Regulation of neural crest migration by the putative phosphatase, paladin

描述(申请人提供)：神经脊是一种多能胚胎细胞类型，它起源于背侧神经管，并迁移到外围形成各种结构，包括大部分头面部骨骼。在分子水平上表征神经脊形成的努力主要集中在转录因子上，这些转录因子指定外胚层中的神经脊细胞。然而，神经脊基因的表达并不保证最终作为神经脊细胞的迁移。我们认为，调节神经脊迁移特性的是差异蛋白活性，而不是差异基因表达。一种可能性是，对神经脊迁移至关重要的蛋白质的磷酸化状态决定了神经脊细胞迁移的能力。我们已经确定了一种可能的磷酸酶Paladin，它在迁移前的神经脊细胞中表达，并在神经脊迁移开始时上调。在鸡胚胎中敲除这种假定的磷酸酶不会影响神经脊的指定时间，但会延迟神经脊迁移的重要转录因子SNALL-2的表达，并抑制神经脊的迁移。根据这些数据/形成了一种假设，即Payed是一种磷酸酶，它调节控制神经脊、EMT和迁移的蛋白质。为了更全面地描述Paladin在神经脊发育中的作用，我们培育了Paladin的突变小鼠。在特定的目标1中，我将描述Paladin突变小鼠神经脊发育和迁移方面的缺陷。在特定的目标2中，我将使用基于功能和活性的分析来确定Paladin是一种磷酸酶还是一种磷蛋白结合的抗磷酸酶。最后，在具体目标3中，我将进行修饰的酵母双杂交来确定Paladin的靶点和结合伙伴。这些研究将使我们更好地了解迁移神经脊细胞是如何产生的，从而更好地了解颅面发育的早期步骤，为我们提供更多预防和治疗神经峰源性出生缺陷的策略。公共卫生相关性：神经脊细胞是一种类似胚胎干细胞的群体，在成年动物体内形成各种不同的结构，包括头盖骨。在1.3%的活产婴儿中，神经脊发育缺陷会导致出生缺陷。我们的目标是了解神经脊细胞是如何形成的，这将有助于我们预防和治疗神经脊衍生的出生缺陷。

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.