Role of FIR in limb morphogenesis

发育中的肢体是一个被广泛研究的形态发生模型，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。在平行研究中，我们正在使用条件突变来源的MEF细胞在细胞水平上分析FIR在细胞生存和增殖中的作用，MEF细胞还携带条件三苯氧胺激活的Cre重组酶和FLOXED-DUAL荧光报告，以允许FACS分离重组(FIR基因敲除)细胞。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. In parallel studies we are analyzing the role of FIR in cell survival and proliferation at the cellular level using conditional mutant derived MEF cells that also carry a conditional tamoxifen-activated Cre recombinase and a floxed-dual fluorescence reporter to allow FACS isolation of recombined (FIR knock-out) cells. 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 function of this Myc-regulator.