The actin cytoskeleton powers a wide range of cellular activities ranging from cell migration to membrane trafficking (Dominguez and Holmes, 2011; Svitkina, 2018). The structure and dynamics of the actin cytoskeleton are regulated by over 100 actin binding proteins (ABPs) (Pollard, 2016). Several families of ABPs crosslink actin filaments (F-actin) to generate F-actin bundles of different densities that underlie distinct membrane protrusions such as microvilli and filopodia (Tseng et al., 2005; Rajan et al., 2023). Filopodia are finger-like protrusions found on motile cells that play an important role in cell migration and cancer cell metastasis (Stevenson et al., 2012). In developing neurons, they represent a hallmark feature of axonal growth cones, contributing significantly to axon guidance and branching (Cajal, 1890; Letourneau, 1983; Armijo-Weingart and Gallo, 2017; Omotade et al., 2017; Vitriol and Zheng, 2012). Dynamic growth cone filopodia constantly survey the environment and respond to a complex array of external axon guidance signals, stabilizing when encountering attractive cues and retracting in response to repellent cues (Dent et al., 2011; Gomez and Letourneau, 2014; Lowery and Van Vactor, 2009; Gallo and Letourneau, 2004). In support of a role for dynamic filopodia in axon guidance, eliminating growth cone filopodia was shown to abolish guidance responses in vitro and in vivo (Bentley and Toroian-Raymond, 1986; Chien et al., 1993; Zheng et al., 1996). Additionally, filopodia are essential for collateral branching, a process vital for both neurodevelopment and injury recovery (Armijo-Weingart and Gallo, 2017). While the importance of filopodia in axon elongation, guidance, and branching is recognized, the molecular mechanisms underlying axon filopodia regulation and function remain to be fully elucidated.

While several families of ABPs can crosslink actin filaments to form different F-actin bundles, fascin is known to generate tight parallel F-actin bundles constituting the core F-actin structure in the filopodia of nerve growth cones and motile cells (Vignjevic et al., 2006; Cohan et al., 2001; Sasaki et al., 1996; Blake and Gallop, 2023). There are three mammalian isoforms of fascin, with fascin1 being the most widely expressed and studied (Jayo and Parsons, 2010). Increased fascin1 expression in cancer cells is associated with increased metastasis and poor clinical prognosis, while knockdown of fascin1 leads to a decrease in cell migration (Hwang et al., 2008; Fu et al., 2009; Wu et al., 2010; Chen et al., 2019). Interestingly, fascin1 exhibits an intriguing expression profile in mammalian brains: it is highly expressed during development, but its expression is substantially decreased in the adult brain, suggesting an important role for fascin in early neurodevelopment (De Arcangelis et al., 2004; Zhang et al., 2008).

Despite its high level of expression in the developing brain and its importance in the formation and dynamics of filopodia in cell culture, the role of fascin1 in neural development and function, especially guided axonal development in vivo, remains unclear. An early study of gross brain morphology in a fascin1 knockout (KO) mouse model revealed an increase in the size of the lateral ventricles and the loss of the posterior region of the anterior commissure, suggesting a potential role for fascin1 in neuronal migration and axon development (Yamakita et al., 2009). In addition, KO neurons exhibited smaller growth cones with fewer and shorter filopodia than wild-type control neurons in culture (Yamakita et al., 2009). These findings, along with the high perinatal lethality exhibited by fascin1 KO mice (~50 %), suggest a crucial role in mouse development (Yamakita et al., 2009). However, no detailed analysis of axon development, neuronal connections, synaptic function, or behavior in fascin1 KO mice was performed. Consistently, a separate fascin1 knockout mouse developed by the University of California, Davis Knock Out Mouse Project (KOMP) showed homozygous fascin1 KO animals exhibited a high degree of lethality (Valenzuela et al., 2003). Interestingly, the heterozygous mice were reported to exhibit neurobehavioral abnormality, further supporting a role for fascin in brain development and function (Valenzuela et al., 2003). In this study, we utilized a combination of mammalian cell culture and Drosophila melanogaster as an in vivo model to investigate the role of fascin in brain development and function. Our findings establish a critically important role for the fascin family of actin bundling proteins in brain development and function.