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Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton

Institut d’Embryologie cellulaire et moléculaire du CNRS et du Collège de France, 49bis, av. de la Belle Gabrielle – 94736 Nogent-sur-Marne cedex, France

View larger version (58K): [in a new window]   Fig. 1. The cephalic neural crest: fate map and Hox gene expression. (A) Presumptive diencephalic, mesencephalic and rhombencephalic territories of the neural fold in the avian embryo at 5 ss, as established by Grapin-Botton et al. (Grapin-Botton et al., 1995). (B) Migration map of cephalic neural crest cells in the avian embryo. The origin of neural crest cells found in the nasofrontal and periocular regions and in the branchial arches is color-coded as in A. Neural crest cells arising from the posterior diencephalon and mesencephalon populate the nasofrontal and periocular region. Posterior mesencephalon also participates in these structures, but in addition populates the anterodistal part of the first branchial arch. The complementary portion of the first branchial arch derives from r1/r2, together with a small contribution from r3. The major contribution to the second branchial arch comes from r4. Neural crest cells arising from r3 and r5 split into strains participating to two adjacent arches: r3 cells migrate to the first and second branchial arches; r5 cells migrate to the second and third branchial arches. r6 cells migrate to the third and fourth branchial arches, r7- and r8-derived cells migrate to the third and to the more caudal branchial arches. (C) Hox gene expression in the chick and quail embryo at E3 when the branchial arches are being colonized by neural crest cells originating from the posterior half of the mesencephalon and the rhombomeres (r1-r8). The arrows indicate the AP origin of the neural crest cells migrating to each branchial arch. Expression of Hox genes is also indicated in the superficial ectoderm, the endoderm and mesoderm. (D,E) Cartilages forming the upper face of a chick embryo at E8 and the contribution of the crest-derived cells according to the level from which they originate. (F,G) Lower jaw skeleton of E8.5 chick embryo: the participation of the crest derived cells is color coded as in A. Skeletal components of hyoid cartilages, which are formed by Hox-expressing crest cells (Hox +), are shown in black. (H) Bilateral surgical ablation of the Hox-negative domain of the skeletogenic neural fold, extending from the mid-diencephalon down to r2 included in 5 ss chick embryo. (I) Frontal view of E8 chick embryo subjected to the bilateral extirpation of the Hox-negative skeletogenic neural folds. In these embryos, which have virtually no face, structures anterior to the second branchial arch fail to develop: the nasal process, mandibular and maxillary buds are rudimentary. (J,K) In this context, the bilateral graft of the posterior diencephalic neural folds can regenerate the excised territory and form a normal face. BA, branchial arch; Ect., ectoderm; Endo., endoderm; Meso., mesoderm; NC, neural crest; NFB, nasofrontal bud; r, rhombomere. A, articular; Bb, basibranchial; Bh, basihyal; Cb, ceratobranchial; E, entoglossum; Eb, epibranchial; Mc, Meckel’s cartilage; Nc, nasal capsule; Q, quadrate; Sc, sclerotic.

View larger version (83K): [in a new window]   Fig. 2. Forced expression of Hoxa2 in the endogenous posterior diencephalic neural fold and the facial phenotype. (A,B) Bilateral co-electroporation of Hoxa2 RCAS (BP)B (red) or control RCAS (BP)B (blue), and GFP constructs (green) in the anterior neural fold of a 5 ss chick embryo. Bilateral extirpation (broken lines) of the cephalic neural fold extending from mid-mesencephalic level down to r2/r3 boundary in the electroporated chick embryo. (C,D) In E3 control and Hoxa2-transfected embryos, in situ hybridization for Hoxa2 shows the endogenous pattern of gene expression at the rhombencephalic level (arrowhead), and in the second branchial arch (open arrow). (D) In Hoxa2-transfected embryos, the ectopic expression is evidenced at the diencephalic level (arrows) where the neural crest cells have started to migrate over the forebrain. Facial morphology and skeleton of a control embryo (E,F) and Hoxa2-transfected embryo (G,G',H) at E7. The latter exhibits severe morphological defects: its face consists in underdeveloped nasal and maxillary buds (G,G') in which no skeletal components of the upper face or lower jaw form as shown in H, representing an embryo treated with Alcian Blue (compare with F). Mxb, maxillary bud; Nlb, nasolateral bud; Nmb, nasomedial bud; T, tongue.

