Proper patterning of the nervous system requires accurate targeting of axons. The dynamic process of axon guidance relies on the interaction between conserved axon guidance receptors and their ligands, which triggers downstream signaling cascades to either attract or repel axons. Disruptions of axon guidance are implicated in several neurological disorders, highlighting its essential role during neurodevelopment. The secreted guidance molecule netrin and its Frazzled (Fra)/Deleted in Colorectal Cancer (Dcc) receptor provide a major attractive force to promote axon growth and guidance during the establishment of multiple neural circuits. Canonically, binding of netrin to Fra/Dcc induces cytoskeletal remodeling locally at the growth cone to drive axon turning and growth. Additionally in Drosophila, Fra functions in a non-canonical Netrin-independent manner by generating intracellular domain fragments (ICDs) through γ-secretase cleavage, which can translocate into the nucleus to regulate gene transcription. However, the upstream regulatory signals that modulate these two distinct Fra/Dcc signaling pathways remain unknown. In Chapter one, we discuss recent advances in the field, focusing on new mechanisms that regulate axon guidance receptor function or their downstream signaling pathways. In Chapter two, we describe the identification of the conserved ADAM metalloprotease Tace/ADAM17 as a bi-directional regulator of Fra/Dcc signaling. We provide evidence that Tace-dependent cleavage of Fra is required to induce the non-canonical pathway, yet the cleavage can also suppress netrin responses in commissural axons to inhibit the canonical pathway. Thus Tace/ADAM17 functions by coordinating and establishing a fine balance between the two distinct signaling outputs of Fra/Dcc, which allows the receptors to signal efficiently. In Chapter three, we defined the Fra interactome with an unbiased proteomic approach and established 85 candidate proteins that potentially interact with Fra in vivo and regulate its signaling properties. Promising candidates include Lar, the CSNs and Rho-5, which are highly enriched in the Drosophila ventral nerve cord and function to regulate axon guidance at the midline. In Chapter four, we discuss the remaining knowledge gaps in our understanding of Fra signaling and propose several directions of future investigations that address these questions and expand upon our findings.