Genes affected by concentration and temporal differences in Shh exposure

描述（由申请人提供）：Sonic Hedgehog （Shh）是一种分泌分子，在多种发育环境中发挥重要作用，Shh通路的不当激活在人类癌症（如基底细胞癌（BCC））中得到了充分的证明。发育中的脊椎动物肢体是解决Shh功能基本问题的绝佳模型。在胚胎发生过程中，Shh是正确的肢体模式所必需的，并参与了手指身份的确定。虽然Shh的空间梯度被认为是数字模式的主要原因，但最近的研究表明，细胞表达Shh的时间长度（或时间梯度）对于确定最后端数字身份也很重要。基于这项研究和其他人的工作，有人提出，手的前后轴（AP）的模式是由Shh浓度的差异和Shh暴露的持续时间的差异精心安排的。虽然已经确定了Shh信号的一些下游靶点，但尚不清楚Shh活动的不同阈值如何协调肢体AP模式并进行分子解释。Shh的这种时间调节只在肢体的发育背景下被记录下来，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数字身份的决定，并在癌症的形成或进展中发挥作用。为了解决这个问题，我们首先将创建小鼠永生化肢体芽衍生细胞系并对其进行表征以确保其肢体身份保留。为了使我们的实验在不同浓度和时间的Shh暴露下标准化，我们将研究已知的Shh信号的直接目标基因的表达诱导和其他已知的对不同Shh持续时间敏感的基因的表达。我们将利用这些条件进行表达谱分析，以发现受Shh信号不同阈值调节的新基因。这些基因将在小鼠和鸡胚胎中进行验证和表征，以确定它们在手指识别中的作用。此外，我们将检查这些基因在小鼠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。虽然之前已经研究过Shh浓度在发育过程中的作用，但Shh暴露时间的影响尚未在全基因组水平上进行研究。发现受Shh信号特定阈值差异调节的新基因将有助于扩大我们对肢体发育背景下这一途径的理解，并提供一个框架，以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.