Samantha Butler - US grants

[unreadable] 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. [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Inductive growth factors are used reiteratively throughout development to direct an extraordinary range of cellular fates and processes. This economy of signaling means that complex organisms can be generated by a relatively small number of signals, but it requires developing cells to differentially interpret the activities of growth factors over time. We have been studying the mechanisms by which the Bone Morphogenetic Proteins (BMPs) are used reiteratively to establish dorsal circuitry in the developing spinal cord. These studies will shed direct light both on our ability to rebuild the spinal cord after injury and our understanding of the congenital diseases that result from deficits in the BMP signaling pathway. The BMPs act from the roof plate (RP) to first pattern the surrounding tissue and then provide guidance information to axons extending away from the dorsal midline. Surprisingly, the mode by which the BMPs induce cell fate remains unclear: although it is widely assumed that they act as morphogens to pattern the dorsal spinal cord, this model has not been clearly demonstrated. Rather, there are many BMPs present in the RP and our preliminary studies suggest that they have distinct effects on the induction of particular neural fates. We will resolve whether the BMPs act quantitatively as morphogens or more qualitatively to specify cell fate in Aim 1. In Aim 2, we will determine how the distinct activitie of the BMPs are translated by dorsal interneurons (INs) to result in different cellular fates and/or processes. Our previous studies have suggested that dorsal INs interpret the distinct activities of the BMPs at both the receptor and second messenger level. A redundant activity common to both type I BMP receptors, BmprIa and BmprIb, permits them to mediate the specification of dorsal cell fates by activating the canonical Smad intermediate, Smad5. In contrast, BmprIb uniquely mediates the guidance activities of the BMPs, by activating the Lim kinase 1 (Limk1)/cofilin pathway and (putatively) Smad1. We will assess both the mechanistic basis by which Smad1, Smad5 and Limk1 are differentially activated by the type I Bmprs and the different mechanisms by which Smad5 and Smad1 then differentially respectively regulate cell fate specification and the control of axon extension in the following aims: Aim 1: Determine the mode by which multiple BMPs direct cell fate in the dorsal spinal cord. Hypothesis: Distinct BMPs qualitatively differentiate progenitor neurons into distinct dorsal IN populations. Aim 2: Determine the mechanism(s) by which dorsal spinal neurons translate the differential activities of the BMPs. Hypothesis: The distinct activities of the BMPs in the dorsal spinal cord are translated by distinct patterns of receptor activation and/or the action of specific second messengers.

CORE D: Abstract The foremost objective of the Structural and Functional Visualization Core is to provide state of the art imaging services to the IDDRC community, including both conventional and confocal microscopy, whole animal MRI acquisition and analysis support. To attain this goal, the core has been redesigned to provide comprehensive imaging services for IDDRC researchers working at on any aspect of the genetic and environmentally-induced developmental diseases affecting nervous system development and function. The research ongoing in the IDDRC spans basic scientists using reductionist approaches to elucidate the mechanisms specific to intellectual developmental disabilities (IDDs) to clinicians assessing therapeutic interventions for patients. To accommodate all of their imaging requirements, we are now providing access to three light microscopy cores, including a microscopy suite dedicated to IDDRC researchers, two MRI facilities and the technical support needed to initiate and complete any imaging analysis. Many IDDRC researchers have imaging systems within their own laboratories, however they are often insufficient to meet their demand and are never comprehensive. Being able to access the IDDRC-supported microscopy cores thus both adds capacity and provides technical resources not otherwise available, permitting researchers to refocus their studies in novel and innovative directions. A second key objective of the Structural and Functional Visualization Core is to develop new technologies for visualizing biological samples and in turn provide them to IDDRC researchers. Here, we focus on [1] developing smaller lighter one-photon miniaturized fluorescent microscopes for live imaging neural activity in freely moving animals (1-3) and [2] refining the methods for CLARITY, a protocol that renders tissue transparent (4, 5), thereby permitting unparalleled visual acuity into the complex circuitry of the brain. These techniques offer the promise of a holistic approach to cutting edge imaging, permitting IDDRC researchers to translate mechanism into therapy. For example, researchers investigating a specific intellectual disorder, such as Dup15q syndrome (see model project), will be able perform MRI on patients to identify the affected region of the brain, implant miniaturized microscopes in rodent models to perform in vivo i m a g i n g to examine how the firing patterns of specific populations of neuron are mechanistically altered by the disease, while concomitantly examining putative aberrant circuit formation using light microscopy coupled with CLARITY. Finally, this core also supports the efforts of all the other cores, offering IDDRC researchers the ability to both probe molecular and cellular function at any level from the sub-cellular to living animals and determine the consequence of therapeutic interventions.

