Cell autonomy of sonic hedgehog signaling in vertebrate

Sonic Hedgehog(Shh)信号对脊椎动物的正常发育和成年健康至关重要。改变Shh通路激活的突变会导致发育异常，包括多指畸形和全前脑畸形。Shh信号在成人中的去抑制有助于多种癌症的进展，包括基底细胞癌。了解活跃Shh信号的后果的一个关键问题是，下游靶标是直接调节的，还是通过二级信号中间产物间接调节的。这项建议试图以鸟类的肢芽作为模型系统来提出这个问题。 劳弗实验室将产生可复制的禽类逆转录病毒，表达Shh，一种膜连接的Shh：CD4融合蛋白，或构成激活Shh途径的突变Smo蛋白。四肢将在不同的靶向机制下感染这些病毒，然后进行整体原位杂交组织化学，以确定最大限度地调节各种Shh靶点所需的条件。靶点包括Shh通路基因、BMP家族信号分子和Hox转录因子。携带相同基因的复制缺陷病毒将在预定条件下感染肢芽。将在切片的肢体组织中分析靶基因的表达，同时进行免疫化学染色，以检测感染细胞。通过比较感染细胞和靶基因表达之间的空间关系，可以确定每种反应的细胞自主性。每种反应性质的可能时间变化也将通过改变病毒感染的发展阶段或暴露于每种信号的持续时间来确定。

Sonic hedgehog (Shh) signaling is critical for normal vertebrate development and adult health. Mutations that alter the activation of the Shh pathway lead to developmental abnormalities including polydactyly and holoprosencephaly. Derepression of Shh signaling in adults contributes to the progression of multiple cancers, including basal cell carcinoma. A key issue for understanding the consequences of active Shh signaling is whether downstream targets are regulated directly, or indirectly through secondary signaling intermediates. This proposal seeks to ask this question using the avian limb bud as a model system. The Laufer laboratory will generate replication-competent avian retroviruses that express Shh, a membrane tethered Shh:CD4 fusion protein, or mutant Smo proteins that constitutively activate the Shh pathway. Limbs will be infected with these viruses under various targeting regimes, and then subjected to whole mount in situ hybridization histochemistry to determine the conditions required for maximal regulation of various Shh targets. Targets include Shh pathway genes, BMP family signaling molecules and Hox transcription factors. Replication-defective viruses carrying the same genes will be used to infect limb buds under the predetermined conditions. Target gene expression will be analyzed in sectioned limb tissue simultaneously immunochemically stained to detect infect cells. Comparison of the spatial relationship between infected cells and target gene expression will allow determination of the cell-autonomy of each response. Possible temporal changes in the nature of each response will also be determined by varying the developmental stage of viral infection, or the duration of exposure to each signal.