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First published online 14 June 2006
doi: 10.1242/dev.02426

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Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles

Gudrun von Scheven, Lúcia E. Alvares^*, Roy C. Mootoosamy and Susanne Dietrich

King's College London, Department of Craniofacial Development, Floor 27 Guy's Tower, Guy's Hospital, London Bridge, London SE1 9RT, UK.

View larger version (59K): [in a new window]   Fig. 1. Head mesoderm develops into muscle according to its local environment. (A) Scheme of operations at HH8-9-; dorsal view of embryos, anterior to the top. Quail-derived head mesoderm from rhombomere 2 levels was grafted orthotopically as control (B), anteriorly next to the anterior midbrain (C) or posteriorly next to otic levels/rhombomere 5-6 (D). In E,G, head mesoderm from midbrain levels was grafted next to rhombomere 2; in F,H, head mesoderm from otic levels/rhombomere 5-6 was grafted next to rhombomere 2. (B-H) Lateral view of the heads of operated embryos at HH19; anterior to the top, dorsal to the left, ventral (branchial arches) to the right. Quail tissues were detected with the QCPN antibody (brown), the lateral rectus EOM with Lbx1 (B-F; blue; note: additional expression in the neural tube), and muscle anlagen in general with Myf5 (G,H, blue). Note that the molecular markers showed normal expression patterns. Scale bar: 200 µm. do, dorsal oblique EOM; dr, dorsal rectus EOM; hy, hyoid arch; lr, lateral rectus EOM; m, midbrain; ma, mandibular arch; nt, neural tube; ov, otic vesicle; r, rhombomere; som, somite; vr, ventral rectus EOM.

View larger version (85K): [in a new window]   Fig. 2. Neural crest cells are dispensable for early head muscle differentiation and specification. (A) Scheme of dorsal neural tube/neural fold ablation at HH8 to remove neural crest cells. Dorsal view, anterior to the top, ablated area in turquoise. (B,F,J,N,R) Heads of unoperated embryos; (C,G,K,O,S) higher magnification of the lateral rectus/MAM region corresponding to boxed area in B,F,J,N,R. (D,H,L,P,T) Operated embryos (E,I,M,Q,U) higher magnification of lateral rectus/MAM region as indicated in D,H,L,P,T. Embryos are at HH19; lateral views, anterior is to the top, dorsal to the left. Embryos are double stained with the RM0270 antibody to trace the nervous system (brown). (B-E) Dlx5 expression. (B,C) In wild-type embryos, Dlx5 is predominantly expressed in the neural crest cells filling the branchial arches. (D,E) Operated embryo. While Dlx5 expression in the derivatives of the nasal and otic placodes and expression in the maxillary and mandibular ectoderm remained, the neural-crest-associated expression is absent (open arrowheads). Moreover, the ophthalmic and the mandibulomaxillary aspect of the trigeminal ganglion are separate. This indicates that the surgical procedure was successful. (F-I) Paraxis expression. Both in the wild type (F,G) and in the operated embryo (H,I), Paraxis labels the developing lateral rectus EOM. (J-M) En2 expression. En2 is expressed in the mandibular arch muscles of both the unoperated (J,K) and the operated (L,M) embryo. (N-Q) Myf5 expression. In the wild type (N,O), muscle anlagen for the mandibular and hyoid arch muscles and the lateral rectus, dorsal oblique, dorsal rectus and ventral retcus EOM are differentiating and express Myf5. This is also the case for neural crest ablated embryos (P,Q). (R-U) MyoR expression. MyoR expression associated with the ventral/branchiomeric muscle anlagen is present in the wild type (R,S) and operated (T,U) embryo. Note that in the absence of neural crest cells, the MyoR expression domains remained proximally unsegregated (small arrows). Scale bars: 400 µm in B,F,J,N,R; 200 µm in C,G,K,O,S; 400 µm in D,H,L,P,T; 200 µm in E,I,M,Q,U. d, diencephalon; do, dorsal oblique EOM; dr, dorsal rectus EOM; hy, hyoid arch; ma, mandibular arch; mmx, mandibulomaxillary aspect of the trigeminal; oph, ophthalmic aspect of the trigeminal (Vth) nerve; ov, otic vesicle; r, rhombomere; VII, facial nerve; vr, ventral rectus EOM.

View larger version (74K): [in a new window]   Fig. 3. The presence of the eye is not required for eye muscle differentiation and specification. (A) Scheme of eye ablation at HH10; dorsal view, anterior to the top. The optic placode and the region of the forebrain delivering the optic cup were removed. Pink: unsegmented head mesoderm. (B-E) Lateral views of HH19 heads; anterior to the top, dorsal to the right for unoperated sides (B,D), dorsal to the left for operated sides (C,E). (B-E) Brown staining: Pax6 antibody staining detecting strong expression in the telencephalon (t), eye, neural tube (nt)/hindbrain. Note, in operated embryos the eye is missing; only the rest of the optic stalk remained (C,E, open arrowhead). (B,C) Lbx1 (blue staining) revealing normal lateral rectus anlagen. (D,E) Also Myf5 expression (blue) is wild type. Scale bar: 200 µm. do, dorsal oblique EOM; dr, dorsal rectus EOM; hm, unsegmented head mesoderm; hy, hyoid arch; lr, lateral rectus EOM; ma, mandibular arch; nt, neural tube; ov, otic vesicle; t, telencephalon; vr, ventral rectus EOM.

