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First published online 24 October 2007
doi: 10.1242/dev.02886

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Delamination of cells from neurogenic placodes does not involve an epithelial-to-mesenchymal transition

Anthony Graham¹, Aida Blentic¹, Sandra Duque^1,* and Jo Begbie^2,

¹ MRC Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK.
² Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.

View larger version (85K): [in this window] [in a new window]   Fig. 1. Placodal neuroblasts exit the characteristic placodal ectoderm through an underlying breach in the basal lamina. (A-D) Transverse confocal section of st17 chick embryo. Boxed regions in A and B are shown at higher magnification in C and D. (A,B) Characteristic morphology of thickened epithelium at the site of neuronal production (lower boxed region). (A) NFM, green; ß-catenin, magenta. (B) ß-catenin staining alone. (C) Non-placodal ectoderm. ß-catenin is localized to the cell membrane (arrows). (D) Placodal ectoderm. ß-catenin is not specifically localized and epithelium appears to consist of multiple cell layers. (E-H) Transverse confocal section of st17 chick embryo. NFM, green; laminin, magenta. (E) Non-placodal ectoderm, anterior to placode. Basal lamina intact beneath the ectoderm for the whole dorsoventral extent of embryo (arrows). (F) Placodal ectoderm showing thickened morphology and NFM-positive cells leaving the epithelium. The boxed region is shown at higher magnification in G,H. (G) Where the neuronal cells exit, there is a breach in the basal lamina. This is seen more clearly when laminin is shown alone (H); the extent of the breach is indicated by arrowheads. Laminin is also seen in neuronal cells as they migrate away. *, placodal epithelium.

View larger version (133K): [in this window] [in a new window]   Fig. 2. Placodes do not express molecules associated with EMT. (A-C) RhoB expression in chick at st18. (A,B) Post-migratory neural crest cells remaining in contact with hindbrain show high levels of RhoB expression. High-level expression is also seen in neural crest cells in the trunk (C), but expression is not specifically elevated in placode (B). (D-F) Snail2 expression at st17. There is no expression in the placode (E); however, a transverse section of trunk shows expression in the neural crest progenitors, as expected (F). (G-I) Snail expression at st18. Expression is seen in cranial mesoderm including post-migratory crest (G,H). No expression of Snail is detected in cranial ectoderm (H) or neural crest progenitors (I). (A,D,G) Lateral view of pharyngeal region (x6); (B,E,H) Transverse section through placode (x16); (C,F,I) Transverse section at hindlimb level (x16). nc, neural crest; ov, otic vesicle; pnc, post-migratory neural crest; I, II, III refer to the pharyngeal arches; *, placodal epithelium; s, somite.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Placodes resemble neuroepithelium. (A) Coronal confocal section through the third and fourth pharyngeal arches demonstrating the break in the basal lamina and the position of the placode (for comparison with B). ß-catenin, green; laminin, magenta. (B) Longitudinal semi-thin section showing that the mesenchymal morphology of surrounding neural crest cells (mc, mesenchymal cells) is distinct from the compact morphology of cells leaving the placode (*). (C) TEM of the boxed region from B showing the round cell morphology of cells leaving the placode. (D,E) GFP labeling of placodal ectoderm and resulting neurons indicating the pseudostratified morphology of the placode. (D) Transverse confocal section showing the whole z-stack. (E) Single optical slice with the outline of labeled cells within placodal ectoderm highlighted schematically. Arrow, neurons forming ganglion; arrowhead, neuroblast leaving placode; *, placodal epithelium. (F,G) GFP labeling of mesencephalon and emigrating neural crest. (F) Transverse confocal section showing the whole z-stack. (G) Single optical slice with the border of the neural tube and the outline of labeled cells highlighted schematically. Arrow, migrating neural crest cells; arrowhead, neural crest cell with mesenchymal morphology leaving the neuroepithelium; *, neuroepithelium.

View larger version (119K): [in this window] [in a new window]   Fig. 4. Cell division occurs at the apical surface of the placodal epithelium. (A,B) Coronal confocal section through the pharyngeal arch region at st16. Islet 1/2, green; PH3, magenta. The boxed region is shown at higher magnification in B. Division occurs at the apical surface of the placode (arrowhead). Proliferation is reduced in the placode as compared with the ventricular zone (vz) of neural tube (pial and ventricular surfaces of neural tube indicated by lines). (C,D) Transverse confocal section through the petrosal placode at st18. Islet 1/2, green; PH3, magenta. The boxed region is shown at higher magnification in D. Little division is seen within the placodal epithelium and where it does occur it is restricted to the apical surface of the placode. (E) Transverse section through the geniculate placode at st18. BrdU, magenta; Phox2a, blue. BrdU labeling confirms that there is little division in the placode and no accumulation of BrdU-positive cells at the point of exit of the placodal cells. *, placodal epithelium; nt, neural tube.

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