA at the gastrula stage. Arrowheads in F,G indicate normal phalloidin staining. (H-L) Cell aggregation assay. (H) Uninjected control neural cells dissociated in CMFM. (I) Neural cells injected with 2 ng DN XNF-ATc3 dissociate faster and more completely than controls. (K) The reaggregation of control neural cells in calcium containing medium (1/3 NMR). (L) Only a few DN XNF-ATc3 expressing cells re-aggregate in 1/3 NMR.

View larger version (61K): [in a new window]   Fig. 3. Activation of XNF-ATc3 generates neural CE defects and anterior truncations. (A) Xenopus embryos were injected at the one-cell stage with 100 pg (middle panels) or 500 pg (right panels) of CA XNF-ATc3. Embryos injected with 100 pg CA XNF-ATc3 resembled control embryos during gastrula stages (first row, vegetal view of stage 11; second row, vegetal view of stage 12.5), but developed neural tube CE defects in 31% of embryos at neurula stages (third row: dorsal view of stage 17). Embryos injected with 500 pg CA XNF-ATc3 exhibited slight defects at stage 12.5 and neural CE defects in 81% of injected embryos. Control embryos developed no neural CE defects. (B) Xenopus embryos were injected at the one-cell stage with 50 pg CA XNF-ATc3 and analyzed for Krox20, Eng, Pax3, Sox2 and HoxB9 expression at the neurula stage. For XAG-1 in situ hybridization embryos were injected with 250 pg CA XNF-ATc3 and 100 pg lacZ (red) for lineage tracing.

View larger version (100K): [in a new window]   Fig. 4. XNF-ATc3 is necessary for neural ectoderm morphogenesis. (A) CA XNF-ATc3 (50 pg) or 250 pg DN XNF-ATc3 were co-injected with 50 pg GFP. Dorsal animal injections target the posterior neural ectoderm while dorsal vegetal injections target the axial mesoderm. The right panel shows the GFP fluorescence in stage 12 Xenopus embryos, demonstrating correct targeting of tissues. (B) Only embryos where the RNA was targeted into the neural ectoderm (NE) show neural CE defects at neurula stages (16-18), while embryos from axial mesoderm (AM) injections appear normal, with the exception that high concentration (500 pg) of CA XNF-ATc3 cause gastrulation delays (data not shown). Neural CE defects are marked with black arrowheads.

View larger version (69K): [in a new window]   Fig. 5. Inhibition of XNF-ATc3 signaling does not inhibit CE in the mesoderm. Animal caps were treated as follows: (A) uninjected control, (B) uninjected control treated with activin, (C) injected with 250 pg CA XNF-ATc3 (CA) and treated with activin, (D) injected with 500 pg CA XNF-ATc3 and treated with activin, (E) injected with 1 ng DN XNF-ATc3 (DN) treated with activin, and (F) treated with 400 nM CsA and activin. Injection of DN XNF-ATc3 did not inhibit CE even at concentrations of 4 ng DN XNF-ATc3. The same was seen in animal caps treated with 4 mM CsA or 400 nM CsA plus FK506, a calcineurin inhibitor that is know to show synergistic effects with CsA (Flanagan et al., 1991; Liu et al., 1991). (G,H) DN XNF-ATc3 also failed to inhibit CE in Keller explants overexpressing 2 ng DN XNF-ATc3 and 200 pg GFP. All seven explants from two experiments elongated. (G) Bright-field of an elongating Keller explant. (H) GFP fluorescence of G.

View larger version (89K): [in a new window]   Fig. 6. NF-AT activity in neuralized animal caps. (A) One-cell stage embryos were co-injected in the animal hemisphere with 20 pg of XBF2 and 0.1 ng or 0.2 ng of CA XNF-ATc3 (CA) or 1 ng of DN XNF-ATc3 (DN) encoding RNAs. (B) RT-PCR analysis was performed with the markers HoxB9, XE10 (EphA2 receptor), Krox20, NCAM and EF1 (loading control). (C) One-cell stage embryos were injected into the animal hemisphere with 0.2 ng of BMP DN receptor (BMP DNR), 0.2 ng of CA XNF-ATc3- or 1 ng of DN XNF-ATc3. BMP-DNR was also co-injected with either CA XNF-ATc3 or DN XNF-ATc3 at the same concentrations. (D) RT-PCR analysis was performed with the markers HoxB9, HoxD1, XAG-1 and EF1 (loading control).

View larger version (72K): [in a new window]   Fig. 7. Inhibition of XNF-ATc3 blocks CE in neural plate explants. (A) Neural plate explants from stage 12-12.5 were cultured and photographed hourly. Upper panels show a control neural plate explant. The middle panels show a neural plate explant of an embryo injected with 2 ng DN XNF-ATc3 and 200 pg GFP. The lower panels show GFP fluorescence. The upper graph illustrates the change in length (L) and the change in width (W) over time. The lower graph shows the change in neural tube length (NT) between 2 and 4 hours. Stars indicate where the DN XNF-ATc3 injected explants are significantly different than the controls in an unpaired Student's t-test. (B) Neural plate explants incubated in 400 nM CsA (lower panels). As a vehicle control, neural plate explants were incubated in 0.33% ethanol (upper panels). The graph describes the change in length (L) and change in width (W) over time. Stars indicate where the CsA-treated explants were significantly different from the control and ethanol treated explants using an unpaired Student's t-test. (C) CsA treatment does not lead to cell fate changes in neural plate explants. In situ hybridization using antisense probes for XAG-1, Krox20 and HoxB9. Left panel shows two control embryos used to stage the explants (left, posterior view; right, anterior view). Middle panel presents control neural plate explants, while the right panel shows explants incubated in 400 nM CsA.

View larger version (72K): [in a new window]   Fig. 8. XNF-ATc3 is expressed in neural tissue. Xenopus embryos analyzed for XNF-ATc3 expression by in situ hybridization: (A) One cell, (B) four cell, (C) stage 11.5, (D) stage 15, (E) stage 21, (F,G) stage 26 and (H) stage 28. Embryos hybridized with the antisense probe are shown on the left, sense control is shown on the right (A-F). Image perspective: (A,B) animal, (C) vegetal, (D,E) dorsal and (G,H) lateral views; (F) ventral view of the head. White arrowhead in E indicates the cranial neural crest; black arrowheads in F mark the cement glands. Abbreviations: b, brain; e, eye; nc, neural crest; o, otic vesicle; s, somites.