View larger version (98K): [in a new window]   Fig. 4. Innervation does not control eye muscle differentiation and specification. Internal views of HH20 bisected chick heads; anterior to the top, dorsal to the left in A and right in B. Embryo stained for Paraxis expression (blue); nervous system traced with RMO270 in brown. (A) Unoperated control side. Note the abducens nerve (cranial nerve VI) with nerve rootlets in rhombomeres 5 and 6 (arrowheads) and axons having entered the Paraxis domain (arrow). (B) Rhombomeres 5/6 ablated side. The abducens nerve is missing (open arrowhead), but Paraxis is expressed normally. Scale bar: 200 µm. r, rhombomere; VI, abducens nerve.

View larger version (69K): [in a new window]   Fig. 5. The neural tube specifies the lateral rectus eye muscle. (A) Schematic dorsal view and (Ai) cross section (dorsal to the top) illustrating the surgical procedures at HH10; head mesoderm in pink, the position of the impermeable barrier (tantalum foil) is indicated. (B,E,H,K,N) Lateral views of unoperated sides; anterior to the top, dorsal to the right. (C,F,I,L,O) Lateral views of operated sides; anterior to the top, dorsal to the left. (D,G,J,M,P) Cross sections at anterior hindbrain/mandibular arch levels; dorsal to the top, operated sides to the right, markers and the position of the barriers are indicated. Note that upon separation of the neural tube from the head mesoderm, the lateral rectus markers Lbx1 (B-D) and Paraxis (E-G) failed to be expressed. Myf5 (H-J) and MyoR (K-M) expression continued. En2 expression (N-P) spread form the mandibular arch muscle anlage medially, encompassing the muscle normally expressing the lateral rectus markers. Thus, muscle specification, but not differentiation, was perturbed. Scale bar: 200 µm. d, diencephalon; da, dorsal aorta; ect, surface ectoderm; fge, foregut endoderm; fp, floor plate of the neural tube; ht, heart; hy, hyoid arch; m, midbrain; ma, mandibular arch; ncc, neural crest cells; not, notochord; nt, neural tube; ov, otic vesicle; r, rhombomere; V, trigeminal ganglion; VII, facial nerve.

View larger version (135K): [in a new window]   Fig. 6. The signal from the neural tube is a soluble factor. Internal views of Paraxis-RMO270 stained, bisected embryo at HH20. (A,D) Unoperated control sides, (B,C,E,F) operated sides before (B,E) and after (C,F) removal of the barrier or filter. Anterior is to the top, dorsal to the left in A,D; to the right in B,C,E,F. Position of the barrier or filter is indicated. (A-C) Separation of neural tube and head mesoderm with tantalum foil, an impermeable barrier. Note that on the control side the abducens nerve is well fasciculated and innervated the Paraxis-expressing lateral rectus anlage (A). On the operated side (B,C), the nerve is defasciculated and did not reach the area that normally would express Paraxis next to rhombomere 2. (D,E) Separation of neural tube and head mesoderm with a polycarbonate filter that admits soluble factors. Note that Paraxis is expressed on the operated side. Moreover, the abducens nerve navigated around the filter and innervated the Paraxis-expressing lateral rectus. Scale bar: 200 µm. r, rhombomere; VI, abducens nerve.

View larger version (70K): [in a new window]   Fig. 7. The signal from the neural tube is not sufficient to trigger lateral rectus development. (A) Scheme of quail-chick neural tube grafting at HH10; dorsal view, anterior to the top. (B-F) Lateral views of operated embryos at HH19, stained for the lateral rectus marker Lbx1 in blue and the quail cell marker QCPN in brown; anterior to the top, dorsal to the left. The anteroposterior orientation of the grafts is indicated. (B) Orthotopic control grafting of rhombomere2; Lbx1 is expressed at the normal location (green arrow). (C) When the neural tube from posterior midbrain to rhombomere 2 levels was grafted in place of midbrain and posterior diencephalon, the ectopic rhombomere 2 did not trigger ectopic Lbx1 expression (open arrowhead). (D) Similarly, no ectopic Lbx1 expression occurred when the graft was transplanted in place of rhombomeres 4-6 (open arrowhead). (E) When the posterior midbrain to rhombomere 2 was replaced with neural tissues from anterior midbrain to diencephalic levels, Lbx1 was expressed at the normal location dorsal to the mandibular arch (green arrow). (F) Grafting of the posterior hindbrain, including parts of the otic placode to midbrain-rhombomere 2 levels, led to the development of an ectopic otic vesicle, and also permitted Lbx1 expression (green arrow). Scale bar: 200 µm. d, diencephalon; hy, hyoid arch; lr, lateral rectus EOM; m, midbrain; ma, mandibular arch; ov, otic vesicle; r, rhombomere.

