Rashid D et al. (2006), A requirement for NF-protocadherin and TAF1/Set...

XB-ART-804

Dev Biol 2006 Mar 01;2911:170-81. doi: 10.1016/j.ydbio.2005.12.027.

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A requirement for NF-protocadherin and TAF1/Set in cell adhesion and neural tube formation.

Rashid D , Newell K , Shama L , Bradley R .

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Neurulation in vertebrates is an intricate process requiring extensive alterations in cell contacts and cellular morphologies as the cells in the neural ectoderm shape and form the neural folds and neural tube. Despite these complex interactions, little is known concerning the molecules that mediate cell adhesion within the embryonic neural plate and neural folds. Here, we demonstrate the requirement for NF-protocadherin (NFPC) and its cytosolic partner TAF1/Set for proper neurulation in Xenopus. Both NFPC and TAF1 function in cell-cell adhesion in the neural ectoderm, and disruptions in either NFPC or TAF1 result in a failure of the neural tube to close. This neural tube defect can be attributed to a lack of proper organization of the cells in the dorsal neural folds, manifested by a loss in the columnar epithelial morphology and apical localization of F-actin. However, the epidermal ectoderm is still able to migrate and cover the open neural tube, indicating that the fusions of the neural tube and epidermis are separate events. These studies demonstrate that NFPC and TAF1 function to maintain proper cell-cell interactions within the neural folds and suggest that NFPC and TAF1 participate in novel adhesive mechanisms that contribute to the final events of vertebrate neurulation.

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Species referenced: Xenopus
Genes referenced: actb actl6a ctnnb1 fubp1 myc pcdh7 set taf1
???displayArticle.antibodies??? Ctnnb1 Ab6 His-tag Ab1 Myc Ab3 Pcdh7 Ab1
???displayArticle.morpholinos??? pcdh7 MO1 set MO1

