Marc A. Wolman, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to understand how molecular guidance cues are integrated to direct fasciculating axons in the zebrafish brain. MLF axons defasciculate following knockdown of Sema3D signaling and fail to initially converge and subsequently extend after TAG-1 knockdown. MLF fasciculation will be analyzed following concurrent manipulation of Sema3D signaling molecules to identify critical components involved in regulating fasciculation. Cell adhesion assays will be used to test whether Sema3D modifies adhesion among cells expressing Npn1A and L1.1 to induce fasciculation. These experiments may reveal a novel mechanism(s) of semaphorin mediated guidance. To discover how growth cones of MLF axons translate Sema3D and TAG-1 into directed cell movements during fasciculation, in vivo timelapse imaging will be used to analyze growth cone motility in the absence of these guidance cues. From this work, a more thorough understanding of how guidance cues function to guide fasciculating axons may provide insight into molecular signaling defects underlying neural developmental diseases and potential approaches for facilitating axon regeneration following injury or disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how developmental mechanisms contribute to neural circuits controlling animal behaviors. The simple neuroanatomy and repertoire of stereotyped locomotor behaviors of larval zebrafish provide an ideal model to study the contribution of developmental genes to the formation and function of neural circuits underlying defined motor behaviors. In this proposal, I will use the zebrafish 'space cadet'mutant as a tool to investigate mechanisms of axon guidance in the context of forming neural circuits that modulate a particular behavior called the "escape response". Larval zebrafish perform this maneuver in response to tactile or acoustic stimuli. Mutations in the space cadet gene result in an errant execution of the escape response, caused by a guidance defect of a small subset of specialized commissural hindbrain neurons, called the spiral fiber neurons, which are part of a highly conserved "brainstem escape network" of neurons that control motor behaviors. In addition, retinal ganglion cell orientation and pathfinding defects within the retina suggest that space cadet plays a critical role in intraretinal pathfinding, a process that is poorly understood. In this proposal, I first will use recombination mapping, DMA sequence analysis, gene expression patterns, and gene misexpression techniques to determine the molecular identity of the space cadet gene to better understand its function in the context of molecular signaling pathways. Second, I will use in vivo timelapse imaging of retinal ganglion cell and spiral fiber axons in wild-type and space cadet mutant embryos to provide insight into the role(s) space cadet plays in mediating neuronal growth cone guidance in vivo. Finally, I will determine whether spiral fiber neurons and their downstream neuronal synaptic partners are required to mediate specific turning behaviors in response to environmental stimuli. Together, these experiments will provide insights into the functional implication of vertebrate axon guidance on animal behavior. My proposed experiments and analyses will result in novel information essential for understanding mechanisms of axon pathfinding and the control of motor behaviors by neural circuits. Moreover, the results from these studies will also provide a foundation on which to address the mechanisms underlying human congenital disorders causing visual and locomotor impairment.

Project Summary. Behavioral selection relies on the nervous system's interpretation of sensory stimuli. The abrupt absence of a stimulus can be as striking as its sudden presence, yet these stimuli require distinction to guide behavior. Sensory circuits code stimulus onset and offset. These neural signals are split into parallel processing pathways via distinct synaptic structures and physiology. Alterations in synaptic structures are known to underlie sensory processing disorders. However, we lack a mechanistic understanding of how molecular cues establish synaptic configurations that split ON and OFF signals. To fully understand this critical feature of sensory processing and define therapeutic targets, we must identify and characterize the molecular cues that promote the development of synaptic structures that mediate the splitting of ON and OFF signals. We exploit the in vivo accessibility of retinal photoreceptor synaptic terminals. Here, signals coding light onset and offset are split into parallel pathways via structurally and functionally distinct synapses with ON and OFF bipolar cells (BCs), respectively. We aim to define molecular cues that establish a photoreceptor's synaptic configurations that differentiate light onset from offset. Mutant analysis in zebrafish revealed a novel postsynaptic cue: pregnancy associated plasma protein aa (pappaa). pappaa mutants show impaired visual responses to light offset, but intact responses to light onset, and lack presynaptic structures at cone-OFF BC contacts. Pappaa, a secreted metalloprotease, is a local stimulator of insulin-like growth factor 1 (IGF1) signaling, yet its roles in synaptic development are unknown. We will test the hypothesis that Pappaa stimulates presynaptic IGF1 signaling, and governs the formation and function of cone-OFF BC synapses by regulating a cone terminal's calcium buffering capacity and driving formation of distinct OFF presynaptic glutamate release mechanisms. In Aim 1, we will define the timing and cell type of Pappaa-IGF1 signaling that is critical for establishing cone-OFF BC synapses. Our results will define whether Pappaa stimulates pre- and/or postsynaptic IGF1 signaling to influence cone presynaptic architecture and whether this pathway supports the development, maintenance, and/or acute function of cone-OFF BC synapses. In Aim 2, we will define if Pappaa regulates endoplasmic reticulum and mitochondrial-mediated calcium buffering. Our results will define whether Pappaa regulates this process in cones to influence cone-OFF BC synaptic structure and function. In Aim 3, we will determine the Pappaa-dependent mechanism of glutamate release at cone-OFF BC synapses. Our results are expected to identify a presynaptic structure governed by Pappaa, which underlies a glutamate release mechanism required to convey light offset. Overall, our results will reveal mechanisms by which a single neuron organizes distinct presynaptic domains to split signals coding stimulus onset and offset.