Post transcriptional Regulation of Oscillatory clock gene expression during somitogenesis

体节时钟是一种分子振荡器，在早期胚胎发生过程中，驱动调节有丝分裂前中胚层（PSM）中体节发生时间所需的周期性基因表达。体节发生是胚胎早期发育过程中的一个关键过程，导致形成节（体节），这些节继续形成骨骼的骨骼和肌肉。分节时钟基因表达所需的关键基因的功能障碍导致发育性疾病，如先天性脊柱侧凸以及几种癌症。对于动态时钟基因表达，三个水平的调控是相关的：转录控制，转录后调控（从剪接到RNA稳定的每一步）和蛋白质降解。一个复杂的系统的转录激活和负反馈回路正在出现，然而，目前的知识控制振荡时钟基因表达的转录后机制是有限的。该项目将系统地确定调节振荡时钟基因表达的转录后机制，使用人类诱导多能干细胞（iPS）分化为PSM细胞的模型系统。该项目将涉及核糖体关联和poly（A）尾长变化以及蛋白质和RNA水平的分析，以研究时钟基因的翻译效率和mRNA稳定性，并将确定时钟基因mRNA中的哪些序列元件决定转录后调控以及在时钟周期中哪些因素与这些元件相互作用。转录后水平的调控因子包括RNA结合蛋白、microRNA和长链非编码RNA。该项目将建立新发现的转录后调节因子在调节时钟基因表达和体节发生中的功能。为此，将利用iPS-PSM分化测定以及类胃体的产生（由形成体节样结构的干细胞形成的3D结构）。该项目将提高对发育障碍的理解，如脊柱侧凸和与时钟基因通路错误表达有关的癌症。该项目将整合各种技术，如干细胞的培养和分化以及类胃体的产生，干细胞的CRISPR Cas9修饰，几种下一代测序和质谱方法以及这些大型数据集的后续分析，生物信息学，各种分子生物学，生物化学和发育生物学技术，包括先进的显微镜成像方法。Sally Lowell教授在她的实验室中常规使用gastruloids和iPS进行PSM细胞分化测定。Dale实验室还常规使用iPS至PSM细胞分化试验。

The segmentation clock is a molecular oscillator that drives the cyclic gene expression required for regulating the timing of somitogenesis in the presomitic mesoderm (PSM) during early embryogenesis. Somitogenesis is a key process during the early development of an embryo resulting in the formation of segments (the somites) that go on to form the bones and muscle of the skeleton. Functional disturbance of key genes required for segmentation clock gene expression leads to developmental diseases such as congenital scoliosis as well as several cancers. For dynamic clock gene expression three levels of regulation are relevant: transcriptional control, post transcriptional regulation (every step from splicing to RNA stability) and protein degradation. A complex system of transcriptional activation and negative feedback loops is emerging, however, current knowledge of the post transcriptional mechanisms that control oscillatory clock gene expression is limited. This project will systematically identify the post transcriptional mechanisms that regulate oscillatory clock gene expression, using a model system of human induced pluripotent stem (iPS) cells differentiated into PSM cells. The project will involve analysis of ribosomal association and poly(A) tail length changes as well as protein and RNA levels to investigate translational efficiency and mRNA stability of the clock genes and will establish which sequence elements in clock gene mRNAs determine post transcriptional regulation and what factors are interacting with these elements during the clock cycle. Regulators at the post transcriptional level include RNA binding proteins, microRNAs and long noncoding RNAs. The project will establish the function of the newly identified post transcriptional regulators in regulating clock gene expression and somitogenesis. For this the iPS-PSM differentiation assay will be utilised as well as the generation of gastruloids (3D structures formed from stem cells that form somite like structures). The project will improve the understanding of developmental disorders such as scoliosis and cancers linked to misexpression of clock gene pathways. This project will incorporate a wide variety of techniques, such as culture and differentiation of stem cells and the generation of gastruloids, CRISPR Cas9 modification of stem cells, several Next Gen Seq and Mass Spectrometry approaches as well as the subsequent analysis of these large data sets, bioinformatics, a variety of molecular biology, biochemistry and developmental biology techniques, including advanced microscopy imaging approaches. Professor Sally Lowell uses gastruloids and iPS to PSM cell differentiation assays routinely in her lab. The Dale lab also uses the iPS to PSM cell differentiation assay routinely.