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Gene Expression during Palate Fusion in vivo and in vitro

¹ Developmental Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK;
² Unit of Paediatric Dentistry, Eastman Dental Institute and Hospital, University College London, UK; and
³ Faculty of Dentistry, Sous Section Odontologie Pédiatrique, Hôpital Civil, Louis Pasteur University, 1 place de l’Hôpital, F-67000 Strasbourg Cedex, France;

View larger version (125K): [in a new window]   Figure 1. Palatal fusion in vivo (A,B,C) and in vitro (D,E,F) examined in transverse sections stained with hematoxylin and eosin. (A) At E13 (n = 3), palatal shelves (ps) lie vertically on both sides of the tongue (T). (B) At late E14 (n = 5), the shelves are elevated horizontally and juxtaposed. The MEE formed the MES (arrow). (C) At E15 (n = 3), the MES was disrupted, and epithelial islands were observed (arrows), which completely disappeared at E16 (unpublished observations). The mesenchyme from both shelves merged at the midline (asterisk), and lateral mesenchymal cells condensed (stars). (D) At 30 hrs in culture (n = 5), the MEE cells formed the multilayered MES (arrow). (E) At 48 hrs (n = 5), MES thinned and was disrupted (arrow), allowing mesenchyme to merge across the midline. (F) At 60 hrs (n = 3), MES disappeared, and the palate was completely fused (asterisk). (G) Diagram summarizing time-frame of the fusion processes in vivo (bold lines) and in vitro (dotted lines). Scale bars: A,B = 500 µm; C,D,E,F = 200 µm. ps = palatal shelf, t = tooth bud, N = nasal surface, O = oral surface, T = tongue.

View larger version (28K): [in a new window]   Figure 2. Expression patterns of genes regulated during palate development in vivo (A,B,C,D,E,F) and in organ culture (G,H,I,J,K,L), assessed by RT-PCR. (A,G) tgfß3, fgfr1; (B,H) fgfr2IIIb; (C,I) twist, snail; (D,J) E-cadherin, keratin 5; (E,K) dsg1; (F,L) vimentin. Error bars represent the standard error of the means (SEM); n = 3. Asterisks indicate significant differences between 2 time-points (P < 0.05).

View larger version (85K): [in a new window]   Figure 3. Expression of snail in vivo (A) and in vitro (B–G) detected by whole-mount in situ hybridization (positive signal is dark or reddish-blue). Palatal shelves (n = 3–7/group) were either cultured in proximity to allow fusion to occur (B-D) or placed apart (E-G). (A) Transverse section of the whole-mount in situ hybridization of an E14.5 (n = 5), showing strong snail expression in the mesenchyme of the fusing palatal shelves, but not in the epithelium of the MES. (B-C) A similar pattern of snail expression is observed in fusing palatal shelves cultured for 30 and 48 hrs, with a weaker signal restricted to the lateral aspect (asterisks) observed at 48 hrs when the MES is being disrupted. (D) snail expression decreases as fusion progresses and is hardly detectable after 60 hrs in culture. (E,F,G) snail expression in palates that are not allowed to fuse decreases over time, as in fusing palatal shelves (B-D). Scale bars: A = 300 µm, B-G = 500 µm. N = nasal aspect, O = oral aspect, t = tooth bud. Color version of this figure is available online as an Appendix.