Requirement of Cul3 for axonal arborization and dendritic elaboration in Drosophila mushroom body neurons

Abstract

Cul3 belongs to the family of cullin proteins, which function as scaffold proteins of E3 ubiquitin ligase complexes. Here we show cell-autonomous involvement of Cul3 in axonal arborization and dendritic elaboration of Drosophila mushroom body neurons. Cul3 mutant neurons are defective in terminal morphogenesis of neurites. Interestingly, mutant axons often terminate around branching points. In addition, dendritic elaboration is severely affected in Cul3 mutant neurons. However, loss of Cul3 function does not affect extension of the axons that rarely arborize. Function of cullin-type proteins has been shown to require covalent attachment of Nedd8 (neural precursor cell-expressed developmentally downregulated), a ubiquitin-like protein. Consistent with this notion, Cul3 is inactivated by a mutation in its conserved neddylation site, and Nedd8 mutant neurons exhibit similar neuronal morphogenetic defects. Together, Cul3 plays an essential role in both axonal arborization and proper elaboration of dendrites and may require neddylation for its proper function.

Figure 1.

l(2L)MB397 exhibits axon terminal morphogenetic defects in MB neurons. A, Schematic diagram of MARCM. Top, Heat shock-induced FLP recombinase (FLPase) mediates the FRT site-specific mitotic recombination, which results in the expression of mCD8-GFP driven by GAL4 in one of the daughter cells attributable to loss of the repressor transgene GAL80. If the mother cell is heterozygous for a mutant gene (X), the daughter cell homozygous for the mutant gene will be positively labeled. Bottom, An Nb or single-cell/two-cell MARCM clone can be generated in Drosophila CNS, depending on whether GAL80 is lost in the newly generated Nb, in the postmitotic neurons (N), or in the ganglion mother cell (GMC). B, C, Wild-type Nb (WT; B) and single-cell (C) clones of MB neurons, generated in NHL, were examined in adults. Note that one Nb clone contains all five MB lobes (outlined by solid lines in B) and that all of the medial lobes (γ, β, and β′) can reach the midline (dashed lines in this and all of the following figures). D, E, l(2L)MB397 mutant Nb (D) and single-cell (E) clones, generated in NHL, were examined in adults. Note that the γ and β′ lobes are significantly shortened (arrow in D), but other lobes have only subtle morphological defects at the termini (arrowheads in D). Truncated γ axons are also observed in single-cell clones (arrows in E). Scale bar (in this and all of the following figures), 20 μm. All unilateral MB clones are oriented such that their medial processes project from left to right toward the midline. All images are processed from composite confocal images. Genotype: B, C, hs-FLP/X;UAS-mCD8-GFP, FRT40A/tubP-GAL80, FRT40A;GAL4-OK107/+; D, E, hs-FLP/X;l(2L)MB397,UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+.

Figure 2.

l(2L)MB397 impedes reextension of γ axons during metamorphosis of the MBs. A, Schematic drawing of γ neuron remodeling. B-I, GAL4-201Y-labeled wild-type (WT; B, D, F, H) and l(2L)MB397 mutant (C, E, G, I) Nb clones were generated in NHL and examined at the wandering larval stage (B, C), 18 h APF (D, E), 48 h APF (F, G), and in adults (H, I). Larval MB neurons have two perpendicular oriented lobes (large arrowheads in B and C) and two short bundles of terminal branches (small arrowheads in B and C). l(2L)MB397 mutant clones exhibit subtle deformation of lobe termini at the wandering larval (wl) stage (arrows in C compared with arrows in B), normal pruning of larval lobes by 18 h APF (arrows in E compared with arrows in D) but incomplete axon regeneration, even after eclosion (arrows in G and I compared with arrows in F and H). Note that late-pupal-born α/β axons have no obvious terminal morphogenetic defects (arrowheads in I compared with arrowheads in H). Genotype: B, D, F, H, FRTG13,UAS-mCD8-GFP,GAL4-201Y/FRTG13,hs-FLP,tubP-GAL80; C, E, G, I, hs-FLP/X;l(2L)MB397,UAS-mCD8-GFP,FRT40A,GAL4-201Y/tubP-GAL80,FRT40A.

Figure 3.

l(2L)MB397 mutant γ axons have few arbors and stall randomly within γ lobes. Single-cell clones of l(2L)MB397 mutant (A-D) and wild-type (WT; E-H) γ neurons, generated in NHL, were examined in adults. Mutant γ axons can extend to the tip of γ lobe (A) or stall at any position with in the γ lobes (arrows in B-D). Few arbors can be found even in fully extended mutant γ processes (A). In contrast, only a small part of wild-type γ axons have no major arbors (E). The majority of wild-type γ axons often have multiple major arbors that are randomly distributed along the γ lobes (arrowheads in F-H). Genotype: A-D, hs-FLP/X;l(2L)MB397,UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; E-H, hs-FLP/X;UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+.

