Characterizing the Roles of Toll7 in the Gryllus Bimaculatus Peripheral Nervous System Development

The study of neuronal development could provide foundational information on neurogenesis and neuroplasticity. The small size and relatively simple nervous system of Orthoptera make them ideal models for neurodevelopmental studies. The peripheral nervous system development is an intricate and precise process that each sensory neurons are able to reach their central nervous system partners in a relatively short amount of time. Although the peripheral nervous system in limb buds and their genetic regulations are well understood in grasshopper embryos, few studies have explored the developing nervous system in a cricket model. Therefore, the first goal of the current experiment is to characterize the normal peripheral nervous system development in cricket embryos. Previous studies in Drosophila have suggested Toll6 and Toll7 receptors could serve as important targets for the neurotrophic-like factors Spaetzle2 and 5. Malfunctioning neurotrophic pathways could lead to abnormal nervous system development. Therefore, the second goal of the current study is to explore the roles of Toll7 in the development of the cricket peripheral nervous system. Immunohistochemical staining using anti-horseradish peroxidase (Anti-HRP) was used to illustrate crickets' embryonic developing peripheral nervous system in the limb buds from developmental stage 7.0 to 11.0. Cricket eggs were injected with Toll7 double stranded RNA (dsRNA) and rhodamine dye to suppress the Toll7 mRNA level. The control eggs were injected with GFP dsRNA and rhodamine dye. The peripheral nervous system development in cricket embryos is largely homologous to that observed in grasshopper embryos. All later-emerged sensory neurons followed the pathway established by the first pioneer neuron Ti1. Ti1 made stereotypical turns following the steering signals on epithelial and guidepost cell surfaces and eventually fasciculate with lateral motor axons from the central nervous system. When examining the peripheral nervous system development with Toll7 knockdown, a decrease in limb bud volume was observed at stage 7.7 and stage 8.0, suggesting Toll7’s potential roles in aiding cell-cell intercalation processes in Orthoptera embryos. Furthermore, a delay in Ti1 pioneer neuron development was observed with Toll7 knockdown at early developmental stages, providing evidence for Toll-Spaetzle pathway’s neurotrophic-like functions. The results of the current experiment provide the first description of the peripheral nervous system development in the cricket limb buds and further evidence of Toll-Spaetzle pathway’s neurotrophic properties.