My research focuses on understanding the molecular mechanisms underlying axon outgrowth and pathfinding. 
1. To characterize the intracellular signal transduction pathways downstream of axon attraction: My early work focused on studying intracellular signal transduction mechanisms mediating neuronal response to guidance cues, e.g., netrin-1. We found that netrin-1 induced tyrosine phosphorylation and activated focal adhesion kinase (FAK), Src family kinases Fyn, and the adaptor protein p130CAS. p130CAS is downstream of the Src family kinases and upstream of the small GTPases Rac1 and Cdc42. Activation of FAK, Fyn, and p130CAS by netrin-1 is crucial for netrin-1-induced axon outgrowth and attraction in vitro and in vivo. The interaction of DCC with DOCK180, a member of guanine nucleotide exchange factors for Rho GTPases, is required for netrin-1 signaling and spinal CA guidance. Based on these results, we proposed a working paradigm for netrin-1 signaling in neuronal guidance, leading from the DCC receptor to FAK and Src family kinases, p130CAS, DOCK180, Rac1, and actin. This body of work helps us understand how the brain establishes its precise wiring during development.
2. To study the coordination of axon guidance signaling: Guidance cues and their receptors is crucial for neurons to maneuver growth cone navigation in the developing nervous system. We have found that DSCAM functions as a new netrin-1 receptor, collaborating with DCC involved in netrin-1-induced axon outgrowth and attraction. C-Jun N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase (MAPK) family and are essential in neuronal degeneration, development, migration, polarity, and regeneration. Our studies have shown that JNK1, not JNK2 or JNK3, is specifically involved in the coordination of DCC and DSCAM in netrin-1-mediated attractive signaling. Interestingly, DSCAM collaborates with UNC5C mediating netrin-1-induced axon repulsion. These studies shed new light on the coordination of netrin signaling in the developing nervous system and further our understanding of molecular mechanisms underlying axon guidance and neuronal migration.
3. To explore cytoskeleton dynamics in axon guidance: Regulation of microtubule and actin dynamics in developing neurons plays an important role in axon guidance and neuronal migration. However, whether microtubule dynamics is directly or indirectly involved in these processes is unclear. Our studies have shown that DCC and DSCAM interact directly with TUBB3, a neuronal β-tubulin isotype, and netrin-1 induces these interactions. Netrin-1 directly modulates microtubule dynamics via TUBB3 in the axonal growth cone. TUBB3 is required for netrin-1-induced axon outgrowth, branching, turning, and pathfinding in the developing nervous system. Interestingly, we found that TUBB3 also directly interacted with UNC5C, and netrin-1 reduced this interaction. UNC5C interacted with polymerized TUBB3 in microtubules, and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of the dorsal root ganglion (DRG) towards the spinal cord in vivo. Netrin-1 differentially increased microtubule dynamics in the growth cone, with more microtubule growth in the distal than the proximal region of the growth cone during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. These data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1-mediated axon repulsion. Altogether, these results demonstrate, to my knowledge for the first time, that guidance receptors could directly interact with a microtubule subunit and provide a novel working model that guidance receptors directly couple microtubule dynamics in neuronal guidance. Missense mutations in TUBB3 result in neurological disorders associated with abnormal neuronal migration and axon guidance. These phenotypic defects are similar to those in netrin-1, DCC, or Robo1 knockout mice. Our studies indicate that missense TUBB3 mutants fail to interact with DCC and UNC5C, and that disease-associated TUBB3 mutations impair netrin/DCC/UNC5C signaling, resulting in specific defects of netrin-mediated axon guidance in the developing nervous system. These data have laid the groundwork for untangling the molecular mechanisms underlying tubulin-mutation-associated tubulinopathies in axon guidance and neuronal migration.
4. To study the role of miRNAs in neuronal guidance during embryonic development: The coordination of different guidance cues and receptors is essential for axon growth cones to navigate properly during embryonic development. A substantial number of guidance receptors, cell-surface and extracellular matrix molecules are differentially expressed in the developing nervous system. MicroRNAs (miRNAs), small non-coding regulatory RNAs, are abundant in the developing nervous system and play an important role in neuronal development through post-transcriptionally regulating gene expression. Our preliminary studies indicate that conditional removal of Dicer, an endoribonuclease of the RNase III family required for miRNA processing, from mouse commissural neurons causes defects of commissural axon projection in vivo, suggesting that miRNAs play an important role in axon guidance. Expression of Robo1 without its 3’ UTR in commissural neurons not only causes repulsive effect of Slit2 on precrossing commissural axon turning in vitro, but defects of commissural axon projection in vivo, as well, indicating miRNAs may be involved in the midline crossing through regulating Robo1 levels during spinal cord development. Our recent results indicate that miR-92, a highly conserved miRNA, suppresses cRobo1 expression in commissural neurons thereby regulating Slit sensitivity to control CA projection and midline crossing. We will further investigate the role of miRNAs in commissural axon guidance during embryonic development: 1) Identify the miRNAs involved in commissural neurons during midline crossing. The regulation of miRNAs in mouse spinal commissural neurons will be examined using miRNA sequencing, miRNA microarray analysis, quantitative real-time RT-PCR, and Northern blot; the temporal and spatial expression of miRNAs in the developing spinal cord will be assessed using in situ hybridization; the potential miRNAs involved in commissural axon guidance will be identified by using bioinformatic analysis, luciferase assays, miRNA activity analysis, and Western blotting. 2) Determine the functional importance of miRNAs in commissural axon guidance. The Slit/Robo1-mediated commissural axon outgrowth and repulsion will be examined by using explants culture and co-culture after in ovo electroporation; the functional role of miRNAs in commissural axon projection in vivo will be assessed by systemic analysis of phenotypes of commissural axon projection in chick embryos after in ovo electroporation of specific miRNAs or miRNA knockdown probes, as well.