Formin-2b (Fmn2b) in the development of neural circuits in zebrafish

Abstract

The formin family member, Fmn2, is a neuronally enriched cytoskeletal remodelling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in regulating cell-substrate adhesion, microtubule dynamics and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo and its impact on associated behaviours remain uncharacterized. As reported in other vertebrates, the zebrafish ortholog of Fmn2, Fmn2b, is also enriched in the developing zebrafish nervous system. Knockdown of Fmn2b resulted in morphological and behavioural defects underlying locomotor circuits. In a custom-made behavioural assay using high-speed video recording of acoustic startle responses in a closed-loop feedback setup, Fmn2b morphants showed defects in short-latency startle responses. The behavioural defects were caused by defects in the development of an excitatory interneuron, spiral fiber neuron, essential for modulation of the acoustic startle response. However, the knockdown of Fmn2b did not compromise other components of the acoustic startle circuit. Given the transient nature of morpholinos, CRISPR-Cas9 based mutants were generated for fmn2b. Further, transgenic lines in the background of homozygous fmn2b mutants were made to examine specific neuronal populations. The fmn2b mutants show early developmental defects and phenocopy morphological defects observed in morphants. Behavioural assessment of fmn2b mutants revealed abnormal spontaneous tail coiling and touch evoked escape response. The behavioural defects were corroborated by outgrowth and branching defects in the motor neurons of fmn2b mutants and were likely due to inadequate innervation of the target myotomes. Preliminary data implicates Fmn2b in the development of visual neural circuits and non-neuronal cytoskeleton regulation during oogenesis and early embryonic development. Our results indicate that Fmn2 is required for specific regulation of axonal outgrowth and pathfinding in vivo, modulating behavioural outputs in larval zebrafish panning different neural circuits. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and will eventually aid our understanding of neurodevelopmental disorders using the zebrafish model.