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First published online November 28, 2005
doi: 10.1242/10.1242/dev.02164

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The dissociation of the Fgf-feedback loop controls the limbless state of the neck

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

View larger version (69K): [in a new window]   Fig. 1. A graft of forelimb lateral mesoderm plus its covering ectoderm in the neck develops into an ectopic limb bud. A scheme of operation is shown on top of the figure. (A-L) Dorsal views (A-I, K-L) or lateral view (J) of the ectopic limb buds observed in the neck. (A'-L') Dorsal views onto the right forelimb buds of the embryos with the ectopic limb buds shown in A-L. Expression of Fgf10 (A,A'), Wnt3a (B,B'), Fgf8 (C,C'), Fgf4 (D,D'), Shh (E,E'), Bmp2 (F,F'), Gremlin (G,G'), Tbx5 (H,H'), Wnt7a (I,I'), Lmx1 (J,J'), En1 (K,K') and Myf5 (L,L') mRNAs are shown in dark blue; arrows. In all the pictures (except J), anterior is to the top and distal is to the right. In J, dorsal is to the top and distal is to the right. Scale bar: 500 µm.

View larger version (77K): [in a new window]   Fig. 2. The ectopic limb buds obtained in the neck are not innervated. Dorsal views of an ectopic limb bud obtained in the neck (A,D) or in the flank (B) and of a hindlimb bud (B) and a forelimb bud (C,D) of an embryo stage HH 25. The axons are stained in brown. (D) The embryo is double stained for Myf5 to reveal the developing muscles. Note that the axons extend into the forelimb and hindlimb buds and into the ectopic limb bud that develops in the flank (arrows in B,C,D) but not in the ectopic limb bud that develops in the neck (arrowheads in A,D). el, ectopic limb bud; fl, forelimb; hl, hindlimb; IX, glossopharyngeal nerve; spn, spinal nerves; V, trigeminal nerve; VII, facial nerve; X, vagal nerve; XII, hypoglossal nerve. Scale bar: 500 µm.

View larger version (55K): [in a new window]   Fig. 3. The neck mesoderm is not recruited to contribute to the bud. A scheme of operation is shown on top of the figure. (A,B) Lateral views of the flank (A) and the neck (B) lateral plate mesoderm of chick embryos at HH20, expressing GFP upon electroporation with the pCaß-IRES-GFP construct at stage HH14 and 12, respectively. A part of the limb lateral plate mesoderm was also electroporated, leading to GFP expression in the anterior part of the hindlimb or the forelimb (arrowheads). (C,D) Lateral views of the ectopic limb buds obtained in the flank (C) or in the neck (D) of stage HH20 embryos after grafting of limb lateral plate mesoderm plus the overlying ectoderm into the areas electroporated with the pCaß-IRES-eGFP plasmid. Fluorescent flank mesodermal cells are present in the ectopic bud (C, arrowheads, enlarged area in window). In the neck, fluorescent cells are found around the base of the ectopic bud (D, arrows) but never inside. As the anterior limb field was co-electroporated, fluorescent cells are also found in the forelimb (D, arrowhead). (E,F) Frontal sections of the buds shown in C,D, respectively. In the flank, fluorescent cells are present in the flank lateral mesoderm and in the mesoderm of the ectopic bud (E, arrowheads). In the neck, fluorescent cells are present in the neck mesoderm around the base of the bud (F, arrows). The co-electroporated limb mesoderm also contains fluorescent cells (F, arrowhead). Significantly, no fluorescent cells contributed to the ectopic bud in the neck. Scale bar: 1000 µm in A-D; 500 µm in E,F.

