The formin Fmn2 is required for the development of an excitatory interneuron module in the zebrafish acoustic startle circuit

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 the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo, and its impact on associated behaviours have not been tested.Using automated analysis of behaviour and systematic investigation of the associated circuitry, we uncover the role of Fmn2 in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2 is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected.Taken together, this study provides evidence for a circuit-specific requirement of Fmn2 in eliciting an essential behaviour in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders.SIGNIFICANCE STATEMENTFmn2 is a neuronally enriched cytoskeletal remodelling protein linked to neurodevelopment and cognitive disorders in humans. Recent reports have characterized its function in growth cone motility and chemotaxis in cultured primary neurons. However, the role of Fmn2 in the development of neural circuits in vivo and its implications in associated behaviours remain unexplored. This study shows that Fmn2 is required for the development of neuronal processes in the acoustic startle circuit to ensure robust escape responses to aversive stimuli in zebrafish. Our study underscores the crucial role of the non-diaphanous formin, Fmn2, in establishing neuronal connectivity and related behaviour in zebrafish.

Antagonistic activities of Fmn2 and ADF regulate axonal F-actin patch dynamics and the initiation of collateral branching

“…Fmn2 is expressed in the developing and the mature nervous systems across vertebrate phyla (Dutta and Maiti, 2015; Leader and Leder, 2000; Nagar et al, 2020; Sahasrabudhe et al, 2016). While Fmn2 has been implicated in neurodevelopmental disorders, intellectual disability, age-related dementia, microcephaly and corpus callosum agenesis in humans (Agis-Balboa et al, 2017; Almuqbil et al, 2013; Anazi et al, 2017; Gorukmez et al, 2020; Law et al, 2014; Marco et al, 2018; Perrone et al, 2012), its function in neurons is only recently being discovered.…”

Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

“…Zebrafish has two paralogs of Formin-2, fmn2a and fmn2b. fmn2b, located on chromosome 12, has higher sequence homology with human Fmn2 compared to fmn2a and has the characteristic Formin Homology 1 (FH1), Formin Homology 2 (FH2) and Formin Spire Interaction (FSI) domains (Nagar et al, 2021). In contrast, the FH1 domain of fmn2a is truncated.…”

“…F-actin nucleation is the canonical function of formins, including Fmn2, and is mediated by the FH2 domain (Goode and Eck, 2007;Quinlan et al, 2007;Yoo et al, 2015). The FH2 domain is conserved across phyla, including zebrafish (Nagar et al, 2021). A conserved isoleucine residue (at position 1226 in mouse Fmn2) is known to be critical for actin nucleation (Quinlan et al, 2007;Roth-Johnson et al, 2014;Kundu et al, 2020).…”

“…The RNA probes and procedure used for whole mount in situ hybridization experiments have been described previously (Nagar et al, 2021).…”

“…CC-BY-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 10, 2021. ; https://doi.org/10.1101/2021.08.10.455777 doi: bioRxiv preprint 6 knockdown of Fmn2b, the zebrafish ortholog of Fmn2, causes impaired outgrowth of the spiral fiber neuron commissures in the hindbrain and results in diminished short latency escape response (Nagar et al, 2021).…”