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First published online 1 September 2004
doi: 10.1242/dev.01337

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Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium

Department of Craniofacial Development, Dental Institute, Kings College London, Floor 28, Guys Hospital, London Bridge, London SE1 9RT, UK

View larger version (31K): [in a new window]   Fig. 1. Examples of DiI and DiO labelling of chick embryos, and the locations of the proximal and distal domains of the oral mandibular primordium. (A) Dorsal view of a stage 9 embryo. (B) Stage 9 embryo viewed from the dorsal side with the head folded back at the sub-blastodermic fold to expose the ventral surface ectoderm. The sub-blastodermic fold, anterior neuropore and the lateral edges of the embryo were used as landmarks, and the DiI/DiO position was recorded relative to these structures. (C) Labelling of the lateral edge of the dorsal side of a stage 10– chick embryo with a DiO crystal (arrow). (D) DiI labelling of the ventral side of stage 8+ head ectoderm. (E) Representation of the chick mandibular primordium at stages 18 to 20. The proximal (p) oral ectoderm where Fgf8 is expressed is shown in red and the distal (d) oral ectoderm where Bmp4 is expressed is shown in green. (F,G) Whole mount in situ hybridisation of Fgf8 (F) and Bmp4 (G). Drawings are not to scale. d, distal ectoderm; p, proximal ectoderm.

View larger version (43K): [in a new window]   Fig. 8. Fgf8 and Bmp4 expression in cultured explants of stage 9 chick embryos. Stage 9 coronal segments, corresponding to approximately one-third of the length of the total head, rostral to the subgerminal fold and encompassing the presumptive mandibular region (as predicted from the fate map), were dissected as shown. (A) Isolation and separation of ventral head tissues. (B) Fgf8 expression in explanted tissue in two lateral domains and a single Bmp4 domain. (C) Isolation of ventral-lateral (p) tissues and ventral-medial tissues (d). (D) Fgf8 is expressed in ventral-lateral explants (p) but not in ventral-medial explants (d). Bmp4 is expressed in both ventral-lateral and ventral-medial explants.

View larger version (30K): [in a new window]   Fig. 2. Dorsal fate map of the lateral edges of stage 8 to 10– chick embryos. Embryos having 4 and 5 somites (stages 8 and 8+), 6 and 7 somites (stages 9– and 9), and 8 and 9 somites (stages 9+ and 10–) were pooled for analysis. The embryos, when labelled, were aligned along the y-axis of the graticule with the anterior neuropore at the zero position and the first somite at position 100. Where label was found to be located in a single area following incubation, the position at which the label was injected along the y-axis is shown by a coloured circle; the different coloured circles represent different fates. Where two structures were labelled following incubation, half circles of each of different colour are placed at the point of labelling. The range of percentage distances along the y-axis between the anterior neuropore and the first somite, where the labels were fated predominantly to the axial level of the maxilla and the mandible, are shown. Also shown is the number of embryos used for each age category and the number of embryos that had more than one structure labelled from an individual DiI/DiO injection.

View larger version (48K): [in a new window]   Fig. 3. (A) In situ hybridisation showing maxilla and proximal mandibular primordial expression of Fgf8. (B-D) A DiI labelled embryo at stage 9, from the dorsal side. DiI was injected on the lateral edge, one-third the distance between the anterior neuropore and the first somite; the embryo was then incubated for an additional 48 hours (until approximately stages 18-20). (B) Frontal view of embryo following 48 hours of incubation. Cells containing DiI are located in the maxilla and the proximal oral mandible primordia. (C) Side view of embryo. (D) Vibratome section (40 µm) showing DiI-labelled cells located on the oral side of maxilla and mandible primordia. The plane of section is shown in B by the white vertical line. (E,F) Chick embryo labelled at stage 9 with DiI from the dorsal side and incubated for 48 hours. DiI was placed on the lateral edge of the embryo halfway between the anterior neuropore and the first somite. (E) Side view of whole embryo, fluorescent cells are located on the aboral side of the first pharyngeal arch. The white line represents the plane of vibratome sectioning. (F) Vibratome section (40 µm) showing that DiI is in the aboral epithelium. (G) Example of DiI labelling of the maxillary primordium of the first pharyngeal arch. (H) Example of DiO labelling of ectoderm rostral to the maxillary primordium of the first arch. mx, maxilla; md, mandible; pp, pharyngeal pouch.

