Coordination of limb bud development : the role of SHH in PD limb bud patterning

The limb bud serves as an excellent model to investigate the signals involved in diverse processes during embryonic development. Limb bud development is controlled by complex regulatory networks that instruct coordinated patterning and proliferation of mesenchymal progenitors along the dorso-ventral (DV), antero-posterior (AP) and proximo-distal (PD) limb axes. Over the last two decades many of the underlying molecular mechanisms instructing limb development have been described. Nevertheless, little is known about how these signals are integrated into the networks controlling limb bud development and how the cells respond to these signals to acquire their identity. Sonic hedgehog (SHH) is known to specify the AP limb bud axis and regulate its expansion as part of a larger self-regulatory signalling system. To analyze the genome-wide effects and to identify novel targets of SHH signalling in the limb bud, we have performed microarray analysis on Shh-deficient limb buds. From this analysis we have identified a novel kinase (Pkdcc), whose expression is up-regulated in Shh-deficient limb buds and that is dynamically expressed during development. Targeted deletion of the Pkdcc gene in mice shows that Pkdcc is essential for embryonic development. Furthermore, the transcriptome analysis uncovers an unexpected function of SHH in PD limb axis development as Shh-deficient mouse limb buds are proximalized. Expression of proximal genes and retinoic acid (RA) pathway activity are up-regulated and distally expanded in Shh-deficient limb buds. In parallel, the expression of the RA inactivating enzyme Cyp26b1 is decreased in the distal mesenchyme. We have investigated the possible SHH-RA interactions using a combination of experimental manipulation, genetics and mathematical simulations. Our findings reveal a SHH-dependent signalling module that normally enhances RA clearance by increasing fibroblast growth factor (FGF) signalling in the apical ectodermal ridge (AER), which in turn up-regulates Cyp26b1 expression in the distal mesenchyme. Disruption or reduction of CYP26b1-mediated RA clearance interferes with distal limb bud development leading to molecular proximalization of Shh-, AER-Fgf- and Cyp26b1-deficient limb buds. In addition, we provide molecular evidence for early specification of the PD axis by a mutually inhibitory interaction of RA with AER-FGFs. Subsequently AP and PD limb bud patterning becomes interlinked via SHH mediated regulation of the AER-FGF/CYP26b1/RA signalling module, which enables spatially coordinated progression of limb bud development.