ZIC2-DEPENDENT CYTOSKELETAL REGULATION RESTRAINS AXONAL GROWTH IN RETINAL GANGLION CELLS

During neuronal circuit development, axons navigate the brain microenvironment by integrating extracellular guidance cues that regulate cytoskeletal dynamics in the neuronal growth cone (GC). In the visual system, retinal ganglion cells (RGCs) either cross the midline to project contralaterally (cRGCs) or avoid it to project ipsilaterally (iRGCs). The transcription factor Zic2, selectively expressed in iRGCs, promotes midline avoidance by upregulating Wnt receptors and EphB1, whose ligands (Wnt5a and ephrinB2) are present at the midline. However, how Zic2 shapes the intrinsic cytoskeletal behaviour of iRGC axons and GCs during midline avoidance remains unclear. Here, we used fluorescent reporters of microtubule (MT) plus-end polymerisation and F-actin remodelling to characterise cytoskeletal dynamics in Zic2-expressing RGCs. Compared with controls, Zic2-expressing RGCs displayed reduced MT plus-end polymerisation events in axons and GCs, together with increased filopodial dynamics. Consistently, Zic2-expressing axons exhibited slower outgrowth. To identify molecular mediators downstream of Zic2, we performed an integrative analysis of RNA-seq and ChIP-seq datasets and shortlisted candidate Zic2-regulated genes. We focused on Pacsin1 and by gain- and loss-of-function experiments we demonstrate that Zic2 induces a Pacsin1-dependent destabilisation of the cytoskeleton. Overall, our results uncover a previously uncharacterised intrinsic cytoskeletal programme downstream of Zic2 that likely enhances GC exploration of midline cues, enabling a more effective response to repulsive signals.