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doi: 10.1242/10.1242/dev.00123

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Neural tube closure requires Dishevelled-dependent convergent extension of the midline

Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA

View larger version (32K): [in a new window]   Fig. 1. The morphogenetic processes involved in Xenopus neurulation. All of the behaviors shown require proper establishment of either apicobasal or mediolateral cell polarity. During early neurulation, the neuroepithelium thickens apicobasally while convergent extension movements shape the neural plate. At mid-neurulation, convergent extension continues, while apical wedging initiates in the neuroepithelium, elevating the folds. Medially directed movement of the epidermis also begins. At late neurula stages, the neural fold fuse dorsally and convergent extension of the dorsal neural tissues shapes the neural tube. Neuroepithelium, blue; dorsal neural tissue and neural crest, yellow; epidermis, light blue; notochord, red. Adapted from Davidson and Keller (Davidson and Keller, 1999).

View larger version (94K): [in a new window]   Fig. 5. Failure of NT closure correlates with defective convergent extension of the embryo. (A) Individual images from time-lapse movies of neurulation showing control, closed-NT and open-NT embryos at stages 11.5, 14, 16, 18 and 22 (first panel shows vegetal view; all subsequent panels show dorsal view). (B) Plot of LWR of AP axis for control and Xdd1 closed-NT embryos. (C) Plot of LWR of AP axis for control and Xdd1 open-NT embryos. Data for B,C are mean LWR±s.d. (control n=16 embryos, eight movies; closed-NT n=10 embryos, six movies; open-NT n=22 embryos, seven movies; data points are for stages 10.5, 11, 12.5 and 13-20). (D) Rate of axis elongation (change in LWR/time) for control, closed-NT and open-NT embryos during early (white bars), middle (gray bars) and late neurulation (black bars). See also Movie 5 at http://dev.biologists.org/supplemental/

View larger version (60K): [in a new window]   Fig. 4. Neural fold apposition in Xdd1 expressing embryos. See also Movies 1-4 at http://dev.biologists.org/supplemental/ (A) Control embryo at stage 16. (B) Xdd1-injected closed-NT embryo at stage 16. (C,D) Xdd1-injected open-NT embryos at stage 16. (a'-d'). Same embryos as in A-D shown at stage 23. (E) Plot of average distance between neural folds for control and Xdd1 closed-NT embryos. (F) Plot of average distance between neural folds for control and Xdd1 open-NT embryos. (G) Plot of rate of neural fold apposition (change in average distance between neural folds/time) for control, closed-NT, and open-NT embryos at mid- and late-neural stages. Data shown in E-G are mean±s.d. from six different movies on different days (control n=9; open-NT n=15; closed NT n=9). (H) Plot of LWR of the AP axis of each embryo in A-D; there is a strong correlation between axis elongation and success of NT closure.

View larger version (117K): [in a new window]   Fig. 2. Expression of mutant Xdsh elicits NT closure defects. (A,B) Control embryos at stage 22 have closed neural tubes. (C) An embryo expressing Xdd1 with an open neural tube. (D) Another embryo expressing Xdd1 with a milder open NT defect. NT is closed normally anterior and posterior to the NT defect. (E) An embryo expressing Xdsh-D2 displaying an NT defect. (E) Another Xdsh-D2 expressing embryo with a severe NT defect in which the entire length of the NT has failed to close.

View larger version (117K): [in a new window]   Fig. 3. Transverse optical sections of NT defects. (A) Control embryo at stage 16. (B,C) Xdd1-injected embryos at stage 16. (D) Control embryo at stage 17. (E,F) Xdd1-injected embryos at stage 17. Arrows indicate neural folds in all panels. Note that at each stage, neural folds in Xdd1-injected embryos are wider than controls at the same stage. Midline tissues are less organized in Xdd1-injected embryos at stage 16, though organization is better at stage 17. In many embryos, a prominent bulge was observed in the midline of the open neural plate (Fig. 3F). Although thinning of the neural plate has been suggested to contribute to tube closure (Poznanski et al., 1997), because it was not consistently observed (Fig. 3E), this midline bulge of the neuroepithelium cannot be the primary cause of the open NT phenotype in Xdd1-injected embryos.

