SummarySonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).

Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.

Idiopathic Congenital Talipes Equinovarus, or clubfoot, is a common developmental disorder of the foot, affecting at least 2 in every 1000 live births in Scotland. The defect is characterised by a twisting of the foot and loss of calf muscle. Diagnosis is usually made on postnatal examination and treatment; usually a series of foot manipulations, takes place within the first year. Treatment can be particularly painful and is not always successful as the defect can recur leading to life-long disability in some cases. Very little is known of the aetiology of clubfoot despite it being such a common problem and therefore our lab set out to elucidate the developmental processes that occur during limb development which result in clubfoot. The chick limb is a well established model system used to study many developmental processes, signalling events and has been used to model several human limb disorders. We have developed a chick model of human clubfoot using a neuromuscular paralysing agent, resulting in a range of clubfoot conditions. Our lab is currently using this chick model to determine the developmental basis of the disorder. Initial phenotypic analyses of the chick clubfoot limb correlate well with those observed in human clubfoot; both exhibit muscle loss, tendon problems and bony abnormalities. We will use this chick model of human clubfoot to test our hypothesis that clubfoot may result from a failure of hindlimb rotation and elucidate the developmental and molecular basis of this congenital defect. The congenital abnormality preaxial polydactyly (PPD) is caused by the ectopic expression of the signalling protein Sonic Hedgehog (SHH) in the developing limb. Targets of FGF signalling, Pea3/Erm, have been shown to play a vital role in restricting Shh to the posterior limb bud. The ZRS, a cis-regulator of Shh expression, has several conserved sub-domains containing binding motifs recognized by PEA3. Single nucleotide mutations in the ZRS allow the enhancer to induce Shh in the anterior as well as posterior limb bud. Point mutations recently identified in two unrelated human families with PPD generate additional PEA3 binding motifs. We report a direct interaction of PEA3 with the ZRS and demonstrate that the addition of a PEA3 binding site is sufficient to generate a loss of posterior restriction and ectopic expression of Shh in the anterior mesenchyme. These data reveal a direct interplay between SHH and FGF signalling. DiGeorge syndrome is the most common microdeletion syndrome in humans frequently caused by a 3 Mb deletion of chromosome 22. The phenotype is characterized by craniofacial, cardiovascular, thymus, parathyroid and psychiatric defects.TBX1 localises within the commonly deleted region on 22q11 and is considered the major genetic determinant of the syndrome. Deletion of Tbx1 in animal models results in pharyngeal and cardiovascular defects, highlighting its role in the development of these structures. We have determined potential Tbx1 target genes through microarray screens, including Hes1, an effec...

intracellular signalling pathways in neuron-like cells and for their ability to stimulate neurite outgrowth of neuron-like cells.Milk proteins that have shown effects on neural-like cell lines are being tested in vitro using a whole gut culture system to examine their effects on the neonatal ENS development.Activin A/TGFb, FGF, Wnt and BMP signalling are crucial for the self-renewal and early differentiation of human embryonic stem cells (hESCs). However, the mechanism of how they coordinate to regulate these two processes is unclear. To address this ques-

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