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Opposing roles of PlexinA and PlexinB in axonal branch and varicosity formation.

Neufeld SQ, Hibbert AD, Chen BE - Mol Brain (2011)

Bottom Line: However, very few molecules have been identified that are involved in determining where and how many synapses form.In contrast, knocking down PlexinB expression decreased morphological complexity by decreasing the number of branches and the overall size of the axonal arbor, but did not reduce the number of varicosities.Our results demonstrate opposing roles for PlexinA and PlexinB in local wiring within a target region, where PlexinA functions to suppress excessive axonal branches and synapses and PlexinB facilitates axonal growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute of the McGill University Health Centre, Centre for Research in Neuroscience, Montréal, Québec, Canada.

ABSTRACT
Establishing precise synaptic connectivity during development is crucial for neural circuit function. However, very few molecules have been identified that are involved in determining where and how many synapses form. The Plexin cell-surface molecules are a conserved family of axon guidance receptors that mediate axon fasciculation and repulsion during neural development, and later in development PlexinA receptors are involved in eliminating axonal branches and synapse numbers. Here we investigate the roles of PlexinA and PlexinB receptors in axonal branch and varicosity formation in Drosophila. We knocked down PlexinA or PlexinB expression using RNAi in identified mechanosensory neurons and analyzed axonal branching patterns and varicosity formations. Reducing PlexinA expression increased the axonal arbor complexity by increasing the number of branches and varicosities along the axon. In contrast, knocking down PlexinB expression decreased morphological complexity by decreasing the number of branches and the overall size of the axonal arbor, but did not reduce the number of varicosities. Our results demonstrate opposing roles for PlexinA and PlexinB in local wiring within a target region, where PlexinA functions to suppress excessive axonal branches and synapses and PlexinB facilitates axonal growth.

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Related in: MedlinePlus

Quantitative analysis of PlexinA and PlexinB branches and varicosities reveals opposing roles in local axonal growth and synapse formation. A, PlexinA RNAi within the pSc neuron increases the number of variable axonal branches, whereas PlexinB RNAi decreases the number of highly stereotyped (skeletal) branches. Significant decreases in the number of highly skeletal pSc branches were also observed in the PlexinA RNAi neurons due to an increase in branch routing errors. Asterisks denote significance of p < 0.05. B, The pSc axonal arbor sizes with PlexinA RNAi are significantly shifted towards larger values. The distribution of pSc arbor sizes with PlexinB RNAi on the other had is shifted towards smaller values. Frequency distributions of the total axonal branch lengths among the three genotypes, 455-Gal4, and 455-Gal4; UAS-PlexA-RNAi, and 455-Gal4; UAS-PlexB-RNAi were significantly different from each other (p < 0.05). The average arbor size for 455-Gal4 control flies was 1045 μm and approximately 30% of flies had total branch lengths of 1000 μm (blue bars). The average arbor size in PlexinB RNAi pSc neurons was 918 μm with 25% of the neurons having total branch lengths of 800 μm (green bars). Reducing PlexinA expression in pSc neurons had the opposite effect, and increased their average total branch lengths to 1219 μm with 35% of arbors at 1200 μm (yellow bars). Histogram bin width, 100 μm. C, Quantification of axonal varicosities is an indirect measure of synaptic contacts. Varicosities (arrowheads) along branches were counted to obtain a lower estimate of synapse number per pSc axon. Scale bar, 5 μm. D, PlexinA knockdown significantly increases varicosities along the pSc axon. Frequency distribution of the number of varicosities in 455-Gal4; UAS-PlexA-RNAi pSc axons (yellow bars) was shifted to the right compared to the 455-Gal4 distribution (blue bars) (p < 0.01), with no significant difference in 455-Gal4; UAS-PlexB-RNAi (green bars) compared to control.
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Figure 4: Quantitative analysis of PlexinA and PlexinB branches and varicosities reveals opposing roles in local axonal growth and synapse formation. A, PlexinA RNAi within the pSc neuron increases the number of variable axonal branches, whereas PlexinB RNAi decreases the number of highly stereotyped (skeletal) branches. Significant decreases in the number of highly skeletal pSc branches were also observed in the PlexinA RNAi neurons due to an increase in branch routing errors. Asterisks denote significance of p < 0.05. B, The pSc axonal arbor sizes with PlexinA RNAi are significantly shifted towards larger values. The distribution of pSc arbor sizes with PlexinB RNAi on the other had is shifted towards smaller values. Frequency distributions of the total axonal branch lengths among the three genotypes, 455-Gal4, and 455-Gal4; UAS-PlexA-RNAi, and 455-Gal4; UAS-PlexB-RNAi were significantly different from each other (p < 0.05). The average arbor size for 455-Gal4 control flies was 1045 μm and approximately 30% of flies had total branch lengths of 1000 μm (blue bars). The average arbor size in PlexinB RNAi pSc neurons was 918 μm with 25% of the neurons having total branch lengths of 800 μm (green bars). Reducing PlexinA expression in pSc neurons had the opposite effect, and increased their average total branch lengths to 1219 μm with 35% of arbors at 1200 μm (yellow bars). Histogram bin width, 100 μm. C, Quantification of axonal varicosities is an indirect measure of synaptic contacts. Varicosities (arrowheads) along branches were counted to obtain a lower estimate of synapse number per pSc axon. Scale bar, 5 μm. D, PlexinA knockdown significantly increases varicosities along the pSc axon. Frequency distribution of the number of varicosities in 455-Gal4; UAS-PlexA-RNAi pSc axons (yellow bars) was shifted to the right compared to the 455-Gal4 distribution (blue bars) (p < 0.01), with no significant difference in 455-Gal4; UAS-PlexB-RNAi (green bars) compared to control.