View larger version (77K): [in a new window]   Fig. 6. Neural tube closure requires Xdsh function in the midline. (A) Targeted injection into dorsal medial animal blastomeres at the 16-cell stage. (B) Targeted injection into the dorsal lateral animal blastomeres. (C) GFP lineage tracing of medial injection at stage 14 reveals delivery to midline. (D) GFP lineage tracing of lateral injection at stage 14 reveals delivery to lateral neural plate. (E) Medial injection results in NT closure defects, and GFP labels floor of open NT (e'). (F) Lateral injection does not elicit NT closure defects and GFP labels dorsal NT and epidermis (f').

View larger version (67K): [in a new window]   Fig. 7. Persistent failure of midline neural convergent extension in Xdd1 injected embryos. (A) Control embryos stained for Xfd12 expression at stage 10.5. (B) Xdd1-injected embryos stained for Xfd12 at stage 10.5. (C) Control embryos stained for Xfd12 expression at stage 11.5. (D) Xdd1-injected embryos stained for Xfd12 at stage 11.5. (E) Expression of Xnetrin in control embryo at mid-neurulation. (F) Expression of Xnetrin in two Xdd1-injected embryos at mid-neurulation; green arrow indicates gap in Xnetrin expression domain. (G) Expression of Xnetrin in control embryo at late neurulation. (H) Expression of Xnetrin in two Xdd1-injected embryos at late neurulation; green arrow indicates gap in Xnetrin expression domain; red arrow indicates area at posterior floor of the open NT, which does not express Xnetrin. (I) Expression of SHH in control embryo at late neurulation (embryo has been cleared). (J) Expression of SHH in two Xdd1-injected embryos at late neurulation; green arrow indicates gap in SHH expression domain; red arrow indicates area in the floor of the open NT, which does not express SHH.

View larger version (71K): [in a new window]   Fig. 8. Xdd1 disrupts convergent extension of dorsolateral neural tissue. (A) Dorsal view Xpax3 expression (blue staining) in a control embryo at mid-neurula stage. (B) Xpax3 expression at mid-neurulation in an embryo injected with Xdd1. It should be noted that embryological and fate-mapping experiments have identified an additional convergent extension event which shapes the dorsal neural tube following overt tube closure (Davidson and Keller, 1999). This later convergent extension event was also disrupted by expression of Xdd1 (not shown). (C) Expression of Xash3 in a control embryo. (D) Expression of Xash3 in an Xdd1-injected embryo; the expression domains are foreshortened. (E) Expression of Sox2 in a control embryo at stage 14. (F) Expression pattern of Sox2 in Xdd1-injected embryos. As a control, we targeted Xdd1 injections to the dorsal, vegetal blastomeres, where expression inhibits predominantly mesodermal convergent extension and does not inhibit NT closure (Wallingford and Harland, 2001); in those embryos, Sox2 expression resembled wild type (not shown).

View larger version (60K): [in a new window]   Fig. 9. Expression of wild-type Stbm elicits neural tube closure defects and failure of neural convergent extension. (A) Control embryo at mid-neurulation with closing NT. (B) Stbm-injected embryo with defective NT closure. (C) Xfd12 expression in control embryo at stage 14. (D) Xfd12 expression reveals defective neural convergent extension in a stage 14 Stbm-injected embryo. (E) Expression of Xpax3 in a control embryo. (F) Expression of Xpax3 in an XStbm injected embryo. (G) Expression of Xash3 in a control embryo. (H) Expression of Xash3 in an XStbm-injected embryos. (I) Expression of Sox2 in a control embryo. (J) Expression of Sox2 in an XStbm-injected embryos.

View larger version (12K): [in a new window]   Fig. 10. Biomechanical contribution of convergent extension to neural tube closure. During normal neurulation, mechanisms that advance the elevated neural folds toward the midline (i.e. apical wedging, pushing by the epidermis, etc.) produce a finite amount of medial movement, arbitrarily defined as `X' in this figure (red bars). Convergent extension narrows the midline by a finite amount, defined as `Y' in this figure (blue bar). So, the distance between the forming neural folds can be defined as X+Y; at the end of normal neurulation this distance is reduced to zero, and the folds can meet and fuse. In embryos that lack PCP function, the distance X is covered by the normally functioning mechanisms (see Fig. 4). However, in the absence of convergent extension, the midline fails to narrow and at the end of neurulation, the folds cannot fuse as they remain separated by a distance roughly equal to `Y.'

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