Abstract: Cytoplasmic inclusions containing the transactivation response element (TAR) DNA-binding protein-43 (TDP-43) aggregates are hallmarks of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (Arai et al., 2006; Neumann et al., 2006). Despite the well-recognized correlation between TDP-43 aggregation and neuronal degeneration, whether this relationship is causal has remained unclear. The recent advent of the optoDroplet technique for controlling protein-protein interaction through light illumination has allowed the generation of droplets containing intrinsically disordered proteins in cells with an unprecedented spatiotemporal precision (Shin et al., 2017). Moreover, the use of this optogenetic approach to explore TDP-43 uncovered the neurotoxicity associated with TDP-43 phase transitions in cultured neurons (Mann et al., 2019; Zhang et al., 2019). Here, we discuss our recent discovery of novel facets of TDP-43, based on the use of an optogenetic TDP-43 variant (opTDP-43) interrogated in zebrafish motor neurons, in which the in vivo dynamic nuclear-cytoplasmic relocation and the clustering of TDP-43 can be observed directly due to the transparent zebrafish body (Asakawa et al., 2020). Our results showed that optogenetically clumped opTDP-43 mislocalizes to the cytoplasm and damages motor neurons before the development of large cytoplasmic aggregates, which are similar to those found in the ALS patients. This unexpected finding raises the possibility that the onset of motor neuron dysfunction caused by TDP-43 in ALS occurs much earlier than previously anticipated; therefore, future efforts should be made to identify the cellular environments and insults that facilitate pathological TDP-43 oligomer formation to better understand, and potentially intervene in, the prodromal phase of ALS and other TDP-43 proteinopathies.