A novel role for receptor tyrosine phosphatase in neuroblast migration

描述（由申请人提供）：受体酪氨酸磷酸酶（RPTPs）在神经系统的神经发生、细胞增殖和神经元定位过程中发挥重要作用，但其作用机制尚不清楚。由于不同RPTP亚型的功能重叠1,2，遗传分析变得复杂，而许多RPTP的真正配体仍然未知3。最近使用Manduca sexta模型系统的研究已经确定了一种特定RPTP的新功能，可能会澄清这些问题。在Manduca发育中的肠神经系统（ENS）中，基板来源的神经母细胞在分化为成熟神经元之前必须广泛迁移，在此过程中，它们沿着预先形成的肌带通路行进，同时避开肠中线。先前的研究表明，中线肌在该系统中表达唯一的Eph受体酪氨酸激酶（MsEph），而神经母细胞表达其同源的Ephrin配体（MsEphrin，一种gpi连接的或a型Ephrin）4。有趣的是，通过MsEphrin的“反向”信号传导阻止了神经母细胞穿过表达mseph的中线细胞，这一反应涉及Src家族激酶（SFK）的局部激活和其主导过程的收缩5,6。相比之下，传统的通过MsEph受体的“正向”信号在这一过程中没有作用，这是Ephrin-A反向信号控制神经母细胞定位的第一个例子。然而，gpi连接的Ephrins通过细胞膜传递信号的机制仍然知之甚少。对MsEphrin共受体的亲和筛选发现了PTP10D，这是一种调节CNS2中线轴突反应的“iii型”RPTP。初步研究表明，PTP10D与MsEphrin在神经母细胞中共表达，抑制PTP10D的表达可引起与阻断MsEphrin信号通路相同的异位中线交叉。有趣的是，PDZ适配蛋白ZO-1（闭塞带-1）也在该筛选中被鉴定出来，并显示在神经母细胞中与MsEphrin和PTP10D共定位。ZO-1最初被归类为紧密连接蛋白7，在体外也可以调节细胞迁移8,9和5

DESCRIPTION (provided by applicant): Receptor tyrosine phosphatases (RPTPs) play important roles during neurogenesis, cell proliferation, and ne ronal positioning in the nervous system, but their mechanisms of action remain poorly understood. Genetic analyses have been complicated by the overlapping functions of different RPTP subtypes1, 2, while authentic ligands for many RPTPs remain unknown3. Recent studies using the model system Manduca sexta have identified a novel function for a specific RPTP that may clarify these issues. In the developing enteric nervous sy tem (ENS) of Manduca, placode-derived neuroblasts must migrate extensively before differentiating into ma- ture neurons, during which they travel on pre-formed muscle band pathways while avoiding the enteric midline. Previous work showed that the midline muscles express the sole Eph receptor tyrosine kinase in this system (MsEph), while the neuroblasts express its cognate Ephrin ligand (MsEphrin; a GPI-linked or type-A Ephrin)4. Intriguingly, "reverse" signaling via MsEphrin prevents the neuroblasts from crossing the MsEph-expressing midline cells, a response that involves the local activation of a Src family kinase (SFK) and retraction of their leading processes5, 6. In contrast, conventional "forward" signaling via MsEph receptors plays no role in this process, providing the first example of Ephrin-A reverse signaling in the control of neuroblast positioning. However, the mechanisms by which GPI-linked Ephrins transduce signals across the membrane remain poorly understood. An affinity screen for MsEphrin co-receptors identified PTP10D, a "type-III" RPTP that regulates midline axonal responses in the CNS2. Preliminary studies showed that PTP10D is co-expressed with MsEph- rin by the neuroblasts, while inhibiting PTP10D expression induced the same pattern of ectopic midline cros- sovers caused by blocking MsEphrin signaling. Intriguingly, the PDZ adapter protein ZO-1 (zonula occludens- 1) was also identified in this screen, and shown to co-localize with MsEphrin and PTP10D in the neuroblasts. Originally classified as a tight junction protein7, ZO-1 can also regulate cell migration in vitro8, 9 and may interact with RPTPs and SFKs in a variety of contexts10-12. The overall objective of this proposal is to determine how PTP10D regulates neuroblast migration in the developing ENS. The central hypothesis is that PTP10D acts as a co-receptor for MsEphrin, coupling this GPI-linked Ephrin with ZO-1 and its downstream effectors. Specific aims will test the role of PTP10D and ZO-1 in MsEphrin-dependent aspects of migration, using published methods to manipulate gene expression and protein interactions in embryo culture. These studies will provide essential new data for an R01 application, with the goal of comprehensively defining the mechanisms of Ephrin-A reverse signaling in the nervous system. Public Heath Relevance: Demonstrating novel roles for RPTPs and ZO-1 in Ephrin-A signaling will provide new tools for investigating why human mutations that affect Ephrin-As result in defective brain growth13, and for developing new therapeutic strategies that address neuro- degenerative conditions in which Ephrin-A reverse signaling may be misregulated14-16.