Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development

Abstract

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. We show that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. We find that the earlier defect coincides with a failure to localize synaptic vesicles, suggesting that it reflects a failure in presynaptic assembly. We then analyze the relationship between syd-1 and Liprin-α in R7s. We find that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: we show that Liprin-α promotes Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. We show that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. We therefore conclude that the two defects are caused by distinct molecular mechanisms. We find that Trio overexpression rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity.

Figure 1.

syd-1 is required for normal R7 terminal bouton morphology. A–C, Medullas of adult mosaic animals in which R7s homozygous for the specified chromosome arms express Syt–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. A, Each wild-type (FRT82) R7 axon terminates at the M6 layer in an ellipsoidal bouton (arrows). B, syd-1^w46 mutant R7 axons often fail to contact the M6 layer (arrow). Those that do contact M6 have abnormally small terminal boutons (double arrowhead), which often project thin extensions, many of which terminate in varicosities (arrowheads). C, Expressing a syd-1 cDNA in syd-1^w46 mutant R7 axons rescues their morphological defects. D, Schematic representation of the syd-1 genomic region. The locations of the P elements used to create the syd-1^CD allele are indicated by green triangles. The locations of the ferrochelatase (FeCH) and syd-1 exons are indicated by blue rectangles. The DNA sequence change in the syd-1^w46 allele is indicated (exon sequence is in blue and intron in black), as is the extent of the syd-1^CD deletion. E–G, Medullas of adult animals in which all R7 neurons are of the same genotype and are labeled with PANR7–Gal4, UAS–N-synaptobrevin–GFP (white). The PANR7 promoter drives such high levels of expression that, despite being fused to an SV protein, GFP labels the whole R7 axon. Scale bar, 5 μm. E, Wild-type (FRT82) R7s terminate in ellipsoid boutons at the M6 layer. F, Like individual syd-1^w46 mutant R7 axons, R7 axons in syd-1^w46/syd-1^CD mutant animals often fail to contact the M6 layer (arrows). G, Like individual syd-1^w46 mutant R7 axons, some R7 axon terminals in syd-1^w46/syd-1^CD mutant animals project thin extensions beyond M6 (arrowheads).

Figure 2.

The syd-1 and Liprin-α loss-of-function R7 phenotypes are not identical. A–D, Medullas of adult mosaic animals in which homozygous R7s express Syt–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. A, Wild-type (FRT82) R7 axons. Like syd-1^w46 mutant R7 axons, R7 axons homozygous for a deletion of syd-1 (syd-1^CD) often fail to contact the M6 layer (B, C, arrows). Those syd-1^CD axons that do contact M6 have abnormally small terminal boutons (B, arrowhead), which often project thin extensions beyond M6 (C, arrowhead). D, Liprin-α^R60 mutant R7 axons have abnormally small terminal boutons (arrowhead) and often fail to contact M6 (arrows), but their terminals do not have extensions. E, The percentages of wild-type, syd-1, and Liprin-α mutant R7 axons that fail to contact M6. Error bars represent SEM. This syd-1^w46 (10.6 ± 1.00%; n = 16 brains) and syd-1^CD (18.9 ± 1.25%; n = 12 brains) mutant R7 defect is rescued by expression of a wild-type syd-1 cDNA (rescue of syd-1^w46, 0.393 ± 0.157%, n = 9 brains, p < 0.0001; rescue of syd-1^CD, 0.913 ± 0.433%, n = 10 brains, p < 0.0001). The difference between syd-1^CD and syd-1^w46 is significant (p < 0.0001), as is the difference between syd-1^CD and Liprin-α^R60 (55.0 ± 2.32%; n = 9 brains; p < 0.0001). The frequency with which R7s in syd-1^w46/syd-1^CD transheterozygous animals fail to contact M6 (14.5 ± 2.32%; n = 7 brains) is not significantly different from that of individual syd-1^w46 mutant R7s (p = 0.079) or of individual syd-1^CD mutant R7s (p = 0.086). Removing adjacent R7s by means of a hypomorphic sev allele has no effect on the frequency with which syd-1^CD mutant R7s fail to contact M6 (19.5 ± 2.28%; n = 97 brains; p = 0.93). F, The percentages of wild-type, syd-1, and Liprin-α mutant R7 axons that have extensions. Error bars represent SEM. Significantly greater percentages of syd-1^w46 (17.6 ± 1.58%; n = 16 brains; p < 0.0001) and syd-1^CD (24.6 ± 1.89%; n = 12 brains; p < 0.0001) mutant R7 terminals have extensions than wild-type R7 terminals (5.75 ± 0.638%; n = 15 brains). This defect is rescued by expression of a wild-type syd-1 cDNA (rescue of syd-1^w46, 7.89 ± 1.05%, n = 9 brains, p < 0.0001; rescue of syd-1^CD, 5.55 ± 0.584%, n = 10 brains, p < 0.0001). The difference between syd-1^CD and syd-1^w46 is significant (p = 0.0078), as is the difference between syd-1^CD and Liprin-α^R60 (0%; n = 9 brains; p < 0.0001). The frequency with which R7s in syd-1^w46/syd-1^CD transheterozygous animals have extensions (14.2 ± 0.59%; n = 7 brains) is not significantly different from that of individual syd-1^w46 mutant R7s (p = 0.19) but is significantly less frequent than that of individual syd-1^CD mutant R7s (p < 0.001). Removing adjacent R7s by means of a sev mutation has no effect on the frequency with which syd-1^CD mutant R7 terminals have extensions (22.5 ± 2.19%; n = 97 brains; p = 0.73).

