PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) SIGNALING AT THE VENTRAL MIDLINE OF THE NEURAL TUBE IS REQUIRED FOR POST-CROSSING AXON GUIDANCE

During development of the neural tube, long-range projections are established by axon guidance tightly regulated in space and time. The commissural dI1 population is a well-established axon guidance model because of its stereotypical growth cone trajectory comprising ventral growth towards the floor plate, crossing to the contralateral side, and a sharp rostral turn. Long-range guidance cues are generally secreted from the floor plate and bind to dI1 growth cones to regulate their growth trajectory. By in situ hybridization-based expression analysis across chicken neural tube development, we have identified the neuropeptide PACAP to be strongly expressed in the dI1 neurons and in a floor plate adjacent population, while its selective receptor (PAC1R) is expressed in the floor plate and the directly adjacent p3 progenitors. This presents an intriguing system with an inverted ligand-receptor expression pattern normally observed in dI1 axon guidance. We therefore performed loss-of-function experiments using in ovo RNAi to investigate PACAP’s role in axon guidance of dI1 interneurons. RNAi of the gene encoding the PAC1R (ADCYAP1R1) at the ventral midline produced significantly more aberrant guidance phenotypes of dI1 neurons, as observed in open book preparations. These aberrancies were exclusively post-crossing phenotypes, indicating that PACAP signaling is required for switching growth cone responsiveness from attraction to repulsion, and possibly for sensing of anterior-posterior signal gradients such as sonic hedgehog or WNT. Live imaging experiments using the recently developed PACAP sensor PAClight1_P78A, are currently conducted to elucidate the spatiotemporal release dynamics of PACAP during the process of dI1 axon guidance.