View larger version (52K): [in a new window]   Fig. 4. Neck ectoderm does not differentiate into an AER. A scheme of operation is shown on top of the figure. (A-H) Dorsal views onto the outgrowths obtained in the neck after grafting of forelimb-derived lateral mesoderm without ectoderm (arrowhead in A-H) and onto the right forelimbs (A'-H') of the operated embryos shown in A-H. (I) Dorsal view of the ectopic bud developing from grafted forelimb mesoderm in the flank, and of the forelimb of the operated embryo. Expression of Fgf10 (A,A',I), Wnt3a (B,B'), Fgf8 (C,C'), Shh (D,D'), Bmp2 (E,E'), Tbx5 (F,F'), Wnt7a (G,G') and Lmx1 (H,H') mRNAs are shown in dark blue. The arrows in A'-H' and I show the wild-type pattern of these genes in limb buds. In all the pictures, anterior is to the top and distal is to the right. Scale bar: 500 µm.

View larger version (52K): [in a new window]   Fig. 5. Neck ectoderm and mesoderm do not respond to Fgf signals. A scheme of operation is shown on top of the figure. Fgf10 beads (A-I) or Fgf8 beads (J-R) were implanted into the flank (A,D,G,J,M,P) or the neck (B,E,H,K,N,Q) of host embryos. Expression of Fgf10 (A-C,J-L), Wnt3a (D-F,M-O) and Fgf8 (G-I,P-R) is shown in dark blue; arrows. Marker gene expression in the forelimb bud of the operated embryos is shown as a control (C,F,I,L,O,R). Note that in the flank, the beads triggered the development of an ectopic limb with normal marker gene expression. In the neck, neither limb budding nor Fgf10, Wnt3a or Fgf8 expression was observed (arrowhead in B,E,H,K,N,Q). Scale bar: 500 µm.

View larger version (113K): [in a new window]   Fig. 6. Fgfr2 expression pattern. Dorsal view (A) and cross-sections of the cervical (B) and presumptive limb (C) region of wild-type chick embryos at HH14. Note that Fgfr2 is expressed in the neural tube, notochord, mesonephros and, most importantly, lateral mesoderm (somatopleure and splanchnopleure) and in the surface ectoderm. ect, surface ectoderm; end, endoderm; ht, heart; mn, mesonephros; not, notochord; nt, neural tube; sompl, somatopleure; sppl, splanchnopleure. Scale bar: 500 µm.

View larger version (80K): [in a new window]   Fig. 7. The MAPK signalling pathway is activated in the neck after implantation of Fgf8 beads but not after implantation of Fgf10 beads. Dorsolateral views (A-F,H) and cross-section (G) of the grafted areas of HH20-21 chick embryos. Fgf10 beads at 1 mg/ml (A,B), Fgf8 beads at 50 (C,D) or 250 µg/ml (E,F,G) and PBS beads (H) were implanted into the flank (A,C,E) or neck (B,D,F,G) of host embryos. Expression of dp-ERK1 and 2 is shown in dark brown. Fgf10 and Fgf8 beads implanted in the flank induce the phosphorylation of ERK1 and ERK 2 (A,C,E, black arrow) and the formation of an ectopic bud. Fgf10 beads implanted in the neck induce neither the phosphorylation of ERK1 and ERK 2 (B, empty arrowhead) nor the formation of a bud. Fgf8 beads implanted in the neck induce the phosphorylation of ERK1 and ERK 2 (D,F,G; black arrowhead) but not the formation of an ectopic bud. Note that dp-ERKs are only present in the mesoderm around the bead (G, black arrowhead). ccv, common cardinal vein; d, dermatome; da, dorsal aorta; drg, dorsal root ganglion; in, intestine; lat mes, lateral mesoderm; m, myotome; not, notochord; nt, neural tube; scl, sclerotome; spn, spinal nerve. Scale bar: 500 µm.

View larger version (29K): [in a new window]   Fig. 8. Interruption of the Fgf feedback loop in the neck. (A) Schematic representation of the Fgf feedback loop that initiates limb budding for a normal limb and for an ectopic limb in the flank; details in the text. (B) In the neck, Fgf signalling is interrupted at distinct points in the neck lateral mesoderm and ectoderm, rendering this region limb-incompetent. Fgfr2b/2c, Fgfr2IIIb/IIIc.

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