Regulation of neural crest migration by the putative phosphatase, paladin

描述（由申请人提供）：神经嵴是一种多能胚胎细胞类型，产生于背神经管并迁移到外周，形成各种结构，包括大部分颅面骨骼。在分子水平上表征神经嵴形成的努力集中在外胚层中指定神经嵴细胞的转录因子上。然而，神经嵴基因的表达并不能保证最终迁移为神经嵴细胞。我们建议，差异蛋白质活性，而不是差异基因表达，调节神经嵴迁移性能。一种可能性是对神经嵴迁移重要的蛋白质的磷酸化状态决定了神经嵴细胞迁移的能力。我们已经确定了一个假定的磷酸酶，paladin，这是由premigrating神经嵴细胞和上调的神经嵴迁移的开始。敲低这种假定的磷酸酶在鸡胚不影响神经嵴规范的时间，但延迟表达的snall-2，一个转录因子的重要神经嵴迁移，并抑制神经嵴迁移。从这些数据/已形成的假设，即支付是一种磷酸酶，调节蛋白质，控制神经嵴EMT和迁移。为了更全面地描述paladin在神经嵴发育中的作用，我们已经产生了paladin的小鼠突变体。在具体目标1中，我将描述paladin突变小鼠神经嵴发育和迁移的缺陷。在《特定目标2》中，我将使用基于功能和活性的分析来确定paladin是磷酸酶还是磷蛋白结合抗磷酸酶。最后，在具体目标3中，我将进行一个修改后的酵母双杂交，以确定目标和paladin的结合伙伴。这些研究将使我们更好地了解迁移性神经嵴细胞是如何产生的，从而更好地了解颅面发育的早期步骤，为预防和治疗神经嵴源性出生缺陷提供更多策略。公共卫生相关性：神经嵴细胞是一种类似胚胎干细胞的细胞群，在成年动物中形成各种不同的结构，包括头骨。神经嵴发育缺陷导致1.3%的活产婴儿出生缺陷。我们的目标是了解神经嵴细胞是如何形成的，这将有助于我们预防和治疗神经嵴源性出生缺陷。

DESCRIPTION (provided by applicant): The neural crest is a multipotent embryonic cell type that arises in the dorsal neural tube and migrates into the periphery to form a variety of structures including most of the craniofacial skeleton. Efforts to characterize neural crest formation at the molecular level have focused on the transcription factors that specify neural crest cells in the ectoderm. However, neural crest gene expression does not guarantee eventual migration as a neural crest cell. We propose that differential protein activity, rather than differential gene expression, regulates neural crest migratory properties. One possibility is that the phosphorylation status of proteins important for neural crest migration determines the ability of neural crest cells to migrate. We have identified a putative phosphatase, paladin, which is expressed by premigratory neural crest cells and upregulated at the initiation of neural crest migration. Knockdown of this putative phosphatase in chick embryos does not affect the timing of neural crest specification, but delays expression of snall-2, a transcription factor important for neural crest migration, and inhibits neural crest migration. From these data / have formed the hypothesis that Paid Is a phosphatase that regulates proteins that control neural crest EMT and migration. In order to more fully characterize the role of paladin in neural crest development, we have generated mice mutant for paladin. In Specific Aim 1, I will characterize defects in neural crest development and migration in paladin mutant mice. In Specific Aim 2, I will use function and activity-based assays to determine if paladin is a phosphatase or a phosphoprotein-binding antiphosphatase. Finally, in Specific Aim 3, I will conduct a modified yeast-two hybrid to identify targets and binding partners of paladin. These studies will allow us to better understand how migratory neural crest cells are generated and therefore better understand the early steps ih craniofacial development, giving us more strategies for prevention and treatment of neural crest-derived birth defects. Public Health Relevance: Neural crest cells are an embryonic stem cell-like population that forms a variety of different structures in the adult animal, including the bones of the skull. Defects in neural crest development cause birth defects in 1.3% of live human births. Our goal is to understand how neural crest cells form, which will help us to prevent and treat neural crest-derived birth defects.