Genetic evidence that FGFs have an instructive role in limb proximal–distal patterning

Abstract

Half a century ago the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal-distal (P-D) axis, but how these signals influence limb patterning is still much debated 1,2 . FGF gene family members are key 4 , with Fgf4, Fgf8, Fgf9, and Fgf17 expressed specifically in the mouse AER 5 . Here, we demonstrate that limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype 6,7 , but when we also removed different combinations of the other AER-FGF genes we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution each FGF can make to total AER-FGF signal. Analysis of the compound mutant limb buds revealed that in addition to sustaining cell survival, AERFGFs regulate P-D patterning gene expression during early limb bud development, thus providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signaling is permissive rather than instructive for limb patterning. We discuss how a twosignal model for P-D patterning can be integrated with the concept of early specification to explain the genetic data presented here. KeywordsAER; apical ectodermal ridge; cell survival; FGF4; FGF8; FGF9; FGF17; limb development; proximal-distal patterning Fgf8 is expressed in prospective AER cells of the nascent limb bud and subsequently throughout the AER until it regresses 8 , whereas Fgf4, Fgf9, and Fgf17 expression commences after the AER is formed, is restricted to the posterior AER, and ceases at least a day before AER regression 5 (Fig. 1a). When AER-FGFs are individually eliminated, only loss of Fgf8 function perturbs skeletal patterning 5-7,9-11 . The other AER-FGFs have been proposed to be essential, but functionally redundant components of a positive-feedback loop between the AER and the patterning center in posterior limb bud mesenchyme that produces Sonic hedgehog (SHH) 5,12,13 . We tested this hypothesis by deleting Fgf4 via Cre-mediated recombination in the AER of embryos homozygous for Fgf9 and Fgf17 null alleles 10,11 (hereafter referred to as F4;9,17-triple knock-out [TKO] mutants; Fig. 1b). Because Fgf4 deletion occurs before Fgf4 expression normally commences 5 (see Fig. 1a), the F4;9,17-TKO limb buds do not produce FGF4, or FGF9 or FGF17. Nevertheless, in F4;9,, the three classically defined limb segments, stylopod (S, upper arm or leg), zeugopod (Z, lower arm or leg), and autopod (A, wrist/hand or ankle/foot), were essentially normally patterned (Fig. 1c) Consistent with this observation, Shh expression appeared normal in F4;9,17-TKO limb buds at embryonic day (E)10.5 by in situ hybridization (not shown) and qRT-PCR (Fig. 1d). Moreover, there was no compensatory upregulation of Fgf8 in F4;9,17-TKO limb buds at E10.5 (Fig. 1d). These data demonstrate that Fgf8 is su...

Vertebrate Limb Development: Moving from Classical Morphogen Gradients to an Integrated 4-Dimensional Patterning System

“…A recent genetic study shows that the other AER-FGFs, in particular FGF9, contribute to this proliferative expansion of the early specified PD axis such that higher AER-FGF levels are required for formation of more distal limb skeletal structures (Fig. 2B,C) (Mariani et al 2008). Taken together, this genetic analysis reveals an instructive role of AER-FGF signaling in the specification and proliferative expansion of the PD limb-bud axis.…”

“…Four Fgf ligands (Fgf4,-8,-9,-17) are expressed by the AER (for review, see Martin 1998) and extensive genetic analysis has provided insight into their overlapping, respectively redundant functions during outgrowth and PD patterning of mouse limb buds. Rather unexpectedly, concurrent inactivation of all three Fgfs expressed predominantly by the posterior AER (Fgf4,-9,-17) does not alter limb-bud development (Mariani et al 2008). …”

Fibroblast growth factor signaling in skeletal development and disease

“…The initiation of the mesenchymal condensation and its differentiation to the chondrogenic lineage are at least partially dependent on FGFR signaling (Murakami et al 2000;Mariani et al 2008;Yu and Ornitz 2008;Kumar and Lassar 2014). In primary chondrocytes and undifferentiated mesenchymal cell lines, FGF signaling increases Sox9 expression (Murakami et al 2000;Shung et al 2012).…”

Wnt/planar cell polarity signaling

“…However, it is clear that in addition to polarized growth, signals emanating from the AER and the flank are critical for determining appropriate fates of cells along the proximal to distal axis. [5][6][7][8]59 Yet, it is likely that this two-signal mechanism is also dependent on distally oriented growth in order to separate the proximal and distal signaling centers, so that the different fates can be assigned along the PD axis. Hence, directed growth and patterning are likely inseparable processes in normal limb development.…”

“…4 Within the past decade, several groups have explored a twosignal hypothesis for proximodistally patterning the limb. [4][5][6][7][8] The ©2 0 1 1 L a n d e s B i o s c i e n c e .…”