The long-term goal of this research is to understand how the zone of polarizing activity (ZPA) functions in space and developmental time to control anterior/posterior (A/P) limb patterning. The chick and mouse developing limbs provide a system where a combination of experimental manipulations can be combined with molecular and genetic approaches to understand how patterned structures are specified, determined and realized. During the past decade, the effector molecules of limb bud signaling centers were defined. However, how the signaling centers are established and the precise targets of the effector molecules are poorly understood. This is especially true for the ZPA, acting through the secreted protein Sonic hedgehog (SHH). There are three aims that cover ZPA function from its initiation, through signaling to target cells, to the function of the interdigits as signaling centers downstream of SHH. Using experimental and molecular techniques, we will: determine the minimal sequence(s) of the chick and mouse conserved Lmbr1 intron 5 region that are necessary for Shh expression in the ZPA; use the oligozeugodactyly mutant limb as a model system to analyze the effects of SHH signaling on target gene expressions and development of limb phenotypes; and test the role of the interdigits as signaling centers functioning through Tbx genes and BMP signaling. To accomplish these aims, we have developed and validated new techniques that include a reporter assay for in vivo enhancer analysis in the chick limb; the use of ultrasound (sonoporation) to express genes ectopically in limited areas of the developing limb; and an in vivo Luciferase reporter assay that permits quantitation of BMP signaling. While the members of my laboratory are highly accomplished in the techniques and experimental designs needed to carry out these aims, we also have engaged experts, with whom we meet regularly, to assist in trouble shooting and data interpretation. Accomplishment of the aims of this proposal has significant implications for understanding human limb malformations. These malformations are among the highest among human live births. Understanding the fundamental mechanisms of normal limb development will lead to insights into why limb malformations occur. This is a necessary step toward preventing and/or curing them through regenerative biological and medical approaches.
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