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First published online June 6, 2008
doi: 10.1242/10.1242/dev.019125

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Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehog-producing cells

¹ CNRS, UPR 2197, Laboratoire de Développement, Evolution et Plasticité du Système nerveux, Institut de Neurobiologie Alfred Fessard, F-91198 Gif-sur-Yvette, France.
² CNRS, UMR 7622, Laboratoire de Biologie du Développement, Paris, France.
³ UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement, Paris, France.

View larger version (122K): [in this window] [in a new window]   Fig. 1. Effect of QT6-Shh cell graft in BA1 presumptive territory on lower-jaw morphogenesis. (A) Graft of QT6-Shh cells (outlined), under the ectoderm, lateral to the posterior mesencephalon, in a 5 ss chick embryo, on its right side. (B) Cross-section at the level of the graft at 10 ss. In situ hybridization for Shh shows the QT6-Shh cells in contact with lateral ectoderm (Ect) and foregut endoderm (End). (C) Lateral right view of the head at E11 showing the normal lower beak (1) and two supernumerary hemi-lower-beaks (2,3) with mirror-image polarities (1/2 and 2/3). (D) Skeleton preparation of the embryo shown in C. The right lower jaw shows a large proximal Meckel's cartilage (arrow) prolonged by three distinct branches of cartilage (1-3). The quadrate and articular (arrowhead) are not perturbed. (E) Higher magnification of D showing that each branch of Meckel's cartilage (1-3) is associated with a dentary bone (white arrows) oriented in mirror image. (F) Higher magnification of structures 1 and 2 viewed through the left side (control side) allows the comparison of the normal left Meckel's cartilage (nMc) and the larger right experimental one (eMc), and shows mirror-image duplication of splenial (black arrows) and dentary bones (white arrows). Scale bar: 50 µm in B.

View larger version (117K): [in this window] [in a new window]   Fig. 2. Localization, at E4, of QT6-Shh cells grafted in BA1. (A) Facial view showing the oral cavity and facial buds of an embryo grafted with QT6-Shh cells on its right side (left on the figure). The experimental mandibular bud (Md) is hyperplasic and presents an outgrowth in its lateroposterior region (arrow). (B) Lateral right view of the same embryo showing the Md and maxillary bud (Mx) on the operated side and the positions of serial, distal-to-proximal, sections (C-E). (C,C') Distal section showing no quail cells with the QCPN mAb. This part of the grafted Md presents a lateroposterior outgrowth (arrow in C'). (D,D') Medial section: few QCPN+ quail cells are present in this Md region (arrowheads in D'). (E-E'') Proximal sections: most of the Shh+ (E'') and QCPN+ (E,E') cells are found in the proximal region of BA1 (arrowheads) close to the boundary with BA2. Some cells are detected in BA2 proximal region (arrow). Scale bar: 100 µm in C-E''.

View larger version (113K): [in this window] [in a new window]   Fig. 3. Gene expression analysis at E3-4 in BA1 grafted with QT6-Shh cells. (A-C) Facial views of whole-mount in situ hybridized chick embryos grafted with QT6-Shh cells in BA1 territory on the right side (left on the figure). The left BA1 (right on the figure) is the control side. Fgf8 (A) and Bmp4 (C) expression is expanded, respectively, in the posterior proximal and distal BA1 ectoderm. Fgf8 is uniformly expanded (one arrow) while Bmp4 shows two extra zones of expression (two arrows). Shh (B) is also ectopically induced in two extra zones in the posterior ectoderm of BA1 (two arrows), while it continues to be normally expressed in the oral endoderm. (D) Facial view at E4 showing a duplicated right BA1 (on the left) (I,I') while BA2 (II) is normal. Orientation of oblique sections in E-I (medial to lateral) are indicated. (E-I) In situ hybridization on adjacent sections evidenced a partial colocalization of Fgf8 (G) and Bmp4 (I) transcripts in the duplicated BA1 ectoderm and a strong Shh expression (H) in the ectodermal fold separating I and I' (arrow). Grafted QT6-Shh cells are visible on this section. (F) Pitx1 is expressed in three distinct zones of BA1. Mes, mesencephalon; Rb, rhombencephalon. Scale bar: 100 µm in E-I'.

View larger version (101K): [in this window] [in a new window]   Fig. 4. Gene expression at E3-4 in BA1 mesenchyme after graft of QT6-Shh cells. (A-D) Expression patterns of Noggin (A), Pitx1 (B), Gli3 (C) and dHand (D) in BA1 mesenchyme at E3 and E4. On the grafted side (left) Noggin, Pitx1 and dHand expression is extended in a mediocaudal direction (arrows). (C) Gli3 is downregulated in the region corresponding to dHand expression (arrow).