View larger version (92K): [in a new window]   Fig. 3. Forced expression of Hoxa2 in the entire Hox-negative neural fold and contribution of Hoxa2 neural fold-derived cells to facial development. (A) The experimental procedure consists in the unilateral co-electroporation of Hoxa2 RCAS (BP)B (red) and GFP (green) constructs in the Hox-negative neural fold, performed alternatively on each side of two different 5 ss chick embryos. The transfected neural folds are excised and bilaterally transposed to homotopic level into a stage-matched recipient chick embryo. Concurrently, the untransfected contralateral neural folds are bilaterally engrafted in a chick embryo that serves as a control. (B) Dorsal view of E3 embryo (HH11) showing the migration of GFP-labeled cells. (C) E3 embryo (HH14) after hybridization for Hoxa2, showing (1) endogenous expression in the second branchial arch (open arrow); and (2) ectopic expression in mesectodermal cells that both fill the first arch and nasofrontal bud and overlay the prosencephalic, mesencephalic and metencephalic vesicles (arrows). At E7, control embryos, in which the contralateral non transfected neural folds have been grafted, (D,D') show a normal development of both upper and lower beaks. By contrast, embryos engrafted with Hoxa2 transfected neural crest (E,E') do not develop facial structures. At E4, embryos in which the diencephalic neural fold has been transfected with the Hoxa2 construct (F), show a severe misdevelopment of the forebrain vesicles compared with control embryos (G). (H) Hoxa2 neural fold-derived cells form glial cells in peripheral nerves, as observed after HNK1 immunolabeling. At diencephalic level (I), neural crest cells give rise to pericytes (that accumulate smooth -actin, detected by 1A4 Ab) to the forebrain vasculature (J), and differentiate into cilliary muscles and corneal endothelium (arrows, K). Di, diencephalon; Mes, mesencephalon; Met, metencephalon. Scale bars: 350 µm in F; 400 µm in G; 50 µm in H; 600 µm in I; 60 µm in J; 75 µm in K.

View larger version (98K): [in a new window]   Fig. 4. Forced expression of Hoxa3 or Hoxb4 and the early steps of neural crest cell migration. (A) To target Hoxa3 or/and Hoxb4 expression in posterior diencephalic neural crest, unilateral electroporations of anterior cephalic neural fold were alternatively carried out on each side in two 5 ss quail embryos. Then, transfected neural folds were bilaterally implanted in a stage-matched chick embryo, which had been subjected to the bilateral ablation of the Hox-negative domain of its skeletogenic neural folds (broken lines). Recipient chick embryos that are bilaterally engrafted with the untransfected contralateral quail neural folds are referred to as quail-grafted controls. In situ hybridization with Hoxb4 probe in control embryo (B) and embryo that has received Hoxb4 transfected neural folds (C): the endogenous expression of Hoxb4 at posterior rhombencephalic level was unperturbed (arrowheads). In experimental embryos engrafted with Hoxb4 neural folds, Hoxb4 transcript expression also features the exogenous neural crest cells that start to spread from the transplant (arrows). Twenty hours after the operation (HH14), quail cell detection show that the migratory behavior of implanted neural crest cells in control embryos (D) is equivalent to that observed in embryos carrying Hoxa3 (E) or Hoxb4 (F) transfected neural fold-derived cells. In all these embryos, quail cells move rostrally to populate the nasofrontal bud (arrows) as well as laterally to colonize the presumptive first branchial arch (arrowheads).

View larger version (107K): [in a new window]   Fig. 5. Effect of ectopic Hoxa3 expression on facial development. At E6, quail-grafted control embryos develop a normal face (A) and show skeletal elements in the first branchial arch (B), consisting of Meckel’s, articular and quadrate cartilages (arrows). At E8, neural crest cells arising from untransfected diencephalic neural folds yield the entire facial and mandibular skeleton (C). By contrast, E6 embryos that have been engrafted with Hoxa3 transfected neural folds (D) show a reduced nasal bud (arrow) and fail to develop the first branchial arch (arrowheads). All the skeletal components of the mandibular bud are missing (E, arrowhead). At E8 (F), the nasal septum has developed rostral to the basipresphenoid and towards the top of the head. Although the posterior part of hyoid structure forms, in which the basihyal (Bh), ceratobranchial (Cb) and basibranchial (Bb) cartilages are recognizible, the lower jaw skeleton is completely absent. (G) At E7, in the forehead territory, telencephalic hemispheres are evidenced in which quail cells are identified as pericytes (H, arrows) of the neuroepithelium capillaries. At diencephalic level (I), the nasal septum that forms at the midline is of quail origin (Alcian Blue and QCPN mAb staining)(J). (K) Quail cells give rise to ciliary muscles and to the corneal endothelium. Bl, blood lacunae; Bs, basipresphenoid; Bv, blood vessel; Ce, corneal endothelium; Cm, ciliary muscle; Mc, Meckel’s cartilage; Ns, nasal septum. B,C,E,F are Alcian Blue stained embryos. Scale bars: 600 µm in G; 15 µm in H; 350 µm in I; 10 µm in J; 60 µm in K.