The establishment of neural circuits requires neurons to extend axons over considerable distances, using molecular cues in the embryonic environment to orient their growth cones. The textbook example of a guidance factor is netrin1, a member of the laminin superfamily first characterized in the spinal cord. Classic studies suggested that netrin1, produced by floor plate (FP) cells, acts by chemotaxis, diffusing over long distances to guide Dcc+ commissural axons towards the FP. Netrin1 was then implicated in guidance decisions throughout the nervous system. However, our studies have recently demonstrated that the key source of netrin1 is neural progenitor cells (NPCs) in the spinal ventricular zone. Rather than acting as a long-range diffusible gradient, our data supports a model in which NPC-derived netrin1 acts locally, forming a directional path for axons that also promotes axonal fasciculation. The bipolar geometry of NPCs permits them to deposit netrin1 at the basal (pial) margins of the spinal cord. This netrin1+ substrate then acts locally to direct fasciculated, ventral axon extension by haptotaxis, the directed growth of cells along an adhesive surface. However, netrin1 may mediate a more complex activity for axons beyond pure adhesion: netrin1 is then deposited onto Dcc+ axons, and these axons then grow precisely around the border of VZ, i.e. a netrin1- expressing domain. Ablating a small region of netrin1 expression causes axons deviate from their trajectory to follow the ectopic netrin1 boundary. We propose to call this collection of activities a ?hederal? growth boundary, from the analogy of a wall supporting the growth of ivy (genus: hedera) that is not itself penetrated by the ivy. Netrin1 can form a local growth substrate, and promote the fasciculated growth of axons around a netrin1-expressing domain. This reinterpretation of the mechanistic basis by which netrin1 functions, explains why little progress has been made using soluble netrin1 as a regenerative factor. To further this goal, we will assess [1] the mechanisms by which spinal NPCs establish a netrin1+ haptotactic substrate that promotes directed, fasciculated axonal growth in Aim 1, [2] whether NPC vs FP cells have distinct roles producing netrins in Aim 2 and [3] whether netrins have haptotactic activities in axon guidance in the forebrain in Aim 3. Aim 1: Determine the mechanism by which spinal NPCs establish netrin1 ?hederal? boundaries Hypothesis: Spinal NPCs traffic netrin1 to the pial surface to establish a haptotactic substrate. Netrin1 then transfers to Dcc+ axons to promote their fasciculated axon growth around netrin1-expressing regions. Aim 2: Determine the range role of FP-derived netrin1 in the spinal cord Hypothesis: FP-derived netrin1 acts over short range to fasciculate commissural axons crossing the midline Aim 3: Determine the role of NPC-derived netrin1/3 in the forebrain Hypothesis: Neural progenitors in the developing forebrain use the netrin family to establish growth boundaries that guide axons between the diencephalon and telencephalon.

CORE E: STRUCTURAL AND FUNCTIONAL VISUALIZATION Samantha Butler, Core Director; Peyman Golshani, Core Co-Director; Neil Harris and Susan Bookheimer; MRI sub-core directors Abstract The Structural and Functional Visualization Core provides comprehensive imaging services to the members of UCLA Intellectual & Developmental Disabilities Research Center (IDDRC), working on any aspect of the genetic and environmentally-induced developmental diseases affecting nervous system development and function. The research ongoing in the UCLA IDDRC spans basic scientists using reductionist approaches to elucidate the mechanisms specific to intellectual developmental disabilities (IDDs) to clinicians assessing therapeutic interventions for patients. To accommodate all of their imaging requirements, we provide access to three light microscopy cores, including a microscopy suite dedicated to IDDRC researchers, human and animal MRI facilities and the technical support needed to initiate and complete any imaging analysis. The Structural and Functional Visualization Core also works to develop new technologies for visualizing biological samples and in turn provide them to IDDRC researchers. In this proposal, we are focused on [1] developing smaller lighter one-photon miniaturized fluorescent microscopes for live imaging neural activity in freely moving animals and [2] refining the methods for CLARITY and iDISCO, protocols that render tissue transparent thereby permitting unparalleled visual acuity into the complex circuitry of the brain. These techniques offer the promise of a holistic approach to imaging, permitting IDDRC researchers to translate mechanism into therapy. For example, researchers investigating a specific intellectual disorder will be able perform MRI on patients to identify the affected region of the brain, implant miniaturized microscopes in rodent models to perform Ca2+ imaging in vivo to examine how the firing patterns of specific populations of neuron are mechanistically altered by the disease, while concomitantly examining putative aberrant circuit formation using light microscopy coupled with CLARITY. Finally, this core also supports the efforts of all the other cores, offering IDDRC researchers the ability to both probe molecular and cellular function at any level from the sub- cellular to living animals and determine the consequence of therapeutic interventions.