Although the molecular networks that operate in the limb bud have received much attention (see Chapter 1), very little is known about the molecular basis of limb bud initiation. A recent clue has emerged from work on chimeric mice combining wild type blastocysts or morulae with pluripotent embryonic stem (ES) cells which constitutively express FGF4. Overexpression of FGF4 leads to dramatic malformations in chimeric embryos, including reduction or deletion of the diencephalon and eyes, and development of multiple small limb bud structures from the flank (Abud et al.,
1996) The latter result suggested the possibility that FGFs may be involved in the initiation of limb budding. This chapter reports the results of experiments carried out in chick embryos to examine whether fibroblast growth factors are involved in initiation of limb development. The major finding is that FGF induces development of additional limbs. This discovery gave rise to a number of related questions that are presented in this chapter. Early development of additional limbs buds was investigated to determine how a limb bud is established from flank cells and how signaling regions arise in the limb. Grafting experiments were performed to investigate the competence of flank cells to express Shh. In light of the ability of FGF to activate Shh in flank cells, the effects of direct application of SHH to the flank were assayed. Finally, FGF and SHH were applied simultaneously to determine whether this affected the anteroposterior polarity of additional limbs.
2. Results
2.1 FGF beads induce additional limbs in chick embryos
Beads soaked in FGF (-1, -2 or -4) and implanted in presumptive flank lead to the development of additional limbs (Figure 4A-F). A major set of experiments was carried-out with FGF2, in which FGF2 beads were placed at different levels along the primary body axis in the lateral plate mesoderm of chick embryos between stages 13 and 17. Stage 13 is well before there is any sign of limb development, and at stage 17 a slight thickening in the lateral plate mesoderm marks the place where buds will form. When beads are placed in lateral plate mesoderm opposite somites 20 to 26 (presumptive flank lies between somites 21 and 25), additional limbs developed in 35 out of 42 embryos (Table 1). Both complete wings (Fig. 4a) and complete legs (Fig. 4c) could develop from the flank (summarized in Table 1 and Fig. 4). The rib cage often appeared to be pushed together distally on the treated side, but vertebral identity was unchanged (Fig. 4c). When beads soaked in FGF2 were placed anterior to somite 15 in the neck region (n = 4), or in the tail bud (n = 3), no additional limbs were produced.
Application of FGF2 to the flank prior to stage 13 did not result in additional limb formation. Embryos treated between stages 10 to 12 (n = 6 cases) did not develop additional limbs, but the wing and leg were sometimes shifted along the body axis and appeared to be drawn together. Loss or fusion of limb elements, cleft neural arches, and rib fusions were also observed in these embryos. Ability of the flank to produce complete additional limbs lasts at least through stage 17, when wing and leg buds are present, but additional limbs could not be induced between stages 19 and 22 (n=7).
also induced additional limbs, but the limbs were less complete than those induced by FGF2, in that they rarely developed digits. Beads soaked in FGF7 (also known as Keratinocyte Growth Factor or KGF) and applied to the flank of stage 14-16 embryos did not lead to the development of additional limbs (n = 10). Nine of 10 embryos receiving FGF7 beads developed normally: however, a single embryo developed with a duplicated pattern in the leg (duplicated fibula and digit pattern || I I II III IV; =
unidentifiable digit).
Beads soaked in PBS and implanted at flank levels had no detectable effects on embryo development (n = 4).
2.2 Identity and morphological pattern of additional limbs
The nature of the limb that developed was related to the position at which the FGF bead was placed along the body axis (Table 1). When FGF beads were placed in the anterior part of the flank (opposite somites 21 and 22), 8 of the 9 limbs that developed were additional wings. FGF beads implanted to the mid-flank (opposite somite 23; n = 9) resulted in the development of either wings (5 cases) or legs (3 cases). The morphology of 2 remaining limbs was not unequivocally characteristic of either wings or leg and was therefore classified as "limb", and one embryo developed two ectopic limbs; an ectopic leg and an ectopic limb. FGF beads placed slightly more posteriorly (opposite somite 24) also resulted in the formation of either wings (3 cases) or legs (3 cases). However, FGF beads placed still more posteriorly in the flank (opposite somite 25) induced only legs (5 cases).
A few implants were made at levels where the wing and leg would normally form. Two of these resulted in additional limbs which articulated near the normal leg and appeared to contain proximal leg elements and
wing digits (Fig. 4b and e). Unexpectedly, one of the FGF beads placed at wing level induced an extra leg.
Only one of the additional limbs with digits had a normal antero posterior polarity and four limbs developed with unidentifiable digits. In all other cases, the a-p axis was clearly reversed (18 of 23 cases; Fig. 4a-c, e and f). Fig. 4a shows a good example; the additional wing has a sequence of digits 432, reading from anterior to posterior, which is reversed compared to the sequence of normal wing digits 234. In Fig. 4c, the additional leg has a reversed sequence of toes IV III II I, compared to the normal leg pattern I II III IV
Many of the limbs which developed wing digits following implantation of FGF beads in anterior or mid-flank (somites 20-23) also had an extra digit
3 inserted into the pattern (6 of 11 cases). This gave, for example, patterns