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First published online 31 March 2004
doi: 10.1242/dev.01106

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Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis

¹ California Institute of Technology, Pasadena, CA 91125, USA
² Kansas State University, Manhattan, KS 66506, USA

View larger version (81K): [in a new window]   Fig. 1. Schematic representation of grafting experiments and resulting E15 quail-chick chimeras. Grafting of midbrain (control, A), cardiac (C) and trunk (E) dorsal neural tube tissues from stage 9-10 quail donors into stage 9 chick hosts results in highly pigmented E15 chimeras B, D and F, respectively. Cardiac neural crest chimeras form a distinct pigmented mass of cells (D, arrow) on the dorsal region of the cornea.

View larger version (116K): [in a new window]   Fig. 2. Grafted quail neural crest contribution to E15 chimeric corneas. All corneas are pigmented on the periphery (A,E,I,M). In addition, cardiac neural crest chimera corneas develop a darkly pigmented mass on their dorsal surfaces (E,I), which in some cases, grows feather buds (I, arrowhead). Cross-sections through representative corneas showing QCPN-positive (red nuclei) and MF20-positive (green) cells counterstained with DAPI. Midbrain control sections (B-D) show QCPN-positive cells in the corneal epithelium (Ep), stroma (st), endothelial cell layer (En) and ciliary muscle (Cm). Cardiac neural crest chimera corneas show numerous QCPN-positive cells and melanocytes in the ectopic mass (F-H), but fewer QCPN-positive cells in the layers of the cornea (J-L). All QCPN-positive cells in trunk neural crest chimera corneas (including those in the ciliary muscle, arrow) are pigmented.

View larger version (92K): [in a new window]   Fig. 3. Cross-sections through trigeminal ganglia of E15 chimeras (A,D,G) showing QCPN-positive nuclei (red) of large and small quail cells (B,E,H). The cells with large QCPN-positive nuclei are dispersed mostly in the dorsal part of the ganglia whereas those with small QCPN-positive nuclei are dispersed throughout the entire ganglia. Some of the QCPN-positive large cells are trkA positive (green) (C,F,I). (J) The percentage of large QCPN-positive nuclei significantly decreases in cardiac and trunk neural crest chimera ganglia. *P<0.001.

View larger version (61K): [in a new window]   Fig. 4. Comparison between E15 trigeminal ganglia from (A) midbrain control, (B) cardiac and (C) trunk neural crest chimera. Although cardiac and trunk chimera ganglia are smaller than the midbrain control, the ophthalmic (op) maxillary (mx), mandibular (mn) and trigeminal motor nerve (m) all look normal. D is a statistical comparison of the relative areas of the ganglia.

View larger version (79K): [in a new window]   Fig. 5. Whole-mount immunostaining of corneas from E15 chimeras. Quail neural crest-derived neurites are QN-positive (red; A,C,E) and all neurites are counterstained with TuJ1 (green; B,D,F). Numerous QN-positive neurites are seen in midbrain control (A,B) but the number decreases in cardiac (C,D) and trunk (E,F, arrowheads) chimera corneas.

View larger version (130K): [in a new window]   Fig. 6. Distribution of grafted quail neural crest cells in the cranial region. All sections are stained with QCPN (red nuclei) to show quail cells, collagen II (green) and counterstained with DAPI (blue). (A-C) Midbrain control neural crest contribute to ciliary ganglion (Cg), trigeminal ganglion (V), maxillary (Mx) and mandibular (Mn) processes, including Meckel's (Mk) cartilage. (D-F) Cardiac neural crest contribution is similar to control, but few cells contribute to Meckel's cartilage (F,F') and many to the quadrate (Qd). (G-I) Trunk neural crest cells contribute to the trigeminal ophthalmic nerve (OpV) and periocular region (arrowhead), but minimally to the mandibular process (arrow).

View larger version (128K): [in a new window]   Fig. 7. Sections through E7 midbrain (A,B) and trunk (C,D) chimera mandibles showing the distribution of QCPN-positive cells after grafting dorsal neural tubes directly into stage 11-12 first branchial arches. QCPN is red, Collagen II is green and the sections are counterstained with DAPI. Midbrain controls show numerous neural crest in the tongue (Tn) and mandibular process, including Meckel's cartilage (Mk). Trunk neural crest form aggregates of cells adjacent to but not within Meckel's cartilage. Bh, Basihyal cartilage.

View larger version (112K): [in a new window]   Fig. 8. Expression of Hoxa2 and Hoxa3 after grafting of cardiac dorsal neural tubes to the midbrain region. At HH12 (12 hours after grafting), Hoxa2 (A-C) and Hoxa3 (E-G) are expressed in the grafted dorsal neural tube and migrating neural crest cells. At HH18 (24 hours after grafting), both Hoxa2 (D) and Hoxa3 (H) are downregulated in the grafted tissue and neural crest. Arrows indicate migrating neural crest cells also immunostained with HNK-1 (red). Arrowheads indicate the midbrain region where cardiac dorsal neural tubes were grafted.