Figure 3.

Loss of Liprin-α but not syd-1 significantly disrupts axon transport, which is required for R7 axons to retain contact with M6. A, The number of ANF–GFP-containing vesicles moving in the anterograde or retrograde direction within larval motor neurons of the indicated genotypes. Error bars represent SEM. In Liprin-α mutants, flux in the anterograde direction (9.48 ± 2.46 vesicles per minute; n = 5 larvae) is significantly different from that in the matched wild-type control (33.26 ± 4.54; n = 7 larvae; p = 0.0011). Flux in the retrograde direction also appears to differ between Liprin-α mutants (4.80 ± 0.66; n = 5 larvae) and wild type (18.26 ± 5.78; n = 7 larvae), but this difference is only marginally significant (p = 0.041). In syd-1 mutants, flux in anterograde (41.52 ± 3.78; n = 5 larvae) and retrograde (16.92 ± 3.42; n = 5 larvae) directions is not significantly decreased compared with those in the matched wild-type control (anterograde, 47.85 ± 10.7, n = 4 larvae, p = 0.28; retrograde, 19.2 ± 6.1, n = 4 larvae, p = 0.38). B, C, Medullas of adult mosaic animals in which homozygous R7s express Mito–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. Unlike wild-type (FRT42) R7 axons (B), imac mutant R7 axons often fail to contact the M6 layer (C, arrow). Mito–GFP is frequently mislocalized in or absent from imac mutant R7 axons (C, arrowhead indicates an imac mutant R7 axon that forms a terminal bouton in M6 from which Mito–GFP is nonetheless excluded).

Figure 4.

syd-1 and Liprin-α are required for the enrichment of SVs and mitochondria at R7 terminals. A–D′, Medullas of adult mosaic animals in which homozygous R7s express Syt–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. In wild-type (FRT82) R7s, Syt–GFP is preferentially localized to the region of the axon that lies below the M3 layer (A, A′), whereas in syd-1^w46 mutant R7 axons, Syt–GFP is more broadly distributed and punctate (B, B′). Syt–GFP is similarly mislocalized in syd-1^CD mutant R7s (data not shown). C, C′, Expressing a syd-1 cDNA in syd-1 mutant R7 axons results in normal localization of Syt–GFP. D, D′, Syt–GFP is similarly mislocalized and punctate in Liprin-α mutant R7 axons. E–H′, Medullas of adult mosaic animals in which homozygous R7s express Mito–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). E, E′, In wild-type (FRT82) R7s, Mito–GFP is preferentially localized to the region of the axon that lies below the M3 layer. F, F′, Mito–GFP remains enriched at R7 terminals even when phototransduction is disrupted by loss of norpA. Note that all R7s in this animal lack norpA and express Mito–GFP (see Materials and Methods). In contrast, in both syd-1 (G, G′) and Liprin-α (H, H′) mutant R7 axons, Mito–GFP is broadly distributed and punctate. Expressing a syd-1 cDNA in syd-1 mutant R7 axons results in normal localization of Mito–GFP (data not shown).