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	Fig. 1. Xenopus TAF1 and NFPC expression in the neural ectoderm. In situ hybridization analysis for TAF1 (A�E, I�M) or NFPC (F�H, N, O), viewed in wholemount (A�I) or in cross section (J�O). At stage 13, TAF1 is expressed throughout the neural plate (np), with highest expression anteriorly. At stages 15 (B) and 17 (C), TAF1 is expressed in the neural folds (nf). After neural tube closure, TAF1 is expressed throughout the neural tube (nt), in the eye (ey) brachial arches (ba), and forebrain (fb) (D, E). In contrast, at stage 17, NFPC expression is restricted to the tips of the neural folds (arrow in panels F and G) and in the ventral ectoderm. After neural tube closure, NFPC is also expressed in the anterior neural tube, eye, and branchial arches (H). (I) A stage 17 embryo stained by in situ for TAF1 (purple) and with an antibody to NFPC (brown) demonstrates co-localization of NFPC and TAF1 at the tips of the neural folds (arrow). Sections through embryos reveal that TAF1 is expressed throughout the neural plate, the somatic mesoderm (sm), and the inner layer of the epidermal ectoderm (J�M), while NFPC is localized to the tips of the neural folds (arrow in panel N) and the inner layer of the epidermal ectoderm prior to neural fold fusion. After the neural tube has closed, NFPC is expressed in the ventral neural tube (O). Other abbreviations: nc, notochord; psm, presomitic mesoderm.
	Fig. 2. NFPC and TAF1 antisense morpholinos disrupt neural tube closure. Embryos were co-injected with NFPCMO (A, D, G), TAF1MO (B, E, H) or CMO (C, F, I) and nlacZ RNA, fixed at stage 17 (A�C), stage 20 (D�F), or stage 25 (G�I), and processed for β-galactosidase activity. In NFPCMO-injected embryos, the neural folds were present at stage 17 (A), but by stage 20 (D) and stage 25 (G), the neural folds have failed to fuse (arrow). Similarly, in embryos injected with TAF1MO, the neural fold on the injected side formed (arrow in panel B) but failed to fuse at stage 20 or 25 (E, H). In contrast, control-injected embryos formed normal neural folds at stage 17 (C), which then fused at stage 20 (F) to form a normal tailbud stage embryo (I).
	Fig. 3. Rescue of neural tube defects by ectopic expression of NFPC or TAF1. (A) Embryos were injected with either NFPCMO or TAF1MO alone or were coinjected with NFPCMO and NFPC RNA, or TAF1MO and TAF1 RNA, then fixed at stage 25, and the percentage of injected embryos with neural tube defects was determined. The percentage of embryos with NTDs was reduced upon co-expression of NFPC or TAF1, respectively. (B�D) Examples of rescued, morpholino-injected embryos from (A). The majority of embryos coinjected with NFPCMO and NFPC mRNA appeared normal (C). While 54% of TAF1MO and TAF1 mRNA-injected embryos appeared normal (D), 46% still exhibited NTDs; however, the defects observed were greatly reduced in severity (white arrows in panel E).
	Fig. 4. NFPC and TAF1 dominant-negative constructs disrupt neural tube formation. Embryos were injected with RNA encoding NFΔE, TAF1ΔN, or TAF1ΔC, along with nlacZ RNA, or were co-injected with NFΔE and NFPC RNA or TAF1ΔN and TAF1 RNA then fixed at stage 25 and stained for β- galactosidase activity. (A) The percentage of injected embryos with NTDs. Both NFΔE and TAF1ΔN resulted in NTDs, the incidence of which was reduced by co-expression of NFPC or TAF1, respectively. In contrast, ectopic expression of TAF1ΔC resulted in few embryos with NTDs. (B�D) Representative embryos from panel A. Compared to embryos injected with TAF1ΔC RNA (D) or nlacZ RNA alone (E), embryos injected with NFΔE or TAF1ΔN RNA (arrows in panels B and C) failed to close the anterior neural tube.
	Fig. 5. NFPC and TAF1 function are required during neural fold formation and neural tube closure. Embryos were co-injected with NFPCMO (A, B, I), TAF1MO (C, D, J), CMO (E, F), or the dominant-negative constructs NFδE (G) and TAF1δN (H) and nLacZ RNA, then fixed at stage 17 or stage 25, stained for β-galactosidase activity, and sectioned. At stage 17, embryos injected with NFPCMO (A) or TAF1MO (C) failed to form a normal neural fold on the injected side. By stage 25, the neural tube failed to fuse, although the epidermal ectoderm had reached the midline (B, D). Similarly, embryos injected with RNA encoding NFδE (G) or TAF1δN (I) failed to fuse the neural tube by stage 25. In contrast, embryos injected with CMO (C, D) formed a normal neural fold and neural tube, as did embryos in which NFPCMO or TAF1MO was targeted to the epidermal ectoderm (I, J). Abbreviations: nc, notochord; nf, neural fold; nt, neural tube; psm, presomitic mesoderm; sm, somite.
	Fig. 6. NFPC-mediated cell adhesion in the neural plate requires TAF1. (A) Embryos at the 16-cell stage were injected into a single dorsal blastomere with RNA encoding the indicated proteins or morpholinos then immunostained at stage 15, and single labeled cells in the neural ectoderm not in contact with another labeled cell were counted. As compared to control embryos injected solely with RNA encoding c-myc (C-MT) or GFP, embryos injected with RNA encoding NFPC-HT exhibited a reduced number of isolated cells in the neural ectoderm. The ability of NFPC-HT to suppress cell mixing was abrogated by coinjecting either the dominant-negative TAF1δN or TAF1MO, indicating that NFPC requires TAF1 to promote cell adhesion. (B) Embryos injected as above and stained by immunofluorescence for the histidine epitope tag to reveal NFPCHT (B and insert in panel C) or the myc epitope tag for TAF1δN-MT (C and D) or C-MT (E). Arrows indicate isolated cells.
	Fig. 7. NFPCMO and TAF1MO alter the organization of the neural folds. (A�F) Embryos were co-injected with NFPCMO (A, D), TAF1MO (B, E), or CMO (C, F), and nlacZ RNA, fixed at stage 17, processed for β-galactosidase activity, then stained with Oregon green-phalloidin to visualize F-actin, and viewed under fluorescence (A�C) or bright field (D�F). While F-actin was localized to the apical neural folds in CMO-injected embryos as well as on the uninjected side in NFPCMO- and TAF1MO-injected embryos (closed arrows in panels A�C), F-actin staining was lost at the tips of the neural folds on the injected side (open arrows in panels A, B). (G�L) Embryos injected with antisense morpholinos and examined by immunofluorescence for β-catenin localization at stage 17. In control embryos and on the uninjected side of NFPCMO- and TAF1MO-injected embryos, the neural folds formed normally. In contrast, the neural fold on the NFPCMO- or TAF1MO-injected side formed abnormally. High power photomicrographs revealed a disorganized neural epithelial on the injected side of NFPCMO- and TAF1MO-injected embryos (arrowheads in panels J, K) as compared to the typical columnar epithelia observed on the uninjected side or in control embryos (long arrows in panels J�L). In all cases, β-catenin was still localized to the cell membrane. In all photos, the injected side is on the right. Abbreviations: nf, neural fold, nc, notochord, nt, neural tube.
	Fig. 8. NFPC and TAF1 are required for convergent�extension after the neural folds have formed. (A) Embryos were injected with NFPCMO, TAF1MO, or CMO into both dorsal blastomeres at the 4-cell stage, and average length/width ratios of the AP axis were obtained at the indicated stages. NFPCMO and TAF1MO did not affect CE until stage 22. (B�D) As compared to the CMO, NFPCMO and TAF1MO-injected embryos at the equivalent of stage 25 exhibited a shortened AP axis.
	set (SET nuclear oncogene) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 28. Lateral view: Anterior left, dorsal up.
	pcdh7 (protocadherin 7 ) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 28. Lateral view: Anterior left, dorsal up.