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Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.

Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR - PLoS Biol. (2009)

Bottom Line: Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material.Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised.Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

Show MeSH
Cell type-specific rescue of draper mutant phenotypes with alternative Draper receptor isoforms.(A) Three isoforms of the Draper receptor have been identified in Drosophila [24]. We designed isoform-specific primers (arrows) to determine the presence of each unique isoform in larvae. Ovals represent EGF-like repeats in the extracellular domain. (B) RT-PCR shows that Draper-I and Draper-III are expressed in body wall muscles. cDNAs for each isoform were used as positive controls, along with a minus RT reaction. (C–E) To assay for the cell-specific function of Draper-I or Draper-III, each isoform was expressed in either glia (with Gli-Gal4) or muscle cells (with C57-Gal4) in draperΔ5  mutant backgrounds to determine which isoform rescued mutant phenotypes, including (C) decreased bouton number, (D) accumulation of ghost boutons, and (E) accumulation of presynaptic debris. draperΔ5 mutant phenotypes are shown in red bars. (C) Expression of Draper-III in glia provides a partial rescue of the decrease in type Ib bouton number observed in draperΔ5 mutants. (D) Expression of Draper-I in glia or Draper-III in muscle or glia provides complete rescue of the accumulation of ghost boutons observed in draperΔ5 mutants. Expression of Draper-III in glia or Draper-I in muscle also provides a partial rescue of ghost bouton number. (E) Expression of Draper-I in glia fully rescues the accumulation of presynaptic debris observed in draperΔ5 mutants. Expression of Draper-III in muscle also provides weak but significant rescue. (F) Model for Draper receptor function at the NMJ. (i) A motorneuron with an increase in activity or other developmental cues produces (ii) more ghost boutons, and an increase in debris that is engulfed by glial extensions. The newly formed ghost boutons will either (iii) stabilize or detach from the main arbor. Detached boutons will either (iv) degrade into debris or be engulfed by the muscle. For (C–E), ***, p<0.001; **, p≤0.01; *, p≤0.05. Red asterisk, compared to draperΔ5 mutants; black asterisk, compared to wild type. For (C–E), n = 9, 9, 8, 8, 8, 8, for genotype as listed left→right, respectively.
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pbio-1000184-g008: Cell type-specific rescue of draper mutant phenotypes with alternative Draper receptor isoforms.(A) Three isoforms of the Draper receptor have been identified in Drosophila [24]. We designed isoform-specific primers (arrows) to determine the presence of each unique isoform in larvae. Ovals represent EGF-like repeats in the extracellular domain. (B) RT-PCR shows that Draper-I and Draper-III are expressed in body wall muscles. cDNAs for each isoform were used as positive controls, along with a minus RT reaction. (C–E) To assay for the cell-specific function of Draper-I or Draper-III, each isoform was expressed in either glia (with Gli-Gal4) or muscle cells (with C57-Gal4) in draperΔ5 mutant backgrounds to determine which isoform rescued mutant phenotypes, including (C) decreased bouton number, (D) accumulation of ghost boutons, and (E) accumulation of presynaptic debris. draperΔ5 mutant phenotypes are shown in red bars. (C) Expression of Draper-III in glia provides a partial rescue of the decrease in type Ib bouton number observed in draperΔ5 mutants. (D) Expression of Draper-I in glia or Draper-III in muscle or glia provides complete rescue of the accumulation of ghost boutons observed in draperΔ5 mutants. Expression of Draper-III in glia or Draper-I in muscle also provides a partial rescue of ghost bouton number. (E) Expression of Draper-I in glia fully rescues the accumulation of presynaptic debris observed in draperΔ5 mutants. Expression of Draper-III in muscle also provides weak but significant rescue. (F) Model for Draper receptor function at the NMJ. (i) A motorneuron with an increase in activity or other developmental cues produces (ii) more ghost boutons, and an increase in debris that is engulfed by glial extensions. The newly formed ghost boutons will either (iii) stabilize or detach from the main arbor. Detached boutons will either (iv) degrade into debris or be engulfed by the muscle. For (C–E), ***, p<0.001; **, p≤0.01; *, p≤0.05. Red asterisk, compared to draperΔ5 mutants; black asterisk, compared to wild type. For (C–E), n = 9, 9, 8, 8, 8, 8, for genotype as listed left→right, respectively.

Mentions: The draper gene gives rise to three different Draper isoforms, each with a unique combination of intracellular and extracellular domains (Figure 8A). Draper-I bears 15 extracellular EGF repeats, whereas Draper-II and -III only contain five [24]. In their intracellular domains, all isoforms contain a potential dCed-6 binding site (NPXY), but the Shark binding site is only present in Draper-I and -II. To determine which of the isoforms might be involved in NMJ development, we first carried out reverse-transcription PCR (RT-PCR) of body wall muscles. Interestingly, we found that Draper-I and III, but not Draper-II were expressed at the neuromuscular system (Figure 8A and 8B). Therefore, we carried out rescue experiments by expressing Draper-I or -III in muscles or glia in a draper mutant background.


Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.

Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR - PLoS Biol. (2009)

Cell type-specific rescue of draper mutant phenotypes with alternative Draper receptor isoforms.(A) Three isoforms of the Draper receptor have been identified in Drosophila [24]. We designed isoform-specific primers (arrows) to determine the presence of each unique isoform in larvae. Ovals represent EGF-like repeats in the extracellular domain. (B) RT-PCR shows that Draper-I and Draper-III are expressed in body wall muscles. cDNAs for each isoform were used as positive controls, along with a minus RT reaction. (C–E) To assay for the cell-specific function of Draper-I or Draper-III, each isoform was expressed in either glia (with Gli-Gal4) or muscle cells (with C57-Gal4) in draperΔ5  mutant backgrounds to determine which isoform rescued mutant phenotypes, including (C) decreased bouton number, (D) accumulation of ghost boutons, and (E) accumulation of presynaptic debris. draperΔ5 mutant phenotypes are shown in red bars. (C) Expression of Draper-III in glia provides a partial rescue of the decrease in type Ib bouton number observed in draperΔ5 mutants. (D) Expression of Draper-I in glia or Draper-III in muscle or glia provides complete rescue of the accumulation of ghost boutons observed in draperΔ5 mutants. Expression of Draper-III in glia or Draper-I in muscle also provides a partial rescue of ghost bouton number. (E) Expression of Draper-I in glia fully rescues the accumulation of presynaptic debris observed in draperΔ5 mutants. Expression of Draper-III in muscle also provides weak but significant rescue. (F) Model for Draper receptor function at the NMJ. (i) A motorneuron with an increase in activity or other developmental cues produces (ii) more ghost boutons, and an increase in debris that is engulfed by glial extensions. The newly formed ghost boutons will either (iii) stabilize or detach from the main arbor. Detached boutons will either (iv) degrade into debris or be engulfed by the muscle. For (C–E), ***, p<0.001; **, p≤0.01; *, p≤0.05. Red asterisk, compared to draperΔ5 mutants; black asterisk, compared to wild type. For (C–E), n = 9, 9, 8, 8, 8, 8, for genotype as listed left→right, respectively.
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Related In: Results  -  Collection

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pbio-1000184-g008: Cell type-specific rescue of draper mutant phenotypes with alternative Draper receptor isoforms.(A) Three isoforms of the Draper receptor have been identified in Drosophila [24]. We designed isoform-specific primers (arrows) to determine the presence of each unique isoform in larvae. Ovals represent EGF-like repeats in the extracellular domain. (B) RT-PCR shows that Draper-I and Draper-III are expressed in body wall muscles. cDNAs for each isoform were used as positive controls, along with a minus RT reaction. (C–E) To assay for the cell-specific function of Draper-I or Draper-III, each isoform was expressed in either glia (with Gli-Gal4) or muscle cells (with C57-Gal4) in draperΔ5 mutant backgrounds to determine which isoform rescued mutant phenotypes, including (C) decreased bouton number, (D) accumulation of ghost boutons, and (E) accumulation of presynaptic debris. draperΔ5 mutant phenotypes are shown in red bars. (C) Expression of Draper-III in glia provides a partial rescue of the decrease in type Ib bouton number observed in draperΔ5 mutants. (D) Expression of Draper-I in glia or Draper-III in muscle or glia provides complete rescue of the accumulation of ghost boutons observed in draperΔ5 mutants. Expression of Draper-III in glia or Draper-I in muscle also provides a partial rescue of ghost bouton number. (E) Expression of Draper-I in glia fully rescues the accumulation of presynaptic debris observed in draperΔ5 mutants. Expression of Draper-III in muscle also provides weak but significant rescue. (F) Model for Draper receptor function at the NMJ. (i) A motorneuron with an increase in activity or other developmental cues produces (ii) more ghost boutons, and an increase in debris that is engulfed by glial extensions. The newly formed ghost boutons will either (iii) stabilize or detach from the main arbor. Detached boutons will either (iv) degrade into debris or be engulfed by the muscle. For (C–E), ***, p<0.001; **, p≤0.01; *, p≤0.05. Red asterisk, compared to draperΔ5 mutants; black asterisk, compared to wild type. For (C–E), n = 9, 9, 8, 8, 8, 8, for genotype as listed left→right, respectively.
Mentions: The draper gene gives rise to three different Draper isoforms, each with a unique combination of intracellular and extracellular domains (Figure 8A). Draper-I bears 15 extracellular EGF repeats, whereas Draper-II and -III only contain five [24]. In their intracellular domains, all isoforms contain a potential dCed-6 binding site (NPXY), but the Shark binding site is only present in Draper-I and -II. To determine which of the isoforms might be involved in NMJ development, we first carried out reverse-transcription PCR (RT-PCR) of body wall muscles. Interestingly, we found that Draper-I and III, but not Draper-II were expressed at the neuromuscular system (Figure 8A and 8B). Therefore, we carried out rescue experiments by expressing Draper-I or -III in muscles or glia in a draper mutant background.

Bottom Line: Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material.Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised.Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

Show MeSH