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

项目总结/摘要 在美国，每年大约有10，000例活产婴儿在颅面区域有缺陷。严重 颅面缺损可导致死亡，而不太严重的缺损则会导致发病， 只会导致部分恢复。识别调节颅面神经细胞命运决定的信号传导因子 发展是了解出生缺陷的病因和确定治疗靶点所必需的。 Wnt/β-catenin信号通路在发育和癌症期间的细胞命运选择中起作用。我们的实验室 证明了在颅间充质中Wnt/β-连环蛋白信号的丢失导致细胞命运从 头骨变成软骨Wnt信号如何促进骨和真皮命运，同时抑制替代细胞命运 在颅间充质中的位置还不清楚以前的研究表明，Erk 1/2激活是 软骨内骨形成和生长以及软骨分化的抑制所需的 胚胎发育我的初步数据表明，Erk 1/2的磷酸化在细胞中减少， E12.5时颅间充质中β-连环蛋白缺失。我假设Wnt/β-catenin信号抑制了 软骨和促进颅骨形成通过激活Erk 1/2信号在小鼠。为了验证这一假设 我提出以下目标。1)确定磷酸化Erk 1/2的缺失是否促进颅间充质 分化为软骨。在这里，我建议利用两种方法来抑制Erk信号在体内，包括遗传 使用En 1Cre和Erk 1/2磷酸化的化学抑制的Erk 1/2损失和异位软骨的测定 E12.5时颅间充质中形成。此外，我将使用CA-Mek过表达Erk 1/2激活 在体外EnlCre; c-catfl/del颅骨间充质中的慢病毒感染。2)检测Erk 1/2的磷酸化 依赖于颅间质中的β-连环蛋白。首先，我将证明脑组织中β-连环蛋白的缺失 间充质导致Erk 1/2磷酸化减少，并确定这种作用是否是由于Wnt 信号此外，β-连环蛋白和Erk 1/2的遗传相互作用将通过杂交EnlCre; catfl/del和Erk 1-/-; Erk 2fl/fl小鼠以获得三重杂合功能丧失胚胎。演示一个函数 这两种信号通路在体内调节颅骨骨细胞命运决定中的联系将改善我们的研究。 了解正常的颅骨发育和相关的出生缺陷，更广泛地说，提供了新的 成人组织中骨修复的途径。成功完成此提案将提供有关如何 Wnt/β-catenin信号调节颅骨发育过程中的细胞命运决定， 在多种细胞类型中决定细胞命运。确定骨发育中Wnt信号传导的新靶点 细胞命运可以为颅面疾病的新治疗靶点或预防性治疗提供选择 并将适用于研究Wnt信号传导和其他疾病背景下细胞命运的更大群体， 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.