Genes affected by concentration and temporal differences in Shh exposure

描述（由申请人提供）：Sonic Hedgehog（Shh）是一种分泌型分子，在几种发育环境中具有重要作用，Shh通路的不适当激活在人类癌症（如基底细胞癌（BCC））中得到了充分证实。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。在胚胎发生期间，Shh是正确的肢体模式所需的，并且参与手指身份的确定。虽然Shh的空间梯度被认为是主要负责数字模式，但最近的研究表明，细胞表达Shh的时间长度（或时间梯度）也可能对确定最后的数字身份很重要。基于这一点和其他人的工作，有人提出，手的前后（AP）轴上的图案是由Shh浓度和Shh暴露持续时间的差异协调的。虽然Shh信号传导的一些下游靶点已经被确定，但尚不清楚Shh活性的不同阈值如何协调肢体AP模式并在分子上进行解释。Shh的这种时间调节仅在肢体的发育背景下被记录，并且在人类癌症中完全未被探索。我们假设Shh通路激活的特定水平或持续时间参与AP指身份的确定，并在癌症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芽衍生细胞系，并对其进行表征，以确保保留其肢体身份。为了用不同浓度和时间的Shh暴露来标准化我们的测定，我们将检查已知为Shh信号传导的直接靶标的基因和已知对不同持续时间的Shh敏感的其他基因的表达的诱导。我们将使用这些条件进行表达谱分析，以发现由Shh信号的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，以确定它们在数字身份中的作用。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形成相关。 虽然Shh浓度的作用以前已经在发育过程中进行了研究，但Shh暴露持续时间的影响尚未在全基因组水平上进行研究。新基因的发现，差异调节的特定阈值的Shh信号将有助于扩大我们的理解这条途径的发展背景下的肢体，也提供了一个框架，以解决可能的Shh参与癌症形成的目标。

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.