Mentions: To examine how reducing PlexinB expression decreased the axonal arbor, we analyzed individually identifiable branches of the pSc axonal arbor. Axonal branches can be classified as being either a skeletal branch that is one of the core branches of the pSc, or a variable branch that is not part of the pSc skeleton. Analysis of the PlexinB RNAi pSc neurons revealed a significant loss of an average 3.3 branches missing from the pSc skeleton (Figure 4A) (p < 0.01). A decrease in skeletal branches was also observed in PlexinA RNAi pSc neurons, but this was due to the frequent branch misrouting phenotypes observed in PlexinA mutants where skeletal branches could no longer be identified with certainty (asterisks in Figure 1), and was accompanied by a large increase in variable branches (8.6 more variable branches than 455-Gal4, p < 0.001) (Figure 4A). PlexinB RNAi axons, however, did not have a significant increase in the number of variable branches. When we measured the total lengths of the skeletal branches in PlexinB RNAi pSc neurons, we also found a significant decrease in size (p < 0.001). The combined decreases in number of axonal branches and size of branches shifted the distribution of total axonal lengths in PlexinB RNAi neurons to smaller values (p < 0.05) (Figure 4B). Conversely, excessive branch formation in PlexinA RNAi neurons significantly shifted the total axonal length distribution towards much larger values than controls (p < 0.01) (Figure 4B).


Opposing roles of PlexinA and PlexinB in axonal branch and varicosity formation.

Neufeld SQ, Hibbert AD, Chen BE - Mol Brain (2011)

