Role of FIR in limb morphogenesis

肢体发育是一个被充分研究的形态发生模型，其中许多控制骨骼模式和生长的调控成分已被阐明，为解释基于突变表型的基因功能提供了强有力的生物学框架。FIR （FBP相互作用抑制因子）是一种高度进化保守的myc转录负调控因子，存在于脊椎动物和果蝇中。果蝇同系物（半品脱）控制发育中的胚胎翅盘的不扩散区。人类的同系物与结直肠癌有关；交替剪接的显性干扰型FIR与这些癌症有关。阐明FIR在哺乳动物组织中的正常功能一直受到FIR零突变胚胎非常早期胚胎致死（在着床之前）的阻碍，限制了评估该基因在正常组织形态发生中的作用的能力。与Levens博士（CCR， NCI）合作，他已经生成了小鼠FIR的条件（封闭）等位基因，我们正在使用几个Cre驱动程序选择性地从早期肢体芽中去除FIR。我们想测试FIR是否在调节肢体生长中起作用，如果起作用，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，在早期肢体芽中胚层中FIR功能的丧失会导致大量细胞凋亡，从而导致肢体骨骼的截断。在果蝇中，半品脱是Wg （wingless, Wnt）通路的靶标。有趣的是，肢体芽中胚层中Wnt信号效应因子β -catenin的缺失也会导致小鼠胚胎的肢体截断表型，这与肢体芽中早期Fgf信号中心（AER，顶端外胚层脊）形成异常有关，而这是肢体生长所必需的。我们正在分析Wnt通路的组成部分，以确定它们是否在FIR突变肢体中发生改变，以及其他对早期肢体细胞存活至关重要的因素和信号中心的分析。令人惊讶的是，我们发现细胞存活对FIR的要求似乎是环境依赖的，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Anne Moon博士（Geisinger诊所）合作，她已经确定CAPER是调节肢体细胞分裂和衰老的因子，并发现了CAPER- fir蛋白-蛋白相互作用。我们正在研究这些相互作用如何以选择性的方式调节细胞存活，以肢体为模型，了解刺猬信号如何改变对FIR功能的需求。这些研究将揭示FIR在调节生长中的各种作用，并将对开发可能针对myc调节因子功能的癌症治疗产生影响。

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.