ABS 018: Forward Genetic Screening in C. elegans Reveals Redundant Pathways with SYD-2 in Synapse Formation and Function

Harshin Vijay ¹ , Spencer Slade ¹ , Presley Bouton ¹ , Sharanya Jairam ¹ , Nathan McDonald ¹

¹ School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA.

Van Wickle (2025) Volume 1, ABS 018

Introduction: Synapses are the specialized intercellular junctions critical for rapid communication between neurons. To build a synapse, the core active zone structures assemble through liquid-liquid phase separation. Proteins responsible for this assembly include the active zone scaffold protein, SYD-2. However, in the absence of SYD-2, limited assembly of synapses and active zones still occurs. Here, we have designed a forward genetic screen in C. elegans to identify genes redundant with SYD-2 involved in synapse formation. We have implemented a strategy to degrade proteins specifically in dorsal motor neurons while keeping ventral motor neurons intact. This strategy was validated by degrading an essential synaptic transmission protein UNC-18 in dorsal motor neurons, which resulted in a coiling phenotype indicative of dorsal-specific neuronal defects. In our forward genetic screen, we have degraded SYD-2 and screened for redundant mutants that impact dorsal motor synapses and result in coiling phenotypes. To date, we have isolated 15+ potential mutants that show the coiling phenotype. In the future, promising mutants will be mapped and sequenced to identify the causative genes. This research aims to provide better insights into the genetic pathways regulating synapse formation and inform strategies to treat synapse degeneration and disfunction.

Methods: SYD-2 was degraded specifically in dorsal motor neurons. SYD-2 was endogenously tagged with mScarlet and a ZF degron, and a Punc-129::Zif-1 transgene was used to express the Zif-1 ubiquitin ligase specifically in dorsal motor neurons. The Punc-129::Zif-1 transgene was validated with a UNC-18-ZF strain to degrade an essential synaptic transmission protein. mScarlet-ZF-SYD-2 degradation was validated with live confocal microscopy of worms immobilized in 1 mM Levamisole. Chemical mutagenesis with EMS was conducted on C. elegans populations to induce random mutations. F2 progeny of mutagenized worms were screened for coiling phenotypes after initiation of reversal by a gentle touch to the head. We hypothesized coiling would arise due to dorsal motor neuron dysfunction resulting from degradation of SYD-2 and an additional mutation. Isolated mutants were re-validated for coiling phenotypes by four independent investigators. Averages of the percentage of worms that coiled are reported.

Results: We developed a targeted genetic screen to identify mutations that enhance motor dysfunction in C. Elegans following SYD-2 degradation in dorsal motor neurons. SYD-2 was endogenously tagged with mScarlet and a ZF degron, and degraded using neuron-specific expression of ZIF-1. EMS mutagenesis followed by behavioral screening for coiling during reversals yielded over 35 mutants with synthetic motor phenotypes. These findings suggest significant redundancy in synapse formation and maintenance pathways. Live imaging confirmed SYD-2 degradation, and all mutants were re-validated by multiple investigators. This screen provides a platform to identify additional components of synaptic stability and neuronal circuit robustness.

Discussion: Over 35 mutants that exhibit specific motor function phenotypes in combination with SYD-2 degradation indicate the existence of redundancies within synapse formation pathways. The developed screening strategy has the potential to uncover additional redundancies in synapse assembly and neuronal function. Future work will focus on mapping the causative mutations in each isolated mutant via whole-genome sequencing. This approach provides a powerful tool for dissecting the genetic architecture underlying synaptic robustness and motor circuit stability.