View larger version (75K): [in a new window]   Fig. 8. The signal from the neural tube is not rhombomere 2-specific. (A) Scheme of neural tube electroporation at HH10. (B-G) Lateral views of electroporated embryos at HH19; anterior to the top, dorsal to the left. (B,E) The targeted area revealed by means of eGFP fluorescence. (C,D,F,G) Paraxis (C,F) or Myf5 (D,G) expression in blue and staining with the anti-GFP antibody in brown. Same embryo shown in B,C and E,F, respectively. (B-D) Control electroporation with the pCAß-IRES-eGFP vector lacking the open reading frame for Hoxb1 allows normal expression of Paraxis and Myf5. (E-G) Misexpression of Hoxb1 in the anterior hindbrain transforms the identity of rhombomeres into that of rhombomere 4 (r1*, r2*). However, Paraxis and Myf5 expression are not perturbed. Scale bar: 250 µm. d, diencephalon; hy, hyoid arch; m, midbrain; ma, mandibular arch; ov, otic vesicle; r, rhombomere; t, telencephalon.

View larger version (115K): [in a new window]   Fig. 9. Differential effect of the mandibular-arch-derived signals Fgf8 and Bmp7. (A) Scheme of bead implantation at HH10, orientation as in Fig. 3A. (Ai,ii) Fgf8 expression at HH10 (Ai, dorsal view, anterior to the top) and HH19 (Aii, lateral view of right side, anterior to the top). Note persistent expression of Fgf8 in the pharyngeal endoderm and at HH19, upregulated expression in the oral ectoderm and in the endoderm plus overlying ectoderm of each pharyngeal pouch (Aii, arrows point towards expression associated with the mandibular arch). (Aii,iv) Bmp7 expression at HH10 (Ai, ventral view, anterior to the top) and HH19 (Aii, lateral view of right side, anterior to the top). Note the persistent expression of Bmp7 in the HH10 endoderm and heart; at HH19 expression is upregulated in the pharyngeal pouches, the oral ectoderm (arrows), and the in- and outflow tract of the heart. Fgf8 and Bmp7 are absent from the area of the lateral rectus EOM underneath the trigeminal ganglion. (B-E) Lateral views of embryos treated with BSA-loaded control beads, (F-O) Fgf8-loaded beads and (P-W) Bmp7-loaded beads; untreated sides of the embryos on the left, treated sides on the right, orientation as in Fig. 3B-E. The embryos were double labelled as indicated. Normal expression domains are marked by arrows, downregulated expression by open arrowheads, upregulated expression by filled arrowheads. The position of the beads is marked by asterisks. Note that Fgf8 beads placed into the head mesoderm did not influence positional values of the hindbrain or the neural crest cells, as evidenced by the unperturbed expression of Hoxa2 (F,G). However, Fgf8 downregulated the lateral rectus marker (H,I) and upregulated the mandibular arch muscle marker (L,M). Unlike neural tube-mesoderm separation experiments, however, Myf5 expression was also downregulated (J,K), while MyoR was upregulated (N,O; compare with Fig. 5). Bmp7 dowregulated Paraxis in the region of the lateral rectus EOM (P,Q) and Myf5 in the region of the lateral rectus and the mandibular arch muscles (R,S). En2 expression was downregulated in the distal part of the mandibular arch but not proximally close to the bead (T,U). MyoR was also downregulated, most prominently in the distal region of the arch (V,W). Scale bars: 250 µm in Ai,Aiii; 100 µm in Aii,Aiv; 200 µm in B-U. di, diencephalon; endo, endoderm; fn, frontonasal Fgf8 signal in ventral forebrain and surface ectoderm; hy, hyoid arch; i, isthmus; m, midbrain; ma, mandibular arch; ov, otic vesicle; pchp, prechordal plate; r, rhombomere; V, trigeminal ganglion; VII, facial nerve.

View larger version (28K): [in a new window]   Fig. 10. Model for the specification of the lateral rectus eye versus mandibular arch muscles. (A) Schematic representation of a chick head at the onset of EOM and MAM-specific marker gene expression; the non-somitic head mesoderm is depicted in grey. (B) Factors from the neural tube (green) and Fgf8 from the branchial arches (blue) signal to the neighbouring head mesoderm. The signal from the neural tube is accompanied by a further, unidentified signal. As a result of the signalling events (C), the lateral rectus EOM anlage (yellow) is specified next to the neural tube; MAM markers are repressed in this area. MAM (magenta) are specified next to the mandibular Fgf8 signals that suppress EOM markers. lr EOM, lateral rectus extraocular muscle, MAM, mandibular arch muscles.