Figure 5.

Comparison of ending points of l(2L)MB397 mutant axons and arborization points of wild-type axons. The distance from the ending points of l(2L)MB397 mutant axons or the branch points of wild-type axons to the midline were measured and compared. In the wild-type γ axons, each dot represents the distance from the first branch point of a single neuron to the midline of the brain. In the l(2L)MB397 mutant γ axons, each dot represents the distance from the ending points of axons to the midline. Only truncated l(2L)MB397 mutant γ axons without major axon arbors were examined. In the case of wild-type β′ axons, the distance from the proximal arborization point (the right cluster) or the first arborization point of the distal cluster of arbors (the left cluster) to the midline were measured separately. Note that ending points of l(2L)MB397 mutant β′ axons fall into two clusters, close to the arborization point of wild-type β′ axons. Numbers in parentheses represent the total number of neurons analyzed.

Figure 4.

l(2L)MB397 mutant β′ axons often terminate around the places at which arborization normally occurs. Single-cell clones of wild-type (WT; A) and l(2L)MB397 mutant (B, C) α′/β′ neurons, generated at the mid-third-instar larval stage, were examined in adults. Wild-type β′ axons form arbors within the proximal one-third segment (arrowhead in A) and the distal one-third segment (arrow in A). Mutant β′ axons stall either near the proximal position of the arborization (arrowhead in B) or close to the distal locus of the arborization (arrow in C). Genotype: A, hs-FLP/X;UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; B, C, hs-FLP/X;l(2L)MB397,UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+.

Figure 6.

l(2L)MB397 carries a splicing mutation in Cul3, and the conserved neddylation site is essential for Cul3 activity. A, l(2L)MB397 fails to complement with the deficiency line whose deficiency region is indicated by the gap in the thick line but complemented with the deficiency lines whose deficiency regions are indicated by gaps in thin lines. Additional complementation tests assign l(2L)MB397 to the Cul3 complementation group. B, l(2L)MB397 carries a g-to-a mutation at the acceptor site of the intron between exon 6 and exon 7 of Cul3. C, The Cul3² mutant Nb clone shows terminal morphogenetic defects in the γ and β′ lobes (arrowhead). Also note the dense axon fascicles within the α and β lobes (arrows). D, All of the medial lobes are fully extended (arrowhead) in the l(2L)MB397 mutant Nb clone in which GFP-tagged wild-type Cul3 is autonomously expressed. E, Partial sequence alignment among yeast Cul1 (Cdc53) and six Drosophila cullin proteins. Consensus neddylation sites are highlighted in red. F, The terminal defects of the γ and β′ lobes (arrowhead) remain in the l(2L)MB397 mutant Nb clones that autonomously express GFP-tagged Cul3^K717R. Genotype: C, hs-FLP,UAS-mCD8-GFP/X;Cul3²,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; D, hs-FLP/UAS-GFP-Cul3;l(2L)MB397,UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; F, hs-FLP/X;l(2L)MB397,UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;UAS-GFP-Cul3^K717R/+;GAL4-OK107/+.

Figure 7.

Nedd8 mutant MB clones show axon terminal morphogenetic defects. Wild-type (WT; A, B) and Nedd8^AN015 mutant (C, D) clones of MB neurons, generated in NHL, were examined in adults. Only a small number of γ neurons exist in the mutant Nb clone (C), and most mutant γ axons stall before reaching the midline (e.g., arrows in C and D). Genotype: A, B, hs-FLP/X;UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; C, D, hs-FLP,UAS-mCD8-GFP/X;Nedd8^AN015,FRT40A/tubP-GAL80, FRT40A;GAL4-OK107/+.

Figure 8.

Poor elaboration of dendrites in Cul3 mutant γ neurons. Close-up views of the dendritic regions of MARCM-labeled adult γ neurons. Wild-type γ neurons (WT; A, B) acquired longer dendritic branches than Cul3 mutant γ neurons (C, D). Arrows in A and B point to the typical claw-like structures at the ends of wild-type dendritic processes. Note that mutant dendrites often stall with dense irregular endings (arrows in D). Genotype: A, B, hs-FLP/X;UAS-mCD8-GFP,FRT40A/tubP-GAL80,FRT40A;GAL4-OK107/+; C, D, hs-FLP/X;Cul3⁸,UAS-mCD8-GFP,FRT40A/tubP-GAL80, FRT40A;GAL4-OK107/+.