View larger version (97K): [in a new window]   Fig. 6. Effect of the ectopic Hoxb4 expression on head morphogenesis. At E6, when compared with quail-grafted-control embryos (A), which exhibit a normal face, the embryos that develop with Hoxb4-expressing diencephalic crest cells (D) have no nasal bud and a reduced first branchial arch. (B,C) At E8, untransfected neural crest cells grafted in control embryos ensure the normal development of the facial and mandibular skeleton. (E,F) In embryos carrying Hoxb4-transfected neural folds, the sella turcica is the most rostral skeletal structure, as the upper beak skeleton is missing. In the first branchial arch, the skeleton is reduced to its proximal elements: quadrate, articular and the proximal region of Meckel’s cartilage. St, sella turcica.

View larger version (85K): [in a new window]   Fig. 7. Contribution of Hoxb4 neural fold-derived cells to facial development. At E6, in chimeric embryos engrafted with Hoxb4 transfected neural folds, quail cells that populate the nasofrontal area (A) contribute to nerve glia (B), become pericyte (arrows) in choroid membrane (C), and form a loose mesenchyme under the olfactory epithelium (D). At E8, in such embryos, the sella turcica remains the most rostral skeletal structure (E) in which the basipostsphenoid is of host mesodermal origin (F) and the basipresphenoid of quail mesectodermal origin (G) (same staining procedure as in Fig. 5I,J). In the first branchial arch (H), quail cells generate both Meckel’s (I) and articular (J) cartilages. Mc, Meckel’s cartilage; Ar, articular. Scale bar: 230 µm in A; 30 µm in B; 30 µm in C; 30 µm in D; 375 µm in E; 10 µm in F; 10 µm in G; 600 µm in H; 12 µm in I; 12 µm in J.

View larger version (93K): [in a new window]   Fig. 8. Effect of the combined ectopic expression of Hoxa3 and Hoxb4 on facial development. (A) At E7, embryos carrying Hoxa3 and Hoxb4 co-electroporated neural folds are devoid of facial structures. (A') Higher magnification showing the underdeveloped nasal, maxillary and mandibular buds. (B) In the forehead of chimeric embryos, graft-derived cells differentiate as pericytes (arrows) for the telencephalic capillaries (C) and for the choroid membrane (D), though in the latter, large blood lacunae (Bl) form. Note the blood vessels (Bv) and blood lacunae are filled with nucleated erythrocytes. (E) In the nasal region, quail cells contribute to ciliary muscles and corneal endothelium (F) and give rise to mesenchyme in the nasal buds (G). Cm, ciliary muscle; Mdb, mandibular bud; Mxb, maxillary bud; Nlb, nasolateral bud; Nmb, nasomedial bud. Scale bar: 360 µm in B; 15 µm in C; 25 µm in D; 700 µm in E; 50 µm in F; 60 µm in G.

View larger version (119K): [in a new window]   Fig. 9. Fate of rhombencephalic r4-r8 neural crest cells rostrally transposed into the Hox-negative domain. (A) Bilateral substitution of the quail rhombencephalic r4-r8 neural fold to the host Hox-negative neural fold (extending from diencephalon down to r2). (B) E7. Frontal view of a chimeric embryo showing the complete absence of upper face and lower jaw. (C) Rhombencephalic quail cells that have colonized the facial primordium contribute Schwann cells to the trigeminal nerve (D). Some rare graft-derived cells are identified as pericytes in vascular wall of blood lacunae (E) and into the choroid membrane capillaries (asterisk, F). (G) Quail cells are poorly present in ciliary muscles and absent from the cornea. Scale bar: 250 µm in C; 50 µm in D; 30 µm in E; 20 µm in F; 30 µm in G.

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