View larger version (101K): [in this window] [in a new window]   Fig. 5. Gene expression analysis at E5 in BA1 grafted with QT6-Shh cells. (A-C) Frontal sections from distal (A) to half-proximal (B,C) regions of E5 mandibular buds (Mds). (A,B) Bmp4 is strongly expressed in the caudal-most ectoderm in the QT6-Shh grafted side (arrow) as in the rostral ectoderm of the Md, where it is normally expressed (arrowheads). (C) Sox9 expression is caudally extended in the mesenchyme of the grafted Md. Mx, maxillary bud. (D) Facial view of a grafted embryo at E5 with the location (double-headed arrow) of a series of transverse sections (E-I) indicated according to the proximodistal axis of the Mds. (E) Shh expression shows the grafted QT6-Shh cells (arrow) in the proximal region close to the oral endoderm expressing Shh. Very few Shh-producing cells are found in the distal mesenchyme (arrowhead). (F) Col2a1 expression shows expansion of Meckel's cartilage (proximal part and induced branch) in the grafted side (arrows). The arrowhead indicates tongue cartilage. (G) Bmp4 transcripts are present in the tip of the tongue mesenchyme (arrowhead) and in the Md distal ectoderm and mesenchyme in the experimental side (arrows). (H,I) Dlx5 and Pitx1 expressions are extended distally and show a colocalization with Bmp4 in the mesenchyme in close contact with lateral ectoderm (arrows). Scale bar: 500 µm in A-C,E-I.

View larger version (117K): [in this window] [in a new window]   Fig. 6. Gene expression analysis at E6 in BA1 grafted with QT6-Shh cells: cell differentiation. (A) Facial view of a grafted E6 chick embryo showing caudal mandibular bud (Md) extension in the grafted side (arrow). (B-G) Frontal serial sections proximodistally at the level of the Mds. (B,B') Bmp4 expression in the distal ectoderm and mesenchyme of the ectopic Md. (C,C') Col2a1 expression indicates an extra Meckel's cartilage (arrow) near the Bmp4 zone of expression. (D,D') Cbfa1 transcripts are present in the mesenchyme of normal and ectopic Mds close to the Bmp4 domain and around Col2A1-expressing Meckel's cartilage anlage. (E) Distal region of the extra Md with a faint expression of Shh in the ectoderm (arrow). (F) Nerve fibers immunostained with HNK1 mAb close to the ectopic Meckel's cartilage (arrows; see outlines in B' and C'). (G) Frontal section in Md proximal half, in the grafted side, shows an extension of muscle (MyoD expression) and HNK1+ nerves (square enlarged in G'). (G') MyoD expression is associated with nerve (HNK1 immunostaining). Scale bar: 500 µm in B-G.

View larger version (102K): [in this window] [in a new window]   Fig. 7. QT6-Shh cells grafted in presumptive BA2 or in the post-optic region do not induce BA1 extra-skeletal pieces. (A) Graft of QT6-Shh cells in the presumptive BA2 territory in a 9 ss chick embryo (dotted circle). (B,C) At E11, the grafted embryo shows a normal beak morphology but presents a larger otic vesicle (arrow). No ectopic skeletal structure is observed. (D-G) Facial views of E3-4 BA2-grafted embryos. (D) Shh expression is reduced in BA2 grafted side (arrows), when compared with the control side. (E) Bmp4, (F) Fgf8 and (G) Pitx1 expression is not affected. (H) QT6-Shh cell graft in the presumptive post-optic region of a 5 ss chick embryo (dotted circle). (I,J) E11 operated chick embryo shows normal morphology and skeletal structures on the grafted side. (K,L) Induction of Bmp4 and Pitx1 expression in the post-optic region (arrows) where these genes are not expressed in control side (K',L').

View larger version (43K): [in this window] [in a new window]   Fig. 8. A model for oral epithelium induction by the foregut endoderm or by the graft of QT6-Shh cells in the chick BA1 territory. (A,B) A 6 ss chick embryo cross-sectioned at the normal oropharyngeal level (A), and at the level of grafted QT6-Shh cells (purple spot) (B). Gene inductions elicited by normal (A') and ectopic (B') sources of Shh (purple) are indicated. (C) Expression of Fgf8 (green), Bmp4 (red) and Shh (purple) in BA1 ectoderm of QT6-Shh grafted (on the left) and control (on the right) sides at E4. Curved arrows indicate growth orientation of the further normal (1) and induced (2 and 3) Meckel's branches. (C') Virtual section of the experimental BA1 (broken line) indicates three distinct lower jaw organizing centers (1-3) presenting Fgf8/Shh/Bmp4 alternations in the normal (1) and induced (2,3) jaw anlagen. I, first branchial arch; I', induced part of I; II, second branchial arch; Ect, ectoderm; End, endoderm; Ao, aortic arch. A'' and B'', normal oral epithelium and induced `oral-like epithelium'.

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