Connecting Polycystin Signaling to Asymmetric Nodal Expression

描述(由申请人提供)：纤毛驱动流是被广泛接受的左右图案模型中的关键元素。然而，我们仍然不知道纤毛或纤毛驱动的流动如何引起结节的不对称表达。长期目标是确定LR轴是如何建立的，以及如何利用产生的不对称信息来指导器官形态发生。实现这一目标的下一步是确定纤毛下游的信号如何建立不对称的节点表达。支持这一假设的中心假设是，纤毛驱动的液体在Kupffer囊泡(KV)中流动，通过PC1L1/PC2通道复合体抑制胚胎左侧Charon的转录。这一假说部分基于初步数据，表明斑马鱼节点基因Spaw在KV衬里的细胞中不表达，因此不是流动下游信号的直接靶点。相反，PC2活性的丧失会影响节点抑制剂Charon在KV的不对称表达。PC2被认为是LR模式中的流量传感器，这表明Charon是流产生信号的目标。因此，胚胎右侧较高水平的Charon表达将抑制右侧的Spaw信号，使Spaw优先向胚胎左侧发出信号。这个项目的基本原理是，一旦完成，这项工作将提供纤毛和不对称节点表达控制之间缺失的联系。这些信息对于充分理解纤毛下游信号如何参与LR模式的形成是至关重要的，而LR模式的中断会导致先天性心脏病(CHD)。中心假说将通过三个具体目标进行检验：1)确定非特异性阳离子通道PC2下游的信号如何调节Charon不对称。2)确定是否需要PC1L1信号来调节Charon不对称性。3)确定产生不对称Charon表达的转录机制。在目标1中，将探索产生Charon不对称所需的PC2的定位和活性。使用KV特异性启动子的钙成像将用于确定PC2是否在正确的地点和时间产生钙信号以影响Charon的表达。在目标2中，PC1L1在斑马鱼中的作用将得到确认。测试C末端尾巴是否因流动而被切割以调节转录的实验将被测试。在目标3中，将确定能够驱动Charon不对称表达的最小增强子片段，并用于鉴定参与不对称Charon表达的转录因子和信号通路。这项拟议的研究意义重大，因为它是理解流动如何影响参与左-右模式建立的信号传递的第一步。这种方法是创新的，因为它是一种定向方法，利用斑马鱼系统中可用的工具和遗传学来解决左右手模式中目前尚未充分探索的一个困难但关键的问题。最终，这项工作将识别信号 参与建立LR轴的网络，并将提供新的目标，作为CHD的基础因素进行调查。

DESCRIPTION (provided by applicant): Cilia driven flow is a key element in widely accepted models for left-right patterning. Yet we still do not know how cilia or cilia-driven flow give riseto the asymmetric expression of nodal. The long-term goal is to determine how the LR axis is established and how the asymmetric information generated is utilized to direct organ morphogenesis. The next step towards realizing this goal is to identify how signaling downstream of cilia establishes asymmetric nodal expression. The central hypothesis underlying this proposal is that cilia-driven fluid flow in Kupffer's vesicle (KV) signals through he PC1L1/PC2 channel complex to repress transcription of charon on the left side of the embryo. This hypothesis is based in part on preliminary data demonstrating that the zebrafish nodal gene southpaw (spaw) is not expressed in cells lining KV and is thus not the direct target of signaling downstream of flow. Instead, loss of PC2 activity affects the asymmetric expression of the Nodal inhibitor charon at KV. PC2 is thought to be the flow sensor in LR patterning, suggesting that charon is the target of flow-generated signaling. Thus, charon expression at higher levels on the right side of the embryo would inhibit Spaw signaling on the right, allowing Spaw to signal preferentially to the left side of the embryo. The rationale for this project is tha upon completion, this work will have provided the missing links between cilia and the control of asymmetric nodal expression. This information is crucial to fully understand how signaling downstream of cilia may participate LR patterning, disruptions in which cause congenital heart disease (CHD). The central hypothesis will be tested through three specific aims: 1) Determine how signals downstream of the non-specific cation channel PC2 regulate charon asymmetry. 2) Determine if PC1L1 signals are required to regulate charon asymmetry. 3) Identify the transcriptional mechanisms that generate asymmetric charon expression. In Aim 1, the localization and activity of PC2 required for generating charon asymmetry will be explored. Calcium imaging with KV specific promoters will be used to determine if PC2 is creating calcium signals in response to flow at the right place and time to affect charon expression. In Aim 2, the role for PC1L1 in zebrafish will be confirmed. Experiments to test whether the C-terminal tail is cleaved in response to flow in order to regulate transcription will be tested. In Aim 3, the smallest enhancer fragment capable of driving asymmetric expression of charon will be determined and used to identify transcription factors and signaling pathways involved in asymmetric charon expression. The proposed research is significant because it is the first step towards understanding how flow influences signaling that participates in establishment of left-right patterning. The approach is innovative, as it is a directed approach utilizing the tools and genetics available in the zebrafish system, to tackle a difficult but critical question in left-rigt patterning that is not currently being explored fully. Ultimately this work will identify signaling networks participating in the establishment of the LR axis and will provide new targets to investigate as factors underlying CHD.