Genes affected by concentration and temporal differences in Shh exposure

描述(申请人提供)：Sonic Hedgehog(Shh)是一种分泌分子，在几种发育背景下具有重要作用，Shh途径的不适当激活在人类癌症(如基底细胞癌)中得到了很好的证明。发育中的脊椎动物肢体是解决Shh功能基本问题的极佳模型。在胚胎发育过程中，Shh是形成正确的肢体模式所必需的，并参与确定手指的身份。虽然Shh的空间梯度被认为是数字图案形成的主要原因，但最近的研究，其中跟踪了Shh表达细胞的后代的命运，表明细胞表达Shh(或时间梯度)的时间长度对于确定最后继的数字身份也可能是重要的。基于这一和其他人的工作，有人提出，穿过手的前后(AP)轴的图案是由Shh浓度和Shh暴露时间的不同来协调的。虽然已经确定了Shh信号的一些下游靶点，但尚不清楚Shh活性的不同阈值如何协调肢体AP模式，以及如何从分子上解释Shh信号。Shh的这种时间调节只在肢体发育的背景下被记录在案，在人类癌症中完全没有被探索过。我们假设Shh通路激活的特定水平或持续时间参与了AP数字身份的确定，并在癌症的形成或进展中发挥了作用。为了解决这个问题，我们首先将创建小鼠永生化的肢芽来源的细胞系，并对它们进行鉴定，以确保它们的肢体特征得到保留。为了使我们的检测在不同的Shh暴露浓度和时间下标准化，我们将检查已知是Shh信号的直接靶点的基因和其他已知对不同持续时间的Shh敏感的基因的诱导表达。我们将利用这些条件进行表达谱分析，以发现受不同Shh信号阈值调控的新基因。这些基因将在小鼠和小鸡胚胎中进行验证和表征，以确定它们在数字识别中的作用。此外，我们将检测这些基因在小鼠BCC肿瘤中的表达，并将其与正常皮肤进行比较，以确定它们的表达是否与癌症的形成有关。虽然Shh浓度在发育过程中的作用已经被研究过，但Shh暴露时间的影响还没有在全基因组水平上进行检验。发现受Shh信号特定阈值差异调控的新基因将有助于扩大我们在肢体发育背景下对这一途径的理解，并提供一个框架，用于解决可能参与癌症形成的Shh靶点。

DESCRIPTION (provided by applicant): Sonic Hedgehog (Shh) is a secreted molecule with essential roles in several developmental contexts, and inappropriate activation of the Shh pathway is well documented in human cancers such as basal cell carcinoma (BCC). The developing vertebrate limb is an excellent model for addressing fundamental questions about Shh function. During embryogenesis, Shh is required for proper limb patterning and is involved in the determination of digit identity. While a spatial gradient of Shh is thought to be primarily responsible for digit patterning, more recent studies, in which the fate of descendants of Shh expressing cells were followed, have suggested that the length of time a cell expresses Shh (or temporal gradient) may also be important for determining the most posterior digit identities. Based on this and others' work, it was proposed that patterning across the anterior-posterior (AP) axis of the hand is orchestrated both by differences in concentration of Shh and in the duration of Shh exposure. While some downstream targets of Shh signaling have been identified, it is unknown how different thresholds of Shh activity coordinate limb AP patterning and are molecularly interpreted. This temporal regulation by Shh has only been documented in the developmental context of the limb and has been completely unexplored in human cancers. We hypothesize that specific levels or duration of Shh pathway activation are involved in the determination of AP digit identity and have a role in cancer formation or progression. To address this, we first will create murine immortalized limb bud-derived cell lines and characterize them to ensure their limb identity is retained. To standardize our assay with different concentrations and times of Shh exposure, we will examine the induction of expression of genes known to be direct targets of Shh signaling and others that are known to be sensitive to different durations of Shh. We will use these conditions to perform expression profiling to discover new genes regulated by different thresholds of Shh signaling. These genes will be validated and characterized in mouse and chick embryos to determine their role in digit identity. Also, we will examine the expression of these genes in mouse BCC tumors and compare it to normal skin to ascertain whether their expression is associated with cancer formation. While the role of Shh concentration has been previously studied during development, the effect of duration of Shh exposure has not yet been examined at a genome-wide level. The discovery of novel genes that are differentially regulated by specific thresholds of Shh signaling will serve to expand our understanding of this pathway in the developmental context of the limb, and also provide a framework with which to address possible Shh targets involved in cancer formation.