During the development of multicellular organisms, pattern formation must be precisely coordinated with proliferation and differentiation. Given that only a relatively small number of signaling pathways are used to direct both pattern formation and cell proliferation during development, it is clear that cell fate specification and cell division are highly context-dependent read-outs of signaling in a given tissue or organ. Activation of a particular signaling pathway, such as the Hedgehog pathway, can stimulate proliferation in one cell type, while activation of the same pathway in another cell type has no effect on proliferation. Moreover, the observation that identical signaling pathways can regulate both pattern formation and cell proliferation provides a mechanism for coordination of these distinct behaviours.
The vertebrate limb is an excellent model system in which to study the interplay between pattern formation and cell proliferation. Limb development is highly amenable to experimental and genetic manipulation in several model organisms, and the main signaling pathways that direct limb development are well characterized (reviewed in [1–3]). Three signaling centers are required for pattern formation and growth in the developing limb bud, two of which we chose to study in this work. One of these is the zone of polarizing activity (ZPA), a small group of cells in the posterior mesenchyme, which controls polarity along the anterior/posterior axis . The secreted signaling protein Sonic hedgehog (Shh) is expressed in the ZPA, and has been shown to mediate the effect of the ZPA during limb development [5–8].
The apical ectodermal ridge (AER) is another major signaling center of the limb bud which runs along its distal margin, and which is the site of expression of several Fgf genes (reviewed in ). The AER is required for outgrowth and patterning of the limb along its proximal/distal axis, and can be functionally replaced by FGF-soaked beads in chicken embryos, indicating that Fgf signaling can mediate AER function [10, 11]. Furthermore, conditional inactivation of both Fgf4 and Fgf8 in the mouse AER leads to failure of proximal/distal outgrowth , thus identifying these members of the Fgf family as the main mediators of AER signaling. Factors from the AER and ZPA form a mutual feedback loop, thereby allowing growth and patterning of the different axes to be coordinated. Thus fgf-4, which is expressed in the posterior AER, can be induced in the anterior AER of the chicken limb bud by ectopic Shh protein [13, 14]. Furthermore, removal of Shh activity from the zebrafish fin buds leads to loss of fgf4 and fgf8 expression in the AER , and, conversely, removal of Fgf4 and Fgf8 activity from the mouse AER leads to loss of shh expression in the ZPA , indicating that each signaling pathway is required for the maintenance of the other pathway.
Members of both the Hh and Fgf family of signaling proteins have been shown to function as mitogens in several contexts. Indeed, Fgf1 and Fgf2 were initially identified as mitogenic factors in fibroblast tissue culture, and subsequently, other members of the FGF protein family were found to have a similar activity . Furthermore, Fgf signaling has also been shown to have mitogenic activity in vivo during embryonic development. Thus FGF-4 is necessary for proliferation of the inner cell mass during early post-implantation development in the mouse , and FGF-8 and FGF-17 are required for proliferation in the mouse dorsal midbrain . Additionally, Fgf signaling promotes proliferation of osteoblasts , of lens cells , and during hematopoiesis .
Like the Fgf family, members of the Hh family function as mitogens in a number of contexts. The Hh signaling pathway has been linked to several cancers, including basal cell carcinoma, pancreatic tumors, and digestive tract tumors, and may be upregulated in as many as 25% of tumors [21–25]. In addition to this oncogenic effect, Hedgehog signaling also directs proliferation during normal development, including in the mouse cerebellum , in the Drosophila eye , in mammalian keratinocyctes , and in the mammalian kidney . In several cases Hh signaling has been shown to stimulate cell-cycle progression by causing transcriptional upregulation of D-type and E-type cyclins in target cells [27, 30–32]. This transcriptional up-regulation of cell-cycle genes in some instances has been shown to occur as a direct response to promoter binding of members of the GLI family, the zinc-finger transcription factors which transduce Hh signaling to the nucleus [27, 32, 33].
Since there is clear evidence that both Shh and Fgf signaling are important for outgrowth of the vertebrate limb bud, and since both signaling pathways are known to have a mitogenic effect during development, this raises the question of the relative contribution of Shh and Fgf signaling to regulation of proliferation in the limb bud. This issue is complicated by the feedback loop operating between the two signals, as inhibition of either signaling pathway leads to loss of the other signaling pathway. Laufer and colleagues have previously addressed this issue by removing the AER from chicken wing buds and adding back either FGF4-soaked beads, or shh-expressing virus [13, 14]. Their results show that Shh alone is insufficient to induce mesodermal proliferation, whereas FGF4 alone is sufficient to do so, leading to them to conclude that the effect of Shh on mesodermal proliferation is indirect, and due to the induction of Fgfs in the AER. However, a recently published study  shows that Shh is sufficient to induce cyclin D1 expression in the mesoderm of chicken wing buds after AER removal. This observation raises a third possibility: that Shh and Fgf signaling both contribute to the regulation of limb bud proliferation.
To distinguish between these possibilities, we have made use of the genetic, embryological, and pharmacological tools of the zebrafish model system to uncouple the activity of Shh and Fgf signaling in the pectoral fin buds, and to investigate their individual effects on proliferation. In order to categorically uncouple the effect of Fgf signaling from Shh, we implanted FGF4-soaked beads into the limb buds of shh mutants. Our data confirm that Fgf signaling is of direct and crucial importance for growth and cell-cycle progression in the limb bud, whereas the effect of Shh on proliferation is indirect, and is mediated via its effect on Fgf expression in the AER.