Figure 5.

syd-1 mutant R7 terminals first lose contact with M6 and only later project extensions. A–D′, Medullas of mosaic pupae in which homozygous R7s express Syt–GFP (green). All R7 and R8 axons are labeled with mAb24B10 (red). Scale bars, 5 μm. At 24 h APF, wild-type (A, A′) and syd-1 mutant (B, B′) R7 axons are indistinguishable: their growth cones have similar morphology, they terminate in the R7 target layer, and Syt–GFP is enriched within their terminals. By 55 h APF (C–D′), syd-1 mutant R7 terminals often fail to contact the R7 target layer (D, arrows), those that do contact M6 have smaller boutons than wild type, and Syt–GFP is mislocalized and punctate. However, neither wild-type nor syd-1 mutant R7 terminals have extensions at this time point. The asterisk in D indicates a syd-1 mutant R7 axon that appears to terminate in M1 but that in fact extends to M6 in focal planes not included in this image. E, The percentages of wild-type and syd-1 mutant R7 axons that fail to contact the R7 target layer at 24, 55, and 72 h APF and in adult. Error bars represent SEM. At 24 h APF, all wild-type and syd-1 mutant R7s contact the R7 target layer (wild type, n = 5 brains; syd-1^w46, n = 9 brains; syd-1^CD, n = 10 brains). However, by 55 h APF, 14.9 ± 1.80% (n = 12 brains) of syd-1^w46 and 15.6% ± 2.03% (n = 11 brains) of syd-1^CD R7s no longer do so. The percentages of syd-1 mutant R7 axons with this defect are not significantly different at 72 h APF (syd-1^w46, 15.0 ± 1.61%, n = 12 brains, p = 0.97; syd-1^CD, 20.7 ± 1.66%, n = 12 brains, p = 0.067), nor does the percentage of syd-1^CD mutant R7s with this defect change significantly between 72 h APF and adulthood [p = 0.39; the difference between the 55 h APF and adult percentages is also insignificant (p = 0.18)]. However, the percentage of syd-1^w46 mutant R7s with this defect may decrease slightly between 72 h APF and adulthood [p = 0.021; the decrease between the 55 h APF and adult percentages is also marginally significant (p = 0.032)]. The adult data used are the same as that in Figure 2E and are redisplayed here for ease of comparison. F, The percentages of wild-type and syd-1 mutant R7 axon terminals that have extensions at 24, 55, and 72 h APF and in adult. Error bars represent SEM. At 24 h APF, wild-type and syd-1 mutant R7 axon terminals are indistinguishable (wild type, n = 5 brains; syd-1^w46, n = 9 brains; syd-1^CD, n = 10 brains). By 55 h APF, a small percentage of wild-type R7s (1.20 ± 0.404%; n = 12 brains) have short extensions beyond their target layer. At this time point, the percentage of syd-1^w46 mutant R7s with extensions (0.528 ± 0.276%; n = 12 brains) is not significantly different from that of wild type (p = 0.18), and the percentage of syd-1^CD mutant R7s with this defect (0.145 ± 0.15%; n = 11 brains) may be slightly lower than that of wild type (p = 0.027). By 72 h APF, a significantly greater percentage of syd-1^w46 (13.5 ± 1.87%; n = 12 brains; p < 0.001) and syd-1^CD (13.6 ± 1.54%; n = 12 brains; p < 0.0001) mutant R7s have extensions than do wild-type R7s. The percentage of syd-1^w46 mutant R7s with this defect does not change significantly between 72 h APF and adulthood (p = 0.11). However, the percentage of syd-1^CD mutant R7s with this defect increases significantly during this period (p < 0.001). The adult data used are the same as that in Figure 2E and are redisplayed here for ease of comparison. G, Of those syd-1^CD mutant R7 axon terminals that have extensions, the percentages whose extensions project forward, laterally, or both (R7s with both have either a single extension that projects both forward and laterally or two extensions of which one projects forward and the other laterally). Removing adjacent R7s by means of a sev mutation has no significant effect on the orientation of syd-1^CD mutant R7 extensions [in the presence of neighboring R7s, 64.3 ± 3.14% extend forward, 17.3 ± 3.23% extend laterally, and 18.5 ± 2.49% have extensions in both directions (n = 14 brains); in the absence of neighboring R7s, 68.6 ± 4.15% extend forward (p = 0.42), 18.6 ± 3.56% extend laterally (p = 0.79), and 12.8 ± 2.71% have extensions in both directions (p = 0.17); n = 7 sets of data binned from multiple brains (for details, see Materials and Methods)].

