Role of FIR in limb morphogenesis

发育中的肢体是一个研究得很好的形态发生模型，其中许多调节骨骼模式和生长的成分已经被阐明，为基于突变表型解释基因功能提供了强大的生物学框架。FIR是一种进化上高度保守的myc转录负调控因子，存在于脊椎动物和果蝇中。果蝇的同系物(半品脱)控制着发育中的胚胎翼盘中的非增殖区。人类同源基因与结直肠癌有关；另一种剪接的显性干扰形式的FIR与这些癌症有关。FIR基因在哺乳动物组织中的正常功能一直受到FIR缺失突变胚胎早期胚胎致死(植入前)的阻碍，限制了该基因在正常组织形态发生中的作用。在与Levens博士(CCR，NCI)的合作下，我们正在使用几个CRE驱动程序来选择性地将FIR从早期的肢芽中移除。我们想测试FIR是否在调节肢体生长方面发挥作用，如果是的话，了解它是如何调节生长的。在初步结果中，我们发现，令人惊讶的是，早期肢芽中胚层FIR功能的丧失会导致大量的细胞凋亡，导致肢体骨骼的截断。在果蝇中，半品脱是Wg(Wingless，Wnt)途径的靶标。有趣的是，肢芽中胚层中Wnt信号效应器β-catenin的缺失也会导致小鼠胚胎中的肢体截断表型，这与肢芽中早期成纤维细胞生长因子信号中心(AER，顶端外胚层脊)的形成异常有关，而AER是肢体生长所必需的。我们正在分析Wnt通路的组成部分，以确定它们在FIR突变肢体中是否发生了变化，同时还分析了对早期肢体细胞生存至关重要的因素和信号中心。令人惊讶的是，我们发现FIR对细胞生存的要求似乎是上下文相关的，而不是普遍的，并且在一些Sonic Hedgehog信号在肢体中活跃的细胞中是可有可无的。我们还与盖辛格诊所的安妮·穆恩博士合作，她发现CAPER是一种调节肢体细胞分裂和衰老的因子，并发现了CAPER-FIR蛋白质-蛋白质相互作用。我们正在研究这些相互作用是如何以一种选择性的方式调节细胞存活的，以肢体为模型，以了解刺猬信号如何改变对FIR功能的要求。这些研究将为FIR在调节生长中的不同作用提供新的线索，并将对开发针对这种Myc调节因子功能的癌症治疗方法产生影响。

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.