Regulation of axonal outgrowth in the developing spinal cord

描述（由申请人提供）：我们的研究目标是了解神经网络在发育过程中是如何建立的，并确定如何再现这一过程以修复受伤或疾病后受损的电路。扩展对其目标,一个轴突必须处理在胚胎环境定向信息,实现信号在正确的时间在发展和主管解释正确的上下文线索。然而，尽管控制轴突方向性的机制已被广泛描述，但如何调节生长锥的生长速度或能力仍未得到解决。我们将通过确定骨形态发生蛋白（BMPs）如何在发育中的脊髓中引导联合轴突(C)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：形态因子，也诱导特定的细胞命运。我们发现，从顶板分泌的bmp通过排斥C轴突远离背中线的初始投射，为C轴突提供定向信号。我们已经确定了两个额外的关键角色BMP信号在C轴突指导:调节C轴突产物的速率和导演C轴突后续指导信号的响应能力。为了研究这些以前未被认识的活性，我们将定义BMPs抑制Aim 1中C轴突生长速率的机制，并确定Aim 2中轴突生长不受调节的发育后果。在目标3中，我们将评估一种由BMP信号调节的综合机制，该机制允许C生长锥随时间改变其对信号的反应。通过推进我们对轴突的基本机制的理解指导在开发期间,这些研究将促进中枢神经系统神经元回路的再生。方法：在这些研究中，我们将采用体外和体内趋同的方法，将生化和组织移植试验与小鼠遗传学和鸡卵电孔相结合，目的如下：目的1：确定BMP信号如何调节Limk1/cofilin来控制C轴突的生长速度。假设：bmp通过上调C神经元中的Lim激酶1 (Limk1)，从而使肌动蛋白聚合的直接调节剂cofilin失活，从而调节C轴突的生长速度。目标2。确定轴突延伸速度调节的机理认识的指导线索假说:轴突的时机决定了反应产物的轴突引导暗示。如果C轴突生长速度增加，则会发生引导错误，因为加速的C生长锥对引导信号的反应本质上发生了改变，或者外部环境没有适当地引导它们。目标3。假设：C生长锥中BMP信号的激活指导C轴突对Netrin1的排斥反应，而不是引诱剂。因此，轴突积累了一个信号事件的历史，为未来的引导决策提供信息，从而允许一些引导线索产生多个不同的轴突轨迹。公共卫生相关性：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。我们已经确定了两个先前未被描述的过程，即神经元内部或内在的过程，它们控制着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，确定轴突生长的内在调节因子将对创伤性损伤后重建轴突回路的研究取得重大进展。

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.