Figure 6.

Overexpressing Liprin-α, Liprin-β, or Trio in syd-1 mutant R7s rescues their defects to different degrees. A–C, Medullas of adult mosaic animals in which homozygous R7s express Syt–GFP (green), and all R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. A, syd-1^CD mutant R7 axons. B, Expressing Liprin-α in syd-1^CD mutant R7 axons partially restores their contact with M6 but does not prevent the formation of extensions. C, Expressing trio in syd-1^CD mutant R7 axons completely restores their contact with M6 and considerably decreases the frequency of extensions. D, The percentages of syd-1^CD mutant R7 axons that fail to contact M6 when overexpressing no transgene, wild-type Liprin-α, wild-type Liprin-β, or wild-type trio. Error bars represent SEM. This syd-1^CD mutant R7 defect (22.3 ± 1.36%; n = 21 brains) is partially rescued by expression of Liprin-α (11.3 ± 1.33%; n = 14 brains; p < 0.0001), fully rescued by expression of trio (2.04 ± 0.501%; n = 15 brains; p < 0.0001), and unaffected by expression of Liprin-β (20.1 ± 1.21%; n = 13 brains; p = 0.15). The difference between rescue by Liprin-α and trio is significant (p < 0.0001). E, The percentages of syd-1^CD mutant R7 axons that have extensions when overexpressing no transgene, wild-type Liprin-α, wild-type Liprin-β, or wild-type trio. Error bars represent SEM. This syd-1^CD mutant R7 defect (23.7 ± 1.52%; n = 21 brains) is partially rescued by expression of trio (15.5 ± 1.59%; n = 15 brains; p < 0.001) but is not rescued by expression of Liprin-α (28.4 ± 1.70%; n = 14 brains; p = 0.052) or Liprin-β (24.6 ± 2.22%; n = 13 brains; p = 0.38). The difference between rescue by Liprin-α and trio is significant (p < 0.0001).

Figure 7.

trio and syd-1 have similar loss- and gain-of-function R7 phenotypes. A–F, Medullas of adult mosaic animals in which homozygous R7s express Syt–GFP (green), and all R7 and R8 axons are labeled with mAb24B10 (red). Scale bar, 5 μm. trio³ mutant R7s often fail to contact the M6 layer (A, arrow) and project thin extensions beyond M6 (B, arrowhead). trio¹ mutant R7s have the same two defects but at a lower frequency (data not shown). C, The percentages of LAR²¹²⁷ mutant R7 axons that fail to contact M6 when overexpressing no transgene, wild-type trio, or wild-type syd-1. Both Trio and Syd-1 significantly rescue this defect. Error bars represent SEM. The LAR²¹²⁷ mutant R7 defect (83.0 ± 2.09%; n = 11 brains) is partially rescued by expression of trio (54.8 ± 1.99%; n = 10 brains; p < 0.0001) or syd-1 (54.6 ± 2.37%; n = 10 brains; p < 0.0001). The difference between rescue by syd-1 or trio is not significant (p = 0.95). D, LAR²¹²⁷ mutant R7 axons fail to contact the M6 layer (arrows). Expressing trio (E) or syd-1 (F) in LAR²¹²⁷ mutant R7 axons restores their contact with M6 to similar degrees.