View larger version (64K): [in a new window]   Fig. 4. DiO and DiI labelling of stage 10 chick embryos, followed by 48 hours of incubation (until approximately stages 18-20). (A) The site at which the DiO crystal was placed, the lateral edge and the proximal ventral ectoderm. (B) Image of embryo taken after 48 hours of incubation using epifluorescence and a GFP2 filter; DiO is located on the proximal mandibular primordium. (C) Image shown in B merged with a visible light image using Photoshop 6 (Adobe, USA). (D) DiI injected into cells on the ventral head ectoderm close to the midline. (E) Image of DiI fluorescence of embryo shown in D after 48 hours. Image taken using an epifluorescent microscope with a rhodamine filter and merged with visible image using Photoshop. Fluorescent cells are located in the distal oral mandibular primordium. (F) Vibratome section (40 µm) illustrating the epithelial location of the DiI. d, distal mandible; epi, epithelium; md, mandible; mes, mesenchyme; mx, maxilla; p, proximal. The location of the DiI is highlighted by the position of the white arrows.

View larger version (31K): [in a new window]   Fig. 5. Fate of DiI/DiO-labelled cells on the ventral surface ectoderm, with diagrammatic representation of the labelling of stage 8-10 embryos. The fate maps of stage 8, 8+ and 9– embryos were pooled (A), as were those of stage 9, 9+, 10– and 10 embryos (B). The position of labelled cells is shown as either grey circles or black triangles. Grey circles represent cells fated to occupy the proximal mandible epithelium and black triangles the distal mandible epithelium in stage 18-22 chick embryos, as shown by the shading of the chick mandible in C.

View larger version (128K): [in a new window]   Fig. 6. Whole-mount in situ hybridisation of chick embryos using a probe for Fgf8. (A,B) Dorsal (A) and side (B) views of a stage 11 embryo. The white arrow and arrowhead indicates the Fgf8 domain believed to demarcate the proximal pharyngeal arch ectoderm. (C,D) Side and ventral views of a stage 13– embryo. The white arrows indicate the Fgf8 domain on the developing first pharyngeal arch. (E,F) Stage 14– embryos with the mandible primordium clearly visible, white arrow pointing to the Fgf8 domain. (G) Ventral aspect of a stage 11 chick embryo showing positions of vibratome sections (40 µm) in H and I, cut at axial levels x and y, respectively. ect, ectoderm; end, endoderm.

View larger version (90K): [in a new window]   Fig. 7. DiO labelling of a stage 11 chick embryo. (A) The head of the embryo is bent back along the sub-blastodermic fold to show the position of the DiO crystal on the ventral surface ectoderm. (B) View of the same embryo from the dorsal aspect. The black arrow shows the position of the midbrain-hindbrain junction. (C,D) The same embryo following 24 hours of incubation. (C) DiO, viewed under fluorescence and a GFP2 filter. (D) The visible and the fluorescent images merged using Photoshop 6 (Adobe, USA). Note that the DiO fluorescence is located on the proximal surface ectoderm of the mandibular primordium. White arrows indicate the DiO crystal.

View larger version (37K): [in a new window]   Fig. 9. Fgf8 expression in the presumptive mandibular ectoderm is lost following removal of the underlying endoderm. (A) Shaded segment is the approximate level at which regions were dissected from stage 9 embryo. (B) Coronal segment; ventral head tissue cut at the midline to expose the foregut endoderm. (C) Fgf8 is expressed at the midbrain-hindbrain junction (MBH) and in two more rostrally located lateral domains (black arrows), corresponding to the presumptive maxillo-mandibular domain. (D) Coronal segment; ventral head tissue cut at the midline to expose the foregut endoderm and the foregut endoderm removed unilaterally. (E) Fgf8 expression in an explant with unilateral endoderm removal. Fgf8 is expressed as normal in the neural tube at the midbrain-hindbrain boundary (MBH). Expression is lost in the lateral domain on which the endodermal removal had taken place (red arrow), but is present on the contralateral side (black arrow).