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. 2023 Apr 11;13(1):5894.
doi: 10.1038/s41598-023-32943-4.

Adult expression of Semaphorins and Plexins is essential for motor neuron survival

Aarya Vaikakkara Chithran  1   2 , Douglas W Allan  2   3 , Timothy P O'Connor  4   5
Affiliations

Adult expression of Semaphorins and Plexins is essential for motor neuron survival

Aarya Vaikakkara Chithran et al. Sci Rep. .

Abstract

Axon guidance cues direct the growth and steering of neuronal growth cones, thus guiding the axons to their targets during development. Nonetheless, after axons have reached their targets and established functional circuits, many mature neurons continue to express these developmental cues. The role of axon guidance cues in the adult nervous system has not been fully elucidated. Using the expression pattern data available on FlyBase, we found that more than 96% of the guidance genes that are expressed in the Drosophila melanogaster embryo continue to be expressed in adults. We utilized the GeneSwitch and TARGET systems to spatiotemporally knockdown the expression of these guidance genes selectively in the adult neurons, once the development was completed. We performed an RNA interference (RNAi) screen against 44 guidance genes in the adult Drosophila nervous system and identified 14 genes that are required for adult survival and normal motility. Additionally, we show that adult expression of Semaphorins and Plexins in motor neurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Knockdown of a subset of axon guidance genes reduces adult survival rate. (a) A summary of the axon guidance genes that were tested for the impact of knockdown on adult survival rate using the GeneSwitch screen. The axon guidance cues are categorized into four groups based on the molecular function GO terms. (b, c) Survival analysis curves of axon guidance genes that showed a significant effect on survival from day 9 post eclosion. We note that Sema2a is not significantly different from the control on day 10 (c), however we included these flies in the graph to group the analysis of the Semaphorin and Plexin genes together. (d, e) Survival analysis curves of axon guidance genes that showed a significant effect from day 14 post eclosion. Data shown are mean ± SEM (p < 0.05, two-way ANOVA and Tukey’s multiple comparison tests; n = 30). ‘*’ indicates the time points when the survival of knockdowns was significantly different from that of the controls.
Figure 2
Figure 2
The TARGET system screen confirmed a reduced adult survival rate for 14 axon guidance genes knocked down. (a–d) Knockdown of Sema1a, Sema1b, PlexA, chic, Sema5c, Sema2a, PlexB, Abl, Apc, Fas3, robo1, msps, Dg and fra exhibited a significant reduction in the adult survival rate. (e) Knockdown of Lis-1 did not show a significant effect on the adult survival rate in the TARGET screen. Data shown are mean ± SEM (p < 0.05, two-way ANOVA and Tukey’s multiple comparison tests; n = 30). ‘*’ indicates the time points when the survival of knockdowns was significantly different from that of the controls.
Figure 3
Figure 3
Motility is impacted by the knockdown of specific axon guidance genes (a) Effect of pan-neuronal knockdown of axon guidance genes (using elav-GeneSwitch-Gal4) on adult climbing behaviour. A significant decrease in adult climbing ability was observed after the knockdown. (b) Similar observations were made when each gene was knocked down specifically in glutamatergic motor neurons using the OK371-Gal4. Data shown are mean ± SEM (‘*’ denotes a significant difference from the control and dsRNA-GFP; p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests; n = 90). (c) Effect of pan-neuronal knockdown of axon guidance genes (using elav-GeneSwitch-Gal4) on adult activity (measured using DAM). The bar graph compares the normalized average activity counts per 12 h for each gene from multiple flies over a period of 3 days in 12 h light and 12 h dark cycle. The black portion (0–12 h) represents the dark cycle, and the white portion (12–24 h) represents the light cycle. Data shown are mean ± SEM (‘*’ denotes a significant difference from the control and dsRNA-GFP; p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests; n = 32).
Figure 4
Figure 4
Knockdown of Semaphorins and Plexins results in motor neuron death. (a) Adult ventral nerve cord showing neuronal GFP expression driven by OK371-Gal4. White arrows point to the location of leg motor neuron cell bodies in T1, T2, and T3 thoracic segments. The scale bar represents 100 µm. (b) Effect of glutamatergic knockdown of Semaphorins and Plexins (using OK371-Gal4) on adult leg motor neuron survival. A decrease in the number of leg motor neurons was observed after the knockdown of Semaphorins and Plexins genes. The scale bar represents 20 µm. (c–e) Quantification of adult leg motor neurons in T1, T2, and T3 respectively, following 9 days of axon guidance gene knockdown. Data shown are mean ± SEM (‘*’ denotes a significant difference from the control and mCherry; p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests; n = 20).
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