Phospho-ERK1/2 in beta-catenin-dependent cranial lineage restriction

项目摘要/摘要 在美国，每年大约有10,000名活产儿在头面部有缺陷。严重者 颅面部缺陷会导致致命，而不太严重的缺陷会导致发病率和大多数手术治疗。 只会导致部分恢复。识别调节颅面部细胞命运决定的信号因子 发育对于理解出生缺陷的病因和确定治疗靶点是必要的。 Wnt/β-Catenin信号通路在发育和癌症过程中的细胞命运选择中发挥作用。我们的实验室有 证实了在颅骨间充质中Wnt/-catenin信号的丢失导致细胞命运从 从头骨到软骨。Wnt信号如何在抑制替代细胞命运的同时促进骨和皮肤的命运 在颅骨间充质中的情况仍不清楚。先前的研究表明，ERK1/2的激活是 软骨内骨形成和生长以及抑制软骨分化所必需的 胚胎发育。我的初步数据表明，ERK1/2的磷酸化在 E12.5时，颅间充质中未见-连环蛋白。我推测Wnt/β-catenin信号被抑制 并通过激活ERK1/2信号通路促进小鼠颅骨的形成。为了检验这一假说 我提出了以下目标。1)确定磷酸化ERK1/2的缺失是否促进了颅骨间充质 向软骨分化。在这里，我建议使用两种方法来抑制体内的ERK信号，包括基因 应用En1Cre和化学抑制ERK1/2磷酸化丢失ERK1/2异位软骨 E12.5在颅骨间充质内形成。此外，我将使用CA-MEK过表达ERK1/2激活 慢病毒感染En1Cre；-Catf1/Del体外颅骨间充质细胞2)检测ERK1/2是否被磷酸化 依赖于颅骨间充质中的-连环蛋白。首先，我将演示-连环蛋白在颅骨中的丢失 间充质导致ERK1/2磷酸化减弱，并确定这一效应是否由Wnt引起 发信号。此外，-连环蛋白和ERK1/2的遗传相互作用将通过杂交En1Cre；- Erk2fl/fl小鼠获得三重杂合子功能丧失胚胎。演示功能 在体内调节颅骨细胞命运的这两个信号通路之间的联系将改善我们的 对正常颅骨发育和相关出生缺陷的了解，以及更广泛地说，提供了新的 成人组织中骨修复的途径。成功完成这项提案将提供有关如何 Wnt/-Catenin信号调节颅骨发育过程中细胞命运的决定 多种细胞类型中的细胞命运决定。寻找Wnt信号在骨发育中的新靶点 细胞命运可以为新的治疗靶点或颅面部疾病的预防性治疗提供选择 并将适用于研究Wnt信号和其他疾病背景下的细胞命运的更大社区， 包括癌症。

Project Summary/Abstract Approximately 10,000 live births have defects in the craniofacial region in the United States annually. Severe craniofacial defects can result in lethality while less severe defects lead to morbidity and most surgical treatments only result in partial recovery. Identifying signaling factors that regulate cell fate decisions in craniofacial development is necessary for understanding the etiology of birth defects and identification of therapeutic targets. The Wnt/β-catenin signaling pathway functions in cell fate selection during development and cancer. Our lab has demonstrated that loss of Wnt/-catenin signaling in the cranial mesenchyme results in a cell fate switch from skull bone to cartilage. How Wnt signaling promotes bone and dermal fate while inhibiting alternative cell fates in the cranial mesenchyme remains unclear. Previous studies have demonstrated that Erk1/2 activation is required for endochondral bone formation and growth as well as repression of cartilage differentiation during embryonic development. My preliminary data suggests that phosphorylation of Erk1/2 is diminished in the absence of -catenin in cranial mesenchyme at E12.5. I hypothesize that Wnt/β-catenin signaling represses cartilage and promotes skull bone formation through activation of Erk1/2 signaling in mice. To test this hypothesis I propose the following aims. 1) Determine if loss of phospho-Erk1/2 promotes cranial mesenchyme differentiation to cartilage. Here I propose to utilize two methods to inhibit Erk signaling in vivo, including genetic loss of Erk1/2 using En1Cre and chemical inhibition of Erk1/2 phosphorylation and assay for ectopic cartilage formation in the cranial mesenchyme at E12.5. Additionally I will overexpress Erk1/2 activation using a CA-Mek lentiviral infection in En1Cre;-catfl/del calvarial mesenchyme in vitro. 2) Test whether phosphorylation of Erk1/2 is dependent on -catenin in the cranial mesenchyme. First I will demonstrate that loss of -catenin in cranial mesenchyme results in diminished Erk1/2 phosphorylation and determine whether this effect is due to Wnt signaling. Additionally, genetic interaction of -catenin and Erk1/2 will be determined by crossing En1Cre;- catfl/del and Erk1-/-; Erk2fl/fl mice to obtain triple heterozygous loss-of-function embryos. Demonstrating a functional link between these two signaling pathways in regulating skull bone cell fate decisions in vivo will improve our understanding of normal skull bone development and associated birth defects, and more broadly, provide new avenues for bone repair in adult tissues. Successful completion of this proposal will provide insights into how Wnt/-catenin signaling regulates cell fate decisions during cranial bone development and can be applied towards cell fate decisions in multiple cell types. Identifying new targets of Wnt signaling in bone development and cell fate could provide options for new therapeutic targets or preventative treatments of craniofacial disorders and would be applicable to a larger community that studies Wnt signaling and cell fate in other disease contexts, including cancer.