Role of FIR in limb morphogenesis

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The developing limb is a well-studied model for morphogenesis in which many of the regulatory components governing skeletal pattern and growth have been elucidated, providing a strong biological framework for interpreting gene function based on mutant phenotypes. FIR (FBP interacting repressor) is a highly evolutionarily conserved negative regulator of myc transcription present in both vertebrates and Drosophila. The Drosophila homolog (half pint) controls the zone of non-proliferation in the developing embryonic wing disc. The human homolog has been implicated in colorectal cancer; alternatively spliced dominant-interfering forms of FIR are associated with these cancers. Elucidation of the normal function of FIR in mammalian tissues has been hampered by the very early embryonic lethal (prior to implantation) of FIR null mutant embryos, limiting the ability to assess the role of this gene in normal tissue morphogenesis. In collaboration with Dr. Levens (CCR, NCI), who has generated a conditional (floxed) allele of mouse FIR, we are using several Cre drivers to selectively remove FIR from the early limb bud. We want to test whether FIR plays a role in regulating limb outgrowth, and if it does, learn how it acts to regulate growth. In preliminary results, we have found that, surprisingly, loss of FIR function in early limb bud mesoderm results in considerable apoptosis leading to truncations of the limb skeleton. In Drosophila, half pint is a target of Wg (wingless, Wnt) pathway. Interestingly, loss of the Wnt signaling effector, beta-catenin, from limb bud mesoderm also causes limb truncation phenotypes in mouse embryos, related to abnormalities in formation of an early Fgf signaling center in the limb bud (AER, apical ectodermal ridge) that is necessary for limb outgrowth. We are analyzing components of the Wnt pathway to determine whether they are altered in the FIR mutant limbs, along with other analyses of factors and signaling centers critical for cell survival in the early limb. Surprisingly, we have found that the requirement of FIR for cell survival appears to be context dependent, rather than universal, and is dispensable in some cells where Sonic hedgehog signaling is active in the limb. We are also collaborating with Dr. Anne Moon (Geisinger Clinics) who has identified CAPER as a factor regulating cell division and senescence in the limb and has discovered a CAPER-FIR protein-protein interaction. We are studying how these interactions regulate cell survival in a selective fashion, using the limb as a model, to understand how hedgehog signaling alters requirements for FIR function. These studies will shed new light on the varied roles of FIR in regulating growth, and will have implications for developing cancer therapies that may target the function of this Myc-regulator.