Quantitative analysis of PlexinA and PlexinB branches and varicosities reveals opposing roles in local axonal growth and synapse formation. A, PlexinA RNAi within the pSc neuron increases the number of variable axonal branches, whereas PlexinB RNAi decreases the number of highly stereotyped (skeletal) branches. Significant decreases in the number of highly skeletal pSc branches were also observed in the PlexinA RNAi neurons due to an increase in branch routing errors. Asterisks denote significance of p < 0.05. B, The pSc axonal arbor sizes with PlexinA RNAi are significantly shifted towards larger values. The distribution of pSc arbor sizes with PlexinB RNAi on the other had is shifted towards smaller values. Frequency distributions of the total axonal branch lengths among the three genotypes, 455-Gal4, and 455-Gal4; UAS-PlexA-RNAi, and 455-Gal4; UAS-PlexB-RNAi were significantly different from each other (p < 0.05). The average arbor size for 455-Gal4 control flies was 1045 μm and approximately 30% of flies had total branch lengths of 1000 μm (blue bars). The average arbor size in PlexinB RNAi pSc neurons was 918 μm with 25% of the neurons having total branch lengths of 800 μm (green bars). Reducing PlexinA expression in pSc neurons had the opposite effect, and increased their average total branch lengths to 1219 μm with 35% of arbors at 1200 μm (yellow bars). Histogram bin width, 100 μm. C, Quantification of axonal varicosities is an indirect measure of synaptic contacts. Varicosities (arrowheads) along branches were counted to obtain a lower estimate of synapse number per pSc axon. Scale bar, 5 μm. D, PlexinA knockdown significantly increases varicosities along the pSc axon. Frequency distribution of the number of varicosities in 455-Gal4; UAS-PlexA-RNAi pSc axons (yellow bars) was shifted to the right compared to the 455-Gal4 distribution (blue bars) (p < 0.01), with no significant difference in 455-Gal4; UAS-PlexB-RNAi (green bars) compared to control.
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Figure 4: Quantitative analysis of PlexinA and PlexinB branches and varicosities reveals opposing roles in local axonal growth and synapse formation. A, PlexinA RNAi within the pSc neuron increases the number of variable axonal branches, whereas PlexinB RNAi decreases the number of highly stereotyped (skeletal) branches. Significant decreases in the number of highly skeletal pSc branches were also observed in the PlexinA RNAi neurons due to an increase in branch routing errors. Asterisks denote significance of p < 0.05. B, The pSc axonal arbor sizes with PlexinA RNAi are significantly shifted towards larger values. The distribution of pSc arbor sizes with PlexinB RNAi on the other had is shifted towards smaller values. Frequency distributions of the total axonal branch lengths among the three genotypes, 455-Gal4, and 455-Gal4; UAS-PlexA-RNAi, and 455-Gal4; UAS-PlexB-RNAi were significantly different from each other (p < 0.05). The average arbor size for 455-Gal4 control flies was 1045 μm and approximately 30% of flies had total branch lengths of 1000 μm (blue bars). The average arbor size in PlexinB RNAi pSc neurons was 918 μm with 25% of the neurons having total branch lengths of 800 μm (green bars). Reducing PlexinA expression in pSc neurons had the opposite effect, and increased their average total branch lengths to 1219 μm with 35% of arbors at 1200 μm (yellow bars). Histogram bin width, 100 μm. C, Quantification of axonal varicosities is an indirect measure of synaptic contacts. Varicosities (arrowheads) along branches were counted to obtain a lower estimate of synapse number per pSc axon. Scale bar, 5 μm. D, PlexinA knockdown significantly increases varicosities along the pSc axon. Frequency distribution of the number of varicosities in 455-Gal4; UAS-PlexA-RNAi pSc axons (yellow bars) was shifted to the right compared to the 455-Gal4 distribution (blue bars) (p < 0.01), with no significant difference in 455-Gal4; UAS-PlexB-RNAi (green bars) compared to control.
Mentions: To examine how reducing PlexinB expression decreased the axonal arbor, we analyzed individually identifiable branches of the pSc axonal arbor. Axonal branches can be classified as being either a skeletal branch that is one of the core branches of the pSc, or a variable branch that is not part of the pSc skeleton. Analysis of the PlexinB RNAi pSc neurons revealed a significant loss of an average 3.3 branches missing from the pSc skeleton (Figure 4A) (p < 0.01). A decrease in skeletal branches was also observed in PlexinA RNAi pSc neurons, but this was due to the frequent branch misrouting phenotypes observed in PlexinA mutants where skeletal branches could no longer be identified with certainty (asterisks in Figure 1), and was accompanied by a large increase in variable branches (8.6 more variable branches than 455-Gal4, p < 0.001) (Figure 4A). PlexinB RNAi axons, however, did not have a significant increase in the number of variable branches. When we measured the total lengths of the skeletal branches in PlexinB RNAi pSc neurons, we also found a significant decrease in size (p < 0.001). The combined decreases in number of axonal branches and size of branches shifted the distribution of total axonal lengths in PlexinB RNAi neurons to smaller values (p < 0.05) (Figure 4B). Conversely, excessive branch formation in PlexinA RNAi neurons significantly shifted the total axonal length distribution towards much larger values than controls (p < 0.01) (Figure 4B).

Bottom Line: However, very few molecules have been identified that are involved in determining where and how many synapses form.In contrast, knocking down PlexinB expression decreased morphological complexity by decreasing the number of branches and the overall size of the axonal arbor, but did not reduce the number of varicosities.Our results demonstrate opposing roles for PlexinA and PlexinB in local wiring within a target region, where PlexinA functions to suppress excessive axonal branches and synapses and PlexinB facilitates axonal growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute of the McGill University Health Centre, Centre for Research in Neuroscience, Montréal, Québec, Canada.

ABSTRACT
Establishing precise synaptic connectivity during development is crucial for neural circuit function. However, very few molecules have been identified that are involved in determining where and how many synapses form. The Plexin cell-surface molecules are a conserved family of axon guidance receptors that mediate axon fasciculation and repulsion during neural development, and later in development PlexinA receptors are involved in eliminating axonal branches and synapse numbers. Here we investigate the roles of PlexinA and PlexinB receptors in axonal branch and varicosity formation in Drosophila. We knocked down PlexinA or PlexinB expression using RNAi in identified mechanosensory neurons and analyzed axonal branching patterns and varicosity formations. Reducing PlexinA expression increased the axonal arbor complexity by increasing the number of branches and varicosities along the axon. In contrast, knocking down PlexinB expression decreased morphological complexity by decreasing the number of branches and the overall size of the axonal arbor, but did not reduce the number of varicosities. Our results demonstrate opposing roles for PlexinA and PlexinB in local wiring within a target region, where PlexinA functions to suppress excessive axonal branches and synapses and PlexinB facilitates axonal growth.

Show MeSH
Related in: MedlinePlus