Regulation of axonal outgrowth in the developing spinal cord

描述(申请人提供)：我们的研究目的是了解神经元网络是如何在发育过程中建立的，长期目标是确定如何重述这一过程，以修复受伤或疾病后受损的电路。为了向目标延伸，轴突必须在胚胎环境中处理方向信息，在发育的正确时间到达信号，并有能力在正确的背景下解释提示。然而，尽管控制轴突方向性的机制已经被广泛描述，但生长锥体的生长速度或能力是如何被调节的仍未解决。我们将通过确定骨形态发生蛋白(BMPs)如何引导发育中的脊髓中的连合(C)轴突来研究这些问题。在我们之前的工作中，我们发现了一类新的引导信号：也可以诱导特定细胞命运的形态因子。我们证明，从顶板分泌的BMP通过排斥C轴突的初始投射远离背中线，为C轴突提供方向信号。我们现在已经确定了BMP信号在C轴突引导中的另外两个关键作用：调节C轴突生长的速率和引导C轴突对后续指导线索的反应。为了研究这些以前未知的活动，我们将定义BMP抑制AIM 1中C轴突生长的速率的机制，并确定AIM 2中未受调控的轴突生长的发育后果。在Aim 3中，我们将评估BMP信号调节的综合机制，该机制允许C生长锥随着时间的推移改变其对信号的反应。通过促进我们对发育过程中轴突引导的基本机制的理解，这些研究将促进中枢神经系统神经元回路的再生。方法：我们将采用体外和体内融合的方法，结合生化和组织移植实验，结合小鼠遗传学和鸡的卵子电穿孔，目的1：确定BMP信号如何调节LIMK1/cofilin，以控制C轴突生长的速度。假设：BMPS通过上调C神经元中LIM激酶1(LIMK1)的表达来调节C轴突的生长速度，从而使肌动蛋白聚合的直接调节因子cofilin失活。目的2.确定轴突延伸速度调节引导线索再认的机制假说：轴突生长的时间决定了轴突对引导线索的反应。如果C轴突生长速度增加，则会发生指导错误，这是因为加速的C轴突生长锥体的反应内在地改变了指导线索，或者外部环境没有到位来指导它们。目的3.确定BMP信号如何调节C轴突对Netrin1的反应假说：C生长锥体中BMP信号的激活引导C轴突对Netrin1做出排斥反应而不是吸引反应。因此，轴突积累了信号事件的历史，通知未来的指导决定，从而允许一些指导线索，以产生多个不同的轴突轨迹。与公共健康相关：我们正在研究神经元在胚胎发育期间向其突触目标发出被称为轴突的投射的机制。我们已经确定了神经元内或神经元固有的两个以前未描述的过程，这两个过程控制着a)轴突生长的速度和b)轴突如何调节它们对胚胎环境中信号的反应。鉴于中枢神经系统中再生的轴突无法克服来自环境的抑制信号的影响，对于试图在创伤后重建轴突回路的研究来说，识别轴突生长的内在调节因素将是一个重大进展。

DESCRIPTION (provided by applicant): Our research aim is to understand how neuronal networks are established during development with the long- term goal of determining how this process can be recapitulated to repair circuits damaged after injury or disease. To extend towards its target, an axon must process directional information in the embryonic environment, reach signals at the correct time in development and be competent to interpret the cue in the correct context. However, although the mechanisms that control directionality for axons have been extensively described, it remains unresolved how either the growth rate or competence of growth cones is regulated. We will examine these questions by determining how Bone Morphogenetic Proteins (BMPs) guide commissural (C) axons in the developing spinal cord. In our previous work, we identified a new class of guidance signals: morphogens that also induce specific cell fates. We showed that BMPs, secreted from the roof plate, provide directional signals for C axons by repelling their initial projections away from the dorsal midline. We have now identified two additional critical roles for BMP signaling in C axon guidance: regulating the rate of C axonal outgrowth and directing the responsiveness of C axons to subsequent guidance cues. To investigate these previously unrecognized activities, we will define the mechanism by which BMPs inhibit the rate of C axon outgrowth in Aim 1 and determine the developmental consequences of unregulated axon outgrowth in Aim 2. In Aim 3, we will assess an integrative mechanism regulated by BMP signaling that permits a C growth cone to change its response to signals over time. By advancing our understanding of the basic mechanisms of axon guidance during development, these studies will facilitate the regeneration of neuronal circuits in the CNS. Approach: We will use convergent in vitro and in vivo methods in these studies, combining biochemical and tissue grafting assays with mouse genetics and chick in ovo electroporation, in the following aims: Aim 1: Determine how BMP signaling regulates Limk1/cofilin to control the rate of C axon outgrowth. Hypothesis: BMPs regulate the rate of C axon outgrowth by upregulating Lim kinase 1 (Limk1) in C neurons and thereby inactivating cofilin, a direct regulator of actin polymerization. Aim 2. Determine the mechanism by which axon extension speed regulates the recognition of guidance cues Hypothesis: The timing of axon outgrowth determines the response of an axon to guidance cues. Guidance errors occur if the rate of C axon outgrowth is increased because either the response of accelerated C growth cones is intrinsically altered to guidance cues, or the extrinsic environment is not in place to guide them. Aim 3. Determine how BMP signaling regulates the response of C axons to Netrin1 Hypothesis: The activation of BMP signaling in C growth cones directs C axons to respond to Netrin1 as a repellent rather than an attractant. Thus, axons accumulate a history of signaling events that informs future guidance decisions, thereby permitting a few guidance cues to generate multiple distinct axon trajectories. PUBLIC HEALTH RELEVANCE: We are examining the mechanisms by which neurons send out projections, called axons, towards their synaptic targets during embryonic development. We have identified two previously undescribed processes within, or intrinsic to, neurons that control a) the rate at which axons grow and b) how axons regulate their response to signals in the embryonic environment. The identification of intrinsic regulators of axon outgrowth will be a significant advance for studies trying to re-establish axonal circuits after traumatic injury, given that regenerating axons in the central nervous system are unable to overcome the effect of